The 2022 On-site Padua Days on Muscle and Mobility Medicine hosts the University of Florida Institute of Myology and the Wellstone Center, March 30 - April 3, 2022 at the University of Padua and Thermae of Euganean Hills, Padua, Italy: The collection of abstracts

H Lee Sweeney; Stefano Masiero; Ugo Carraro

doi:10.4081/ejtm.2022.10440

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

LECTURE: The crucial role of force transmission in muscle function: Focus on cytoskeleton and myotendinous junction Part A: Cytoskeletal specialization and mechanoprotection in fast and slow skeletal muscles. Insights from single-fiber proteomics and comparison with cardiac muscle

Stefano Schiaffino ^1,^✉, Leonardo Nogara ^1,², Jane-Lise Samuel ³, Martina Baraldo ^1,², Bert Blaauw ^1,^2,^✉, Carlo Reggiani ², Marta Murgia ^2,⁴

Abstract

The mechanical stress produced by active contraction of skeletal muscle may cause disruption of sarcolemma and sarcomeres, especially when contraction is combined with stretching (eccentric muscle contractions). However, force-induced muscle damage is normally prevented or limited by the robust scaffold of the muscle cytoskeleton and by a variety of “force buffers” or “shock absorbers” embedded in the cytoskeleton. In addition, disrupted muscle structures can be fixed by specialized repair systems, such as membrane resealing systems. Not surprisingly, mutations of genes coding for cytoskeleton and mechanoprotection proteins cause muscle pathology and myopathies. Proteomic approaches for the first time provide a global view of the proteins present in single muscle fibers¹ and specific myofiber domains, such as the myotendinous junction². We have recently observed that fast and slow fibers in human skeletal muscle differ in the molecular composition of the cytoskeleton and mechanoprotection systems.³ Interestingly, data mining of a recent proteomic study of the human heart⁴ reveals that some of these differences are also found in the fast atrial compared to the slow ventricular myocardium. These findings point to cell type-specific adaptations to contraction-dependent mechanical stress in both skeletal and cardiac muscle.

Key Words: Skeletal muscle, cardiac muscle, cytoskeleton, mechanoprotection, proteomics

References

1.Murgia M, Toniolo L, Nagaraj N, Ciciliot S, Vindigni V, Schiaffino S, Reggiani C, Mann M. Single Muscle Fiber Proteomics Reveals Fiber-Type-Specific Features of Human Muscle Aging. Cell Rep. 2017. Jun 13;19(11):2396-2409. doi: 10.1016/j.celrep.2017.05.054. [DOI] [PubMed] [Google Scholar]
2.Karlsen A, Gonzalez-Franquesa A, Jakobsen JR, Krogsgaard MR, Koch M, Kjaer M, Schiaffino S, Mackey A, Deshmukh AS. The proteomic profile of the human myotendinous junction. iScience. 2022; doi: 10.1016/j.isci.2022.103836. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Murgia M, Nogara L, Baraldo M, Reggiani C, Mann M, Schiaffino S. Protein profile of fiber types in human skeletal muscle: a single-fiber proteomics study. Skelet Muscle. 2021. Nov 2;11(1):24. doi: 10.1186/s13395-021-00279-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Doll S, Dreßen M, Geyer PE, Itzhak DN, Braun C, Doppler SA, Meier F, Deutsch MA, Lahm H, Lange R, Krane M, Mann M. Region and cell-type resolved quantitative proteomic map of the human heart. Nat Commun. 2017. Nov 13;8(1):1469. doi: 10.1038/s41467-017-01747-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Part B: The myotendinous junction: the weakest link of the muscle-skeleton force transmission chain?

Abigail L Mackey ^1,^✉

Abstract

For limb movement to occur, force must be transmitted from the myofilament cross-bridges to the bones. A critical link in this chain is the interface between the muscle fibres and tendon – the myotendinous junction (MTJ). Connecting these two tissues with greatly differing mechanical properties is no small engineering feat. To transmit, and withstand, high tensile forces, the myofibre sarcolemma and its basement membrane are highly folded as the tip of the myofibre interdigitates with the tendon on the other side of the MTJ (Figure 1), thus increasing the total contact area between muscle and tendon. In addition to a structural specialization, the MTJ is characterized by the presence of proteins not expressed elsewhere in the myofiber (or tendon). For example, collagen type XXII is found in the basement membrane only at the myofiber tips (Koch et al., 2004),¹ where it has been shown in the zebrafish to play a vital role in MTJ integrity (Charvet et al., 2013).² Single nuclei RNA sequencing in animals has revealed a distinct transcriptional profile of the myonuclei at the tips of the fibers (Dos Santos et al., 2020),³ further underlining the specialization of this region. We recently determined the proteomic composition of the human MTJ (Karlsen et al., 2022),⁴ with the presence of several obscure proteins raising additional questions about the biology of the tips of the muscle fibres and their attachment to the tendon. Paradoxically, despite this high degree of specialisation, the MTJ remains susceptible to strain (rupture) injury – healing from which is often incomplete, thus representing a major unsolved clinical challenge.

Figure 1. — Confocal microscope single slice image of the tip of a single human muscle fibre, teased from isolated fascicles of semitendinosus muscle obtained during anterior cruciate ligament reconstruction surgery. The intricate folds of the myofibre sarcolemma are clearly visible (dystrophin, magenta) at the tip, where it interdigitates with the tendon (left, unstained). Myofibre sarcomere striations are displayed in green (phalloidin), and nuclei in blue (Hoechst). Scale bar 20 microns.

Key Words: Myotendinous junction, force transmission, muscle injury, human tissue

References

1.Koch M, Schulze J, Hansen U, Ashwodt T, Keene DR, Brunken WJ, Burgeson RE, Bruckner P, Bruckner-Tuderman L. (2004). A novel marker of tissue junctions, collagen XXII. J Biol Chem. 279, 22514-22521. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Charvet B, Guiraud A, Malbouyres M, Zwolanek D, Guillon E, Bretaud S, Monnot C, Schulze J, Bader HL, Allard B, Koch M, Ruggiero F. (2013). Knockdown of col22a1 gene in zebrafish induces a muscular dystrophy by disruption of the myotendinous junction. Development 140, 4602-4613. [DOI] [PubMed] [Google Scholar]
3.Dos Santos M, Backer S, Saintpierre B, Izac B, Andrieu M, Letourneur F, Relaix F, Sotiropoulos A, Maire P. (2020). Single-nucleus RNA-seq and FISH identify coordinated transcriptional activity in mammalian myofibers. Nat Commun 11, 5102. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Karlsen A, Gonzalez-Franquesa A, Jakobsen JR, Krogsgaard MR, Koch M, Kjaer M, Schiaffino S, Mackey AL, Deshmukh AS. (2022). The proteomic profile of the human myotendinous junction. iScience 25, 103836. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION I: Mechanisms of muscle atrophy: from UPS implication in rodent models to human biomarkers

Daniel Taillandier ^1,^✉, Dulce Peris Moreno ¹, Cécile Polge ¹

Abstract

The ubiquitin proteasome system (UPS) is a major player of skeletal muscle wasting, a common characteristic of many diseases (cancer, sepsis, heart failure, kidney diseases, etc.) that negatively impacts treatment and life prognosis. In the early 2000, the notion of “atrogenes” (atrophy-related genes) was proposed for genes that were systematically up regulated during catabolic situations in rodents, which included 2 founding members, the E3 ubiquitin ligases MAFbx/Atrogin-1 and MuRF1/TRIM63.¹ Interestingly, MuRF1 is so far the only E3 ligase known for targeting several sarcomeric proteins (a-actin, MYHC, Troponin I, telethonin) and we recently identified the E2 ubiquitin conjugating enzymes that bring the catalytic activity for MuRF1-dependent protein degradation.^2,3 Besides rodent models, we and others demonstrated that MAFbx and MuRF1 are also gold standard atrogenes in human pathologies and we recently found that numerous proteins may be part of a muscle atrophy program in chronic kidney disease and lung cancer patients.⁴ In addition, we have identified blood markers that reflect skeletal muscle loss in patients suffering from several pathologies.

References

1.Peris-Moreno D., Taillandier D., Polge C. MuRF1/TRIM63, Master Regulator of Muscle Mass. Int. J. Mol. Sci. 2020, 21, 6663. doi:10.3390/ijms21186663 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Polge C., Cabantous S., Deval C., Claustre A., Hauvette A., Bouchenot C., Aniort J., Béchet D., Combaret L., Attaix D., Taillandier D. A Muscle-Specific MuRF1-E2 Network Requires Stabilization of MuRF1-E2 Complexes by Telethonin, a Newly Identified Substrate. J. Cachexia Sarcopenia Muscle 2018, 9, 129–145. DOI: 10.1002/jcsm.12249 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Peris-Moreno D., Malige M., Claustre A., Armani A., Coudy-Gandilhon C., Deval C., Béchet D., Fafournoux P., Sandri M., Combaret L., Taillandier D, Polge C. UBE2L3, a Partner of MuRF1/TRIM63, Is Involved in the Degradation of Myofibrillar Actin and Myosin. Cells 2021, 10, 1974. 10.3390/cells10081974 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Aniort J., Stella A., Philipponnet C., Poyet A., Polge C., Claustre A., Combaret L., Béchet D., Attaix D., Boisgard S., Filaire M., Rosset E., Burlet-Schiltz O., Heng A-E, Taillandier D. Muscle wasting in patients with end-stage renal disease or early-stage lung cancer: common mechanisms at work. J. Cachexia Sarcopenia Muscle 2019, 9, 129–145. DOI: 10.1002/jcsm.12376. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes

Pascal Maire

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Novel players in muscle mass regulation

Marco Sandri ^1,^✉

Abstract

Maintaining the integrity of organelles and proteins despite the cellular stresses that arise from environmental pressure is essential for life. The time-dependent accumulation of cellular damage is widely considered to be the general cause of cellular degeneration. Decline of the protein quality control, called the loss of proteostasis, contributes to impairment of macromolecule function and is considered among the primary hallmark of aging and disease onset. All cells take advantage of an array of mechanisms to preserve the stability and functionality of their proteins or to remove them when they are irreversibly damaged. The two the most important cellular housekeeping and pro-survival pathway are autophagy and ubiquitin proteasome (UPS) whose main action is to remove damaged proteins/organelles and generate molecules that sustain cellular core metabolism. Autophagy, like the ubiquitin-proteasome system, strictly regulates diverse cellular functions through its selectivity. I will describe the mutual regulation between the UPS and autophagy and their involvement in protein turnover and disease onset in post mitotic tissues.

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Loss of MuRF1 prevents skeletal muscle wasting and weakness, and slows the rate of tumor growth, in mice bearing pancreatic tumors

Daria Nereid ^1,², Orlando Latino ³, Christopher Lopez ¹, David W Hammers ⁴, H Lee Sweeney ^4,^✉, Glenn A Walter ⁵, Sarah M Judge ¹, Andrew R Judge ¹

Abstract

Skeletal muscle wasting and dysfunction in the context of cancer represents a major debilitating condition which contributes to a deterioration in physical function and reduced quality of life, and limits the ability of cancer patients to withstand anti-cancer treatments. Unfortunately, there are no currently available medical treatments for cancer-induced muscle pathology which is due, in part, to an incomplete understanding of the molecular mechanisms which are causative in the condition. The E3 ubiquitin ligase MuRF1 is required for muscle atrophy in several pathophysiological conditions which associate with muscle atrophy. Here we tested the requirement of MuRF1 for cancer-induced muscle atrophy using MuRF1 KO (MuRF1-/-) mice, or wild type (WT) controls, and an orthotopic pancreatic cancer (KPC) model. WT mice reached experimental endpoint 14 to 17 days following KPC cell injection, as defined by significant body weight loss (-12%) and deterioration of body condition score, with an average tumor mass of 1.9 ± 0.5 g. At this same time point, MuRF1-/- mice were weight stable (-0.6%), and showed ~84% lower tumor mass (0.3 ± 0.1 g) compared to WT mice. When allowing tumor growth to progress in MuRF1-/- mice, it took 21-36 days to reach a comparable tumor mass to that of WT mice at days 14-17. Moreover, when WT and MuRF1-/- mice presenting matching tumor sizes were compared, MuRF1-/- mice showed preserved muscle mass, with no muscle wasting observed in the triceps surae (-0.1% in MuRF1-/- vs. -9% in WT mice) and attenuated wasting observed in tibialis anterior (-5% in MuRF1-/- vs. -18% in WT mice). In addition to preserving muscle mass, MuRF1 deletion also protected against cancer- induced muscle weakness. Indeed, the soleus and diaphragm of WT mice showed a 32% and 46% reduction in specific tetanic force, respectively, whereas no such muscle weakness was observed in MuRF1-/- mice (+20% and +3% in soleus and diaphragm respectively). Skeletal muscle proteome and ubiquitinome analyses were conducted to examine the mechanisms through which MuRF1 deletion conferred its protection against cancer-associated muscle wasting and dysfunction. These analyses revealed that KPC-tumor-burden induces, in a MuRF1-independent manner, an early and sustained increase in the abundance of proteins involved in the acute phase response as well as in extracellular matrix remodeling, while proteins involved in protein translation and oxidative phosphorylation showed a MuRF1- dependent reduction in their abundance at time points reflective of cachexia initiation and progression. Identification of proteins ubiquitinated in a MuRF1-dependent manner further revealed an early and sustained enrichment in proteins related to muscle contraction, proteasome complex and glucose homeostasis. In conclusion, our work suggests that MuRF1 is required for the normal muscle wasting and weakness that occurs in KPC tumor bearing mice, and is also required for normal tumor growth. This suggests that MuRF1-dependent muscle protein breakdown may be required to supply the tumor with nutrients needed for its growth.

Key Words: Cancer cachexia, muscle atrophy, E3-ubiquitine ligase, pancreatic cancer

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Insights into the mechanisms of muscle wasting at high age

Daniel J Ham ¹, Anastasiya Börsch ¹, Giulia Milan ¹, Nitish Mittal ¹, Mihaela Zavolan ¹, Markus A Ruegg ^1,^✉

Abstract

Sarcopenia is defined as the loss of muscle mass and function and is considered a major contributor to loss of life quality at high age. The mammalian target of rapamycin complex 1 (mTORC1) is well known to regulate cell size by controlling cap-dependent protein translation in response to growth factors and nutrients. In skeletal muscle, the mTORC1 inhibitor rapamycin prevents load-induced hypertrophy. In contrast to expectations, mTORC1 signaling is high in sarcopenic muscle, its constant activation causes atrophy in most muscles and prolonged treatment with rapamycin (i.e. inhibition of mTORC1) prolongs lifespan in many species. Based on these observations, we have addressed the role of PKB/Akt-mTORC1 signaling in age-related sarcopenia. We find that i) mice depleted of the mTORC1 inhibitor TSC1 in skeletal muscle show a phenotype reminiscent of sarcopenia and ii) inhibition of mTORC1 by rapamycin has an overly anti-sarcopenic effect. Molecular analysis also shows a strong overlap in the gene signatures of aging and mTORC1 activation. We also show that caloric restriction (CR), the best-studied anti-aging intervention thought to act via inhibition of the nutrient-sensing mTORC1, has an anti-sarcopenia effect. Interestingly, molecular analysis reveals that the CR effect is largely distinct from the effect of rapamycin-mediated mTORC1 inhibition. Consistent with this, the combination of CR and rapamycin has additive effects on the prevention of sarcopenia. Finally, we also provide evidence that hyperactivation of PKB/Akt is detrimental to muscle function in skeletal muscle with high mTORC1 activity. In summary, our data indicate that great caution should be exercised with intervention strategies designed to increase muscle size by interfering with the pathways that control the balance between protein synthesis and protein degradation

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Molecular mechanisms of cancer-induced muscle wasting

Andrew R Judge ¹, Andrew C D’Lugo s ¹, Jose G Trevino ², Sarah M Judge ^1,^✉

Abstract

Purpose of this study was to assess the effect of a course of homeopathic preparations using acupuncture points on the severity of Cancer cachexia is a multifactorial condition characterized by skeletal muscle loss that impairs longevity and quality of life of the vast majority of cancer patients.^1,2 However, the ability to develop therapeutic strategies to counter cachexia is impeded by the limited understanding of the underlying mechanisms of cachexia in human cancer patients. The purpose of this study was therefore to identify the proteomic signature of skeletal muscle obtained from cachectic pancreatic ductal adenocarcinoma (PDAC) patients, who exhibit one of the highest rates of cachexia.^3,4 Muscle biopsies of the rectus abdominis were obtained from cachectic PDAC patients (n=8) undergoing tumor resection surgery as well as age and sex-matched non-cancer controls (n=6). Total proteome profiling of skeletal muscle protein isolate was performed using 10-plex tandem mass tag labeling and liquid chromatography tandem mass spectrometry (LC-MS/MS). MS/MS spectra were evaluated using SEQUEST and differentially expressed proteins were identified through the UniProt Homo Sapiens database [adjusted P value (FDR) ≤0.01; -1.25≥fold change≥1.25]. The total number of identified peptides and proteins are shown in Figure 1, which further shows that 383 proteins were identified to be differentially expressed in the skeletal muscle of PDAC patients compared to controls. To identify altered biological processes within the skeletal muscle of cachectic PDAC patients, differentially expressed proteins (DEP) were analyzed using several bioinformatic platforms. The results of these bioinformatic analyses will be presented, along with immunohistochemical analyses to further support changes in select proteins of interest.

Key Words: Cachexia, pancreatic cancer, proteomics

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Neuromuscular basis of disuse muscle atrophy and weakness

Marco Narici ^1,^✉, Elena Monti ¹, Martino Franchi ¹, Fabio Sarto ¹, Sandra Zampieri ¹, Carlo Reggiani ¹

Abstract

Muscle atrophy is an inevitable consequence of chronic disuse. Until recently, in non-pathological conditions, the main cause of muscle wasting induced by inactivity was considered to be the lack of mechanical loading, resulting in a depression of protein synthesis and an increase in protein breakdown. However, there is now increasing evidence that neurodegenerative processes, triggered by inactivity, contribute to the loss of muscle mass and function. Indeed, instability of the neuromuscular junction (NMJ) and onset of denervation/reinnervation processes have been recently found after short-term and long-term inactivity in humans caused by bed rest or unilateral lower limb suspension (ULLS) (Salanova et al. 2011, Arentson-Lantz et al. 2016, Demangel et al. 2017, changes seems extremely fast as initial signs of fibre denervation/reinnervation, evidenced from the presence of neural cell adhesive molecule (NCAM) positive myofibres, can be observed after just 3-days (Demangel et al. 2017) and 10 days of bed rest (Monti et al. 2021) (Fig. 1). These changes are also accompanied by instability of the NMJ, as shown either by increased serum levels of c-terminal agrin fragment (CAF) after 10-day bed rest and after 10-day ULLS, or by a decreased expression of Homer proteins in muscle biopsies, after 60-day bed rest (Salanova et al 2011). These findings of NMJ instability and partial fibre denervation were associated with an impairment of sarcoplasmic reticulum (SR) calcium handling, since a significant depression in the caffeine-induced release of Ca2+ by the SR was found after 10-day bed rest (Monti et al. 2021), suggesting an impairment of excitation-contraction coupling. These findings support the view that the early onset of NMJ instability and impairment in SR function are eligible mechanisms contributing to the greater decline in muscle force than in muscle size observed in these bed rest studies and in most models of muscle disuse.

Key Words: Skeletal muscle, inactivity, muscle atrophy, neuromuscular junction, denervation

Acknowledgements

Funding by ASI, MARS-PRE Project n. DC-VUM-2017-006, is acknowledged.

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Activation of Akt-mTORC1 signaling reverts cancer-dependent muscle wasting

Alessia Geremia ^1,², Roberta Sartori ¹, Martina Baraldo ^1,², Leonardo Nogara ^1,², Valeria Balmaceda ¹, Georgia Ana Dumitras ^1,², Stefano Ciciliot ¹, Marco Scalabrin ^1,², Hendrik Nolte ³, Bert Blaauw ^1,^2,^✉

Abstract

Loss of muscle mass is a debilitating aspect in numerous forms of cancer. Here, we use various transgenic mouse strains to determine the role of Akt-mTORC1 signaling in skeletal muscle during cancer cachexia. We find that loss of Raptor only in muscle fibers does not aggravate cancer-related muscle wasting. On the contrary, activation of Akt-mTORC1 signaling is sufficient to completely revert muscle wasting, normalize the muscle transcriptome, and prevent protein degradation.

Key Words: Cancer cachexia, mTOR, Raptor, Akt, muscle growth

References

Geremia A, Sartori R, Baraldo M, Nogara L, Balmaceda V, Dumitras GA, Ciciliot S, Scalabrin M, Nolte H, Blaauw B. Activation of Akt-mTORC1 signaling reverts cancer-dependent muscle wasting. Journal of Cachexia, Sarcopenia and Muscle, in press. [DOI] [PMC free article] [PubMed] [Google Scholar]
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SESSION II: Mitochondrial fusion proteins and their role in metabolism and in inflammation

Antonio Zorzano

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Dynamic regulation of muscle mass by dark matter DNA

Jorge Ruas

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The role of IL-6 signaling in muscle growth, atrophy, and wasting

Antonio Musarò ^1,^✉

Abstract

Interleukin-6 (IL-6) is a soluble mediator with pleiotropic actions on different tissues and organs. The multifaceted nature of IL-6 action can be ascribed to the multi-level regulation of the different signaling pathways that it is able to induce. Although IL-6 is produced by several cell types and it can signal through different receptor complexes, inducing a variety of intracellular pathways, its expression and signal- transduction is strictly controlled (Figure 1). IL-6 expression is regulated at the transcriptional and post-transcriptional level, while the induction of the signaling cascade is negatively regulated by systems of internalization/degradation of receptor subunits and by the action of negative feedback molecules. Thus, acting as a warning molecule, the abundance and persistence of IL-6 need a fine regulation in order to limit the powerful action of the cytokine at the physiologic stimuli. IL-6 overexpression is able to exacerbate the dystrophic muscle phenotype, fostering pathologic changes in skeletal muscle and closely approximating the disease progression of DMD patients,^1,2 whereas short inhibition of IL-6 activities, by a neutralizing antibody directed against IL6R, is beneficial to dystrophic muscle decreasing inflammation−induced myonecrosis, favoring the maintenance of muscle function, and preventing exercise-induced fiber damage and loss of muscle strength.³ In addition, overexpression of circulating IL-6 enhances the generation and accumulation of free radicals, influencing the local redox homeostasis, inducing the establishment of prooxidative conditions in skeletal muscle tissue.⁴

Key Words: IL-6, muscle homeostasis, muscle regeneration

References

1.Pelosi L, Berardinelli MG, Forcina L, Spelta E, Rizzuto E, Nicoletti C, Camilli C, Testa E, Catizone A, De Benedetti F, Musarò A. (2015). Increased levels of interleukin-6 exacerbate the dystrophic phenotype in mdx mice. Human Molecular Genetics. 24, 6041-6053. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Diaphragm ABCB6 overexpression preserves respiratory function following doxorubicin chemotherapy treatment

Mustafa Ozdemir ¹, Matthew P Bomkamp ¹, Vivian Doerr ¹, Branden L Nguyen ¹, Ashley J Smuder ^1,^✉

Abstract

Doxorubicin (DOX) is a highly effective chemotherapeutic agent used to treat a wide variety of cancers. However, its clinical use is associated with respiratory muscle weakness, resulting in fatigue, dyspnea and exercise intolerance.¹ While no therapeutic countermeasures exist to prevent the development of DOX-induced respiratory dysfunction, previous preclinical work has demonstrated that acute endurance exercise preconditioning prior to DOX administration was sufficient to preserve diaphragm muscle force production, maintain fiber cross-sectional area and prevent ventilatory deficits during a hypercapnic challenge. These protective effects were associated with the exercise-induced upregulation of the mitochondria-localized ATP-binding cassette transporters in the diaphragm, including ABCB6, which is hypothesized to play a role in maintaining mitochondrial redox balance and heme homeostasis.² To determine if ABCB6 overexpression, independent of exercise, is sufficient to prevent DOX-induced respiratory dysfunction, female Sprague-Dawley rats were randomly assigned to receive either rAAV9-MHCK7-rABCB6 to overexpress ABCB6 in the diaphragm or saline. These groups were further divided to receive saline or DOX (20mg/kg i.p.) treatment. Performance of an exercise tolerance test forty-eight hours following treatment revealed significant reductions in run time to fatigue in both DOX-treated groups. However, ventilatory function in response to hypercapnia was only reduced in the saline-saline rats compared to the saline-DOX rats. Additionally, ABCB6 overexpression attenuated the DOX-induced reduction in diaphragm muscle force production. These findings support a role for ABCB6 in the preservation of respiratory function following acute exposure to DOX.

Key Words: Chemotherapy, diaphragm, respiratory, ABCB6, mitochondria

References

1.Singal PK, Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl J Med. 1998. Sep 24;339(13):900-5. doi: 10.1056/NEJM199809243391307. [DOI] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Skeletal muscle homeostasis in an experimental model of hind limb ischemia

Mattia Scalabrin ^1,^✉, Annabel Critchlow ¹, Viktor Engman ¹, Nadira Yudalsheva ², T Scott Bowen ¹

Abstract

Low extremity arterial disease (LEAD) is the most common form of peripheral vascular disease and is often a direct consequence of the atherosclerotic process. Characterised by chronic decreases in lower limb blood flow, this progressive disease results in symptoms ranging from pain, muscle weakness, to severe exercise intolerance. Several animal models have been used to study the angiogenesis and muscle remodelling process in LEAD in order to progress novel therapies for patients. However, conflicting information is available on the effect of this condition on lower hind limb skeletal muscle remodelling, thus hindering the development of treatments to prevent muscle dysfunction in LEAD. Using a mouse model of severe lower hindlimb ischemia (femoral artery ligation), we found ischemic muscle at 7 days (n=5) demonstrated a loss of wet-mass and fibre cross-sectional area (CSA) (t(8)=2.51, p<0.05) alongside reduced type IIx fibre proportion (-20%) vs. non-ischemic muscle (t(8)=2.159, p<0.05; n=5). Muscle wasting was associated with excessive autophagy rather than other catabolic pathways, with autophagy and mitophagy markers significant upregulated. At 28 days following ischemia (n=5) vs control limb (n=5), muscle regeneration allowed increased muscle mass and fibre CSA (p>0.05) with markers of autophagy upregulated at 7 days returned to baseline values. Furthermore, expression of Sestrins was dysregulated at 7 days and normalised at 28 days following ischemic insult. In conclusion, these data indicate severe lower hindlimb ischemia is associated with early-activation of autophagy that may mediate early-onset muscle wasting. Whether hypoxia-associated ischemia triggers activation of Sestrin 2 and subsequent autophagy-dependent remodelling remains uncertain.

Key Words: Ischemia, skeletal muscle, autophagy, Sestrins

References

McDermott MM, Ferrucci L, Gonzalez-Freire M, Kosmac K, Leeuwenburgh C, Peterson CA, Saini S, Sufit R. Skeletal Muscle Pathology in Peripheral Artery Disease: A Brief Review. Arterioscler Thromb Vasc Biol. 2020. Nov;40(11):2577-2585. doi: 10.1161/ATVBAHA.120.313831. Epub 2020 Sep 17. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Exercise-dependant remodelling of the sarcotubular system: the role of temperature and pH

Barbara Girolami ¹, Matteo Serano ¹, Laura Pietrangelo ¹, Feliciano Protasi ^1,^✉

Abstract

Skeletal muscle function is regulated by intracellular Ca²⁺ levels. Two main mechanisms control movements of Ca²⁺ ions from intracellular stores (i.e. the sarcoplasmic reticulum, SR) and from extracellular space: i) excitation-contraction (EC) coupling; and ii) store-operated Ca²⁺ entry (SOCE). SOCE allows recovery of extracellular Ca2+ during prolonged muscle activity, when the SR undergoes depletion. We recently discovered that prolonged exercise leads to formation of Calcium Entry Units (CEUs),¹ intracellular junctions located at the I band that are formed by two distinct elements: SR-stacks and transverse tubules (TTs)^1,2. Assembly of CEUs during exercise promotes the interaction between STIM1 and Orai1, the two main proteins that mediate SOCE, and increases muscle resistance to fatigue in presence of extracellular Ca²⁺.

The molecular mechanisms underlying the exercise-dependent remodeling of SR and TT leading to CEU assembly remain to be fully elucidated.³ Here, we first verified whether CEUs can assemble ex-vivo (in absence of blood supply and innervation) subjecting excised EDL muscles from mice to an ex-vivo incremental fatigue protocol (80Hz tetanus stimulation lasting 45 minutes): the data collected demonstrate that CEUs can assemble ex-vivo in isolated EDL muscles. We then evaluated if intracellular parameters that are affected by exercise, such as temperature and pH, may influence the assembly of CEUs. We found that higher temperature (36°C vs. 25°C) and lower pH (7.2 vs. 7.4) promotes formation of CEUs increasing the % of fibers containing SR stacks, the n. of SR stacks/area, and also the elongation of TTs at the I band. Importantly, increased assembly of CEUs at higher temperature (36^oC) or at lower pH (7.2) correlated with increased fatigue resistance of EDL muscles in presence of extracellular Ca²⁺, suggesting that CEUs assembled ex-vivo are functional.

Key Words: calcium entry unit (CEU), store-operated Ca²⁺ entry (SOCE)

References

1.Boncompagni S, Michelucci A, Pietrangelo L, Dirksen RT, Protasi F. Exercise-dependent formation of new junctions that promote STIM1-Orai1 assembly in skeletal muscle. Sci Rep. 2017. Oct 27;7(1):14286. doi: 10.1038/s41598-017-14134-0. Erratum in: Sci Rep. 2018 Nov 27;8(1): 17463. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Form vs. function: strategies to deliver IGF-I for muscle therapeutics

Elisabeth R Barton ^1,^✉

Abstract

Skeletal muscle is a post-mitotic tissue, yet has the ability to regenerate through the activation of muscle satellite cells and the coordinated actions of multiple growth factors. A key player in the regeneration process is Insulinlike Growth Factor-I (IGF-I).¹ IGF-I helps to resolve damage by promotion of satellite cell proliferation and differentiation, suppression of proinflammatory cytokines, and fiber formation. A long-standing goal in our laboratory has been to optimize IGF-I forms to improve its actions in skeletal muscle. While the most well characterized form of IGF-I is the small mature 7kD protein, complexity of alternative splicing and posttranslational modification leads to several additional forms of this growth factor. These larger forms have been shown to have more efficient storage in muscle tissue as well as enhanced receptor activation,^2,3 and raise the possibility that improvements in therapeutic potential could occur through the use of a different IGF-I form. To understand the necessity of glycosylated and mature IGF-I forms, gene editing was employed to remove glycosylation sites in the C-terminal portion of IGF-IA, and to eliminate the ability for cleavage of the C-terminal extensions of all IGF-I forms (Figure 1).⁴ Mice were viable, with normal force generation. To determine the effects on regeneration after acute injury, cardiotoxin injections were performed in male and female mice from all genotypes, and monitored for changes in fiber size. In both 2ND and 3RA genotypes, there was an enhanced recovery of fiber size following injury. This suggests that the pro-IGF-I forms, common to both genotypes may contribute to accelerating the resolution of damage in muscle. Future studies will take advantage of these forms to develop more effective therapeutics for muscle.

Key Words: Muscle regeneration, IGF-I forms

References

1.Vassilakos G, Barton ER. Insulin-Like Growth Factor I Regulation and Its Actions in Skeletal Muscle. Compr Physiol. 2018. Dec 13;9(1):413-438. doi: 10.1002/cphy.c180010. [DOI] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Metabolic link between mitochondrial and contractile abnormalities

Leonardo F Ferreira ^1,^✉, Ravi Kumar ¹, Guilherme Bresciani ¹, Vinicius Mariani ¹

Abstract

Impaired mitochondrial function and abnormal muscle bioenergetics are evident in several diseases and aging and have been associated with loss of muscle contractile function. However, a direct link between metabolic dysfunction and loss of muscle force or power has not been established. Mitochondrial respiration impairments lead to accumulation of metabolites of the tricarboxylic acid cycle that are upstream of the electron transport system such as succinate and succinyl-derivatives.¹ High fat diet, diabetes, hypoxia/ischemia, cancer, and even exercise disrupt the metabolism succinate and succinyl-derivatives resulting in several fold increase in succinate.^1-5 We have found that postmenopausal heart failure with preserved ejection fraction (HFpEF) causes muscle mitochondrial dysfunction and weakness,⁶ which are accompanied by elevated plasma [succinate] that reflects increased cellular concentration and export from the most metabolically active tissues: adipose, liver, and skeletal muscle. This presentation will focus on preliminary findings showing that: 1) HFpEF disrupts the abundance of proteins involved in mitochondrial succinate transport and lysine succinylation, resulting in increased myofibrillar protein succinylation; 2) acute exposure of intact skeletal muscle and permeabilized single fiber to succinate, which causes succinylation of myofibrillar proteins, impairs isometric and isotonic contractile function; and 3) the effects of succinate in vitro are independent of ROS from mitochondrial complex II. Overall, our findings suggest that succinate and lysine succinylation are a novel link between metabolic and contractile dysfunction in health and disease.

Key Words: Heart failure, succinate, weakness, peak power

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Higher abundance of deletions and strand break damage within specific mitochondrial ETC genes are associated with functional performance in older adults

Sunil K Saini ², Anna Picca ¹, Emanuele Marzetti ¹, Christiaan Leeuwenburgh ^2,^✉

Abstract

The goal of this study was to investigate if higher abundance of deletions and strand break damage within specific Mt ETC genes are associated with functional performance in older adults. Healthy older adults (65.2 ± 6.7 years; n = 25) were enrolled in this study(1). The participants completed a 6-minute walk test with an average 6-minute walking distance of 420 (± 149) meters and a usual-paced walking speed over 4 meters of 0.86 (± 0.28) meters per second. We quantified in muscle-biopsy specimen the abundance of mtDNA deletion and SB damage and associated these with the recorded walking speed. Specifically, mtDNA regions were selected based on previous findings from our group as potential hotspots for damage (2). We determined (a) the abundance of the 4977 bp deletion, a common deletion present in human mtDNA, which results in loss of a mtDNA fragment that includes genes coding for ATPase6, ATPase8, cytochrome oxidase III, NADH dehydrogenase subunit 3 (ND3), ND4, ND4 subunit L (ND4L), and ND5; and (b) the abundance of mtDNA strand break damage within specific mtDNA regions that code for subunits of Complex I (ND1, ND2, ND4, ND5), Complex III (Cytochrome B6), Complex IV (Cytochrome Oxidase subunit II), and Complex V (ATPase subunit 6/8), as well as SB in the regulatory non-coding D-loop region. Please note that the highly sensitive mtDNA strand break method spans through regions coding for several ETC genes. We plotted the robust correlations of mitochondrial SBs in specific mtDNA regions with walking performance in 25 older adults (Fig). For example, a greater abundance of density of mtDNA SB damage (SB damage normalized to mtDNA copy number) within NADH dehydrogenase subunit 1/2 (ND1/2) and 4/5 (ND4/5) was strongly associated with poor walking performance (r = -0.59 and r = -0.70). Our data showed the powerful association and sensitivity of mtDNA SB in specific Mt ETC regions with walking performance. Conclusio n: This set of data indicates that mtDNA mutations in ETC components (strand breaks to ND1/2 and ND4/5) are strongly related to slower walking speed in older adults.

Key Words: Sarcopenia, Mitochondria, Mutations, Deletions Mt genome

References

Saini SK, McDermott MM, Picca A, Li L, Wohlgemuth SE, Kosmac K, Peterson CA, Tian L, Ferrucci L, Guralnik JM, Sufit RL, Leeuwenburgh C. Mitochondrial DNA damage in calf skeletal muscle and walking performance in people with peripheral artery disease. Free Radic Biol Med. 2020. Nov 20;160:680-689. doi: 10.1016/j.freeradbiomed.2020.09.004. Epub 2020. Sep 8 [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Molecular Antagonism between DUX4 and DUX4c Highlights a Potential Pathomechanism in Facioscapulohumeral muscular dystrophy

Massimo Ganassi ^1,^✉, Nicolas Figeac ¹, Magalie Reynaud ¹, Huascar Pedro Ortuste Quiroga ¹, Peter S Zammit ¹

Abstract

Aberrant expression of the transcription factor DUX4 from D4Z4 macrosatellite repeats on chromosome 4q35, and its transcriptome, associate with pathogenesis in Facioscapulohumeral muscular dystrophy (FSHD)(1, 2). In skeletal muscle cells, forced DUX4 expression halts cell proliferation and induces cell death (Figure 1). DUX4 binds DNA via homeodomains that have high sequence similarity to those of DUX4c, a closely related transcriptional regulator encoded by a single, inverted, mutated D4Z4 unit located centromeric to the D4Z4 macrosatellite array(3). However, DUX4c function and contribution to FSHD pathogenesis is unclear. To explore interplay between DUX4, DUX4c and DUX4-induced phenotype, we investigated whether DUX4c interferes with DUX4 function in human myogenesis. Constitutive expression of DUX4c rescued the DUX4-induced inhibition of proliferation and blunted the onset of cell death in human myoblasts (Figure 1). Functionally, DUX4 forces the nuclear translocation of β-CATENIN and disrupting canonical WNT signalling. However, concomitant overexpression of DUX4c restores β-CATENIN cytoplasmic/nuclear shuttling and the downstream transcriptional program (Figure 1). We identified a subset of genes involved in the β-CATENIN pathway that are differentially regulated between DUX4 and DUX4c and can separate FSHD from healthy muscle biopsies based on the expression pattern. Constitutive expression of DUX4c robustly suppresses expression of DUX4 target genes confirming molecular dueling. In line, FSHD myoblasts display upregulation of DUX4 and downregulation of DUX4c compared to aged-matched unaffected myoblast (Figure 2). Finally, blockade of WNT/β-CATENIN pathway rescue proliferation defect of FSHD myoblasts (Figure 2). Together, our study demonstrates that DUX4 alters cell viability via β-CATENIN activation and that DUX4c can counteract aspects of DUX4-mediated toxicity in human muscle cells, informing future interventions to ameliorate FSHD pathology.

Figure 2. — Human FSHD myoblasts display increased DUX4 expression, but reduced DUX4c level compared to unaffected myoblasts. Inhibition of B-catenin pathway rescues the proliferation defect of human FSHD myoblasts.

Key Words: FSHD, DUX4, DUX4c, β-CATENIN, apoptosis, myoblast, proliferation

References

1.Banerji CRS, Zammit PS. Pathomechanisms and biomarkers in facioscapulohumeral muscular dystrophy: roles of DUX4 and PAX7. EMBO Mol Med. 2021;13(8):e13695. Epub 2021/06/22. doi: 10.15252/emmm.202013695. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The p97/Nploc4 ATPase complex plays a role in muscle atrophy during cancer and amyotrophic lateral sclerosis

Andrea David Re Cecconi ¹, Mara Barone ¹, Simona Gaspari ¹, Massimo Tortarolo ¹, Caterina Bendotti ¹, Luca Porcu ², Giulia Terribile ¹, Rosanna Piccirillo ^1,^✉

Abstract

The p97 complex participates in the degradation of muscle proteins during atrophy upon fasting or denervation interacting with different adaptors.¹ We investigated whether and how it might also be involved in muscle wasting in cancer, where loss of appetite occurs, or amyotrophic lateral sclerosis (ALS), where motoneuron death causes muscle denervation and fatal paralysis.² The mRNA levels of p97 were induced in tibialis anterior (TA) of three distinct cancer cachectic mouse models but not in the non-cachectic 4T1 tumor. Similarly, p97 was high both in mRNA and protein in muscles from SOD1G93A mice. Electroporation of a shRNA for murine p97 into muscle reduced the fiber atrophy caused by colon adenocarcinoma C26 and ALS. When we interrogated a microarray we had previously generated for the expression of p97 adaptors, we found few of them induced in cachectic TA from C26-mice. By qPCR, we validated their inductions in TA of cachectic and ALS models and selected Nploc4 as the one most induced. Electroporation of a Crispr/Cas9 vector against Nploc4 into muscle reduced the fiber atrophy caused by C26 and ALS. Since Disulfiram (DSF) uncouples p97 from Nploc4,³ we treated atrophying myotubes with DSF, and found accumulated polyubiquitinated proteins and reduced degradation of long-lived proteins. DSF halves Nploc4 in the soluble muscle fraction and given to C26-mice limited the body and muscle weight loss, with no effect on tumor. The p97/Nploc4 complex plays a crucial role in muscle atrophy during these disorders and disrupting this complex might serve as a novel drug strategy.

Key Words: Muscle wasting, cancer cachexia, Amyotrophic Lateral Sclerosis, Nploc4, protein degradation

References

1.Piccirillo R, Goldberg AL. The p97/VCP ATPase is critical in muscle atrophy and the accelerated degradation of muscle proteins. EMBO J 2012;31:3334–3350. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Deciphering the cachexia-inducing signature

Roberta Sartori ^1,^2,^✉, Adam Hagg ³, Sandra Zampieri ^2,⁴, Camilla Pezzini ^1,², Stefano Merigliano ⁴, Paul Gregorevic ³, Marco Sandri ^1,²

Abstract

More than half of cancer patients are facing with a multifactorial syndrome characterized by excessive body weight loss, due to fat and lean mass loss, named cachexia.¹ The debilitating frailty experienced by cachectic individuals impairs quality of life increases morbidity and complications from cancer surgery, reduces both tolerance and responsiveness to cancer treatment regimes, complicating patient management, and ultimately accounting for up to 30% of deaths associated with advanced cancer.^2,3 As the underlying mechanisms of this multifactorial syndrome are incompletely defined, effective therapeutics have yet to be developed. The loss of muscle mass and strength is considered the most important clinical event in cancer cachexia, and a key predictor of poor outcomes (4, 5) However, cachexia is not an inevitable consequence of cancer. Variation has been noted with regards to the prevalence and severity of cachexia and the causes of such variation are unknown. Cachexia onset is the result of the interaction between cancer cells and the organism that hosts cancer growth. Tumor’s secreted pro-inflammatory factors depend from the type of cancer cells and from the interaction with organisms’ cells. The combinatorial action of soluble secreted mediators (secretome) by cancer cells and cells within the tumor microenvironment, activates different catabolic signaling pathways within different tissues, including skeletal muscle. The signatures present in one organism could explain inter-cancer and inter-individual variations in susceptibility to cachexia.

Key words: Duchenne Muscular dystrophy, Simvastatin, Gene therapy, AAV, minidystrophin Key Words: Cancer cachexia, muscle wasting, secreted mediators, signaling pathways

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Session III: The Challenges of AAV gene therapy in Duchenne Muscular Dystrophy

Francesco Muntoni ^1,^✉

Abstract

A number of different microdystrophin constructs are at various stages of clinical development. Early data on microdystrophin protein production in the biopsies of individuals treated in the higher dose categories are encouraging, but as the programs proceed, a number of issues that the field needs to address are becoming more evident:

Expectations. The degree of functional improvement following AAV gene therapy has attracted a lot of attention as if this was an essential aspect of the outcome, but in reality the protection of transduced muscle from progressive damage is a more relevant outcome.
Durability. As the transduced protein is a Becker-like microdystrophin, complete protection from further damage may be challenging, although the levels at which this AAV transgene is produced is significantly higher than those that BMD patients typically produce.
Efficacy in all tissues. Many of these microdystrophins lack domains that, in BMD patients with similar deletions, have been associated with higher risks of developing a more severe cardiomyopathy, suggesting different requirements between skeletal and cardiac muscles.
Dilution. As AAV does not integrate, and as muscle grows, and the number of myofiber nuclei increases between birth and early puberty, the optimal timing for using this approach requires careful assessment.
Immune responses. While AAV do not cause disease in the human, the doses that are administered systemically to these children are enormous and different types of innate and adaptive immune responses have been observed, together with the occurrence of autoimmunity against specific epitopes in rare genotypes

In my presentation I will provide information regarding these key issues.

Key Words: AAV gene therapy, Duchenne muscular dystrophy, tolerability and efficacy

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Alternative gene therapy approaches to DMD

Kevin M Flanigan ^1,^✉

Abstract

Significant enthusiasm exists for delivery of engineered microdystrophins as a treatment for Duchenne muscular dystrophy, and multiple clinical trials are underway using that approach. However, alternative gene therapy approaches exist, including delivery of surrogate genes or modifying gene structure at the genomic or mRNA level. Particularly promising are those that are directed toward restoration of a full-length and completely functional protein. One such therapy, directed toward boys who carry a duplication of DMD exon 2, has reached clinical trial (Clinicaltrials.gov NCT04240314). The AAV9 vector contains four non-coding U7snRNAs targeted to exon 2 definition elements, and preclinical studies have demonstrated dose-dependent expression of the full-length dystrophin protein, absence of toxicity, and absence of off-target splicing events. Three subjects have been dosed, including an infant, with biomarker evidence of an effect and with evidence of clinical stabilization. This promising approach can be extended to correct other common mutations, as well as considered as a personalized therapy for mutations that will allow full-length dystrophin restoration.

Key Words: Dystrophin, U7snRNA therapy, exon 2 duplications

References

Wein N, Dunn DM, Waldrop MA, Gushchina LV, Frair EC, Weiss RB, Flanigan KM. Absence of Significant Off-Target Splicing Variation with a U7snRNA Vector Targeting DMD Exon 2 Duplications. Hum Gene Ther. 2021. Nov;32(21-22):1346-1359. doi: 10.1089/hum.2020.315. [DOI] [PubMed] [Google Scholar]
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Zambon AA, Waldrop MA, Alles R, Weiss RB, Conroy S, Moore-Clingenpeel M, Previtali S, Flanigan KM. Italian DMD Network and the United Dystrophinopathy Project. Phenotypic Spectrum of Dystrophinopathy Due to Duchenne Muscular Dystrophy Exon 2 Duplications. Neurology. 2022. Feb 15;98(7):e730-e738. doi: 10.1212/WNL.0000000000013246. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Limitations to micro-dystrophin gene therapy for DMD

Cora Hart ¹, David Hammers ¹, Matthew Lee ¹, Michael Matheny ¹, H Lee Sweeney ^1,^✉

Abstract

Adeno-associated viruses (AAVs) containing versions of truncated dystrophin (micro-dystrophins) are being delivered to patients with Duchenne muscular dystrophy (DMD) in clinical trials. DMD is a progressive, childhood onset muscle wasting disease caused by mutations in the DMD gene that result in the loss of dystrophin protein in all muscle types.¹ These clinical gene therapies aim to overexpress a truncated version of dystrophin in striated muscle capable of achieving partial correction of the disease. We have examined this strategy with two different micro-dystrophins in a severe mouse model of DMD, the D2.mdx mouse,^2,3 using doses of AAV comparable to those used in the clinical trials. While significant correction of the skeletal muscle disease is observed, which slows skeletal muscle disease progression, a lethal acceleration of cardiac disease progression occurs with one of the micro-dystrophins, while the second has little or no impact on the heart. The detrimental impact on the heart appears to be caused by high levels of micro-dystrophin that result in competition between micro-dystrophin and utrophin at the cardiomyocyte membrane and by excessive protein accumulation. The micro-dystrophin with no cardiac impact does not outcompete utrophin to the same extent as the deleterious construct. While the significance of these observations for patients currently being treated with AAV-micro-dystrophin therapies is unclear, it suggests that micro-dystrophin treatments for the heart may need to be carefully titrated, and potentially dissociated from treatment for skeletal muscle, in order to achieve optimal benefits for both types of muscles.

Key Words: Duchenne muscular dystrophy, AAV, gene therapy, cardiomyopathy

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Micro-dystrophin-mediated utrophin displacement from cardiomyocyte sarcolemma in the D2.mdx mouse model of DMD

Young il Lee ^1,^✉, Michael K Matheny ¹, Cora C Hart ¹, David W Hammers ¹, H Lee Sweeney ¹

Abstract

Duchenne muscular dystrophy (DMD) results from mutations in the DMD gene and the subsequent loss of functional dystrophin protein.^1,2 In its absence, utrophin – the autosomal paralogue of dystrophin – is upregulated and compensates, albeit incompletely, for the loss of dystrophin.^3,4 Adeno-associated virus (AAV)-mediated gene delivery provides the means to restore the expression of missing proteins with the aim of reversing the pathology of such monogenic disorders. The size of the dystrophin gene, the largest in the human genome, however, makes it impossible to re-introduce full-length dystrophin. This limitation is partly overcome by the use of heavily truncated iterations of the dystrophin protein (micro-dystrophin, μDys).^5,6 AAVs containing versions of micro-dystrophin are now being delivered to patients with DMD in clinical trials. Such micro-dystrophins, however, are unlikely to fully compensate for the loss of dystrophin, a protein now recognized for its scaffolding/signaling roles in addition to its traditional role in mechanical force transmission. However, there has been no consideration of the fact that the cardiac disease progression is greatly slowed by the high degree of utrophin expression in the heart,^7,8 and that there could be a displacement of utrophin by micro-dystrophin expressed at high levels. Indeed, in examining the potential therapeutic efficacy of a micro-dystrophin, μDys ΔR3-R21_ΔCT (missing spectrin-like repeats 3 through 21 as well as the C-terminal end of the dystrophin protein) 5 in the D2.mdx, ^9,10 mouse model of DMD, we find that AAV-mediated overexpression of micro-dystrophin leads to the displacement of utrophin from the cardiomyocyte sarcolemma. This appears to, in essence, phenocopy dystrophin/utrophin double mutants that exhibit an exaggerated cardiac dysfunction.⁷. As the heavily truncated micro-dystrophin, μDys ΔR3-R21_ΔCT, is unlikely to functionally replace full length utrophin, its overexpression may be detrimental to cardiac function.

Key Words: Duchenne muscular dystrophy, dystrophin, utrophin, adeno-associated virus, microdystrophin, heart

References

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10.Hammers DW, Hart CC, Matheny MK, Wright LA, Armellini M, Barton ER, Sweeney HL. The D2.mdx mouse as a preclinical model of the skeletal muscle pathology associated with Duchenne muscular dystrophy. Sci Rep. 2020. Aug 21;10(1):14070. doi: 10.1038/s41598-020-70987-y. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

NOX4 inhibition reduces skeletal muscle fibrosis in a severe murine model of Duchenne muscular dystrophy

David W Hammers ^1,^2,^✉

Abstract

The muscular dystrophies (MDs) are a class of genetic muscle diseases often characterized by progressive degeneration and replacement of muscle with fibrotic tissue. Limited treatment options exist for MD patients, and no therapeutics specifically target the progression of intramuscular fibrosis. NAD(P)H oxidase (NOX) 4 has been previously implicated in the development of fibrosis in other organs, but not skeletal muscle. The specific aim of the current report was to investigate NOX4 as a potential anti-fibrotic target in dystrophic skeletal muscle, using the severe D2.mdx mouse model of Duchenne muscular dystrophy (DMD). It is shown that NOX4 is increased in the muscles of D2.mdx mice and localizes primarily to interstitial regions between muscle fibers. In agreement with the hypothesis that this increase in NOX4 contributes to muscle fibrosis, genetic ablation of Nox4 prevents the progressive accumulation of fibrosis in skeletal muscle. Furthermore, it is demonstrated that pharmacological targeting of NOX4 using the small molecule clinical candidate NOX1/4 inhibitor, GKT137831, also prevents the progression of muscular fibrosis in the diaphragm and gastrocnemius of D2.mdx mice. Mechanistically, NOX4 loss-of- function results in decreased accumulation of periostin and periostin-associated interstitial cells within dystrophic muscle. These data indicate NOX4 is an effective anti-fibrotic target to combat progression of muscle fibrosis in DMD and, potentially, other MDs.

Key Words: Muscular dystrophy, fibrosis, NADPH oxidase, extracellular matrix, regeneration, pharmacology, myogenesis

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The co-administration of simvastatin does not boost the benefit of gene therapy in the mdx mouse model for Duchenne muscular dystrophy

Nathalie Bourg ^1,^2,^*, Ai Vu Hong ^1,^2,^*, William Lostal ^1,^2,^*, Abbass Jaber ^1,², Nicolas Guerchet ^1,², Guillaume Tanniou ^1,², Nathalie Daniele ^1,², Isabelle Richard ^1,², David Israeli ^1,^2,^*

Abstract

Duchenne muscular dystrophy (DMD) is the most common and a cureless muscle pediatric genetic disease, which is caused by the lack or the drastically reduced expression of dystrophin. Experimental therapeutic approaches for DMD are mainly focused on attempts to restore the expression of dystrophin. While significant progresses were achieved, therapeutic benefit of treated patients is still unsatisfactory. Efficiency in gene therapy for DMD is hampered not only by incompletely resolved technical issues, but likely also due to the progressive nature of DMD. It is indeed suspected that some of the secondary pathologies, which are evolving over time in DMD patients, are not fully corrected by the restoration of dystrophin expression. We recently identified perturbations of the mevalonate pathway and of cholesterol metabolism in DMD patients. Taking advantage of the mdx model for DMD, we then demonstrated that some of these perturbations are improved by treatment with the cholesterol-lowering drug, simvastatin.¹ In the present investigation,² we tested whether the combination of the restoration of dystrophin expression with simvastatin treatment, could have an additive beneficial effect in the mdx model. We confirmed the positive effects of minidystrophin, and of simvastatin, when administrated separately, but detected no additive effect by their combination. Thus, the present study does not support an additive beneficial effect by combining dystrophin restoration with a metabolic normalization by simvastatin.

Key Words: Duchenne Muscular dystrophy, Simvastatin, Gene therapy, AAV, minidystrophin, microdystrophin combined therapy, lipid metabolism

References

1.Amor F, Vu Hong A, Corre G, Sanson M, Suel L, Blaie S, Servais L, Voit T, Richard I, Israeli D. Cholesterol metabolism is a potential therapeutic target in Duchenne muscular dystrophy. J Cachexia Sarcopenia Muscle. 2021. Jun;12(3):677-693. doi: 10.1002/jcsm.12708. Epub 2021. May 26. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Therapeutic potential of combined cycling and isometric strength training in patients with DMD: preliminary findings

Tanja Taivassalo ^1,^✉, Andrew Carvill ¹, Donovan Lott ¹, Kim Stubbs ¹, Warren Dixon ¹, Sean Forbes ¹, Krista Vandenborne ¹, Glenn Walter ¹, Lee Sweeney ¹

Abstract

Duchenne muscular dystrophy (DMD) is a devastating, rapidly progressive neuromuscular disease caused by mutation of the dystrophin gene and is characterized by severe muscle weakness and fatigability, early loss of ambulation and premature death. The search for effective therapies is ongoing; understanding the potential of exercise alone, or as an adjuvant to developing therapies, is likely to be beneficial but is limited by (i) a lack of understanding of exercise prescription parameters (type, intensity, target muscle groups) that are safe and effective for patients with DMD; and (ii) a lack of accessibility to exercise equipment suited to the limitations of dystrophic muscle. High intensity, eccentric actions are known to be damaging, however our group recently reported moderate-intensity isometric exercise to be safe and increase muscle strength¹ in boys with DMD. Dynamic cycling was shown to benefit boys and delay loss of muscle function^2,3, however improvements in muscle strength and endurance were not detected likely relating to the low intensity of the cycling exercise which as motor-assisted. Our objective was to engineer a therapeutic exercise device for patients with DMD (Fig. 1), allowing for safe and appropriate overload of muscle (using both isometric strengthening and aerobic cycling with assistive and resistive capabilities) to induce remodeling and attenuate disease progression. The device is used in-home with remote video supervision by the study team to allow high-resolution real-time quantification of dynamic and isometric force profiles and capture physiological measures (i.e. heart rate and perceived exertion). Our goal is to develop guidelines and advance exercise as therapy for DMD. Outcome measures to assess intervention safety and efficacy include standard clinical function tests, magnetic resonance imaging of leg muscle (cross sectional area, fat fraction and T2 quantification), muscle strength and aerobic capacity. In this ongoing study (ClinicalTrials.gov NCT04322357), one boy with DMD on daily steroids (age 8-yrs) completed the 6-month training program (34 sessions of cycling and 20 sessions of strength training). Preliminary findings are shown in Figure 1.

Supported by Department of Defense Grant number: W81XWH1910330

Key Words: Duchenne muscular dystrophy, aerobic exercise, strength training, physiological adaptation, combination therapy

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Uncovering the epigenetic control of paracrine crosstalk between motor neurons and skeletal muscles in SMA

Stéphanie Astord ¹, Sonia Pezet ¹, Benoit Giroux ¹, Fiorella Carla Grandi ², Maria Grazia Biferi ¹, Piera Smeriglio ^1,^✉

Abstract

Spinal muscular atrophy (SMA) is a genetic disorder characterized by motor neuron degeneration and progressive muscle weakness, atrophy and premature the survival of motor neuron 1 (SMN1) gene with an incidence of 1 in 10,000 live births representing the most common genetic cause of infant mortality. Loss of motor neuron function is thought to be the initial event which triggers muscle atrophy due to the lack of physical nerve-muscle contact at the neuromuscular junction (NMJ). But, recent findings reveal that muscle atrophy in SMA precedes motor neuron death. However, the crosstalk between the muscle and the motor neuron in the pathogenesis of the disease has not yet been deciphered. We aim to clarify the motor neuron function and its contribution to skeletal muscle pathology using the multi- omics analysis of DNA epigenetics and transcriptomic profiles. Our data intriguingly supports an alternative paradigm where paracrine crosstalk between motor neuron and skeletal muscle plays a key role in the survival and function of both tissue compartments. We found that i) spinal cord tissue of the SMAdelta7, a severe SMA mouse model, exhibited a genome-wide decrease in the DNA demethylation mark 5hmC; ii) reduced 5hmC mark supresses the expression of a subset of genes, identified to play an important role in paracrine signalling regulating musclefunction. Our data support the crucial role of modulating DNA epigenetics in MN’s; demonstrating that the effects of altered 5hmC levels in SMA go beyond the MN compartment to impair muscle function, by affecting the crosstalk signals independent of NMJ function.

Key Words: Epigenetics, hydroxymethylation, tissue crosstalk, spinal muscular atrophy

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Video killed the imaging star

Francesco Girardi ^1,^✉

Abstract

Biological systems are extremely dynamic and heterogeneous. Because of their fascinating nature, scientists have been trying to visualize cells using microscopy since the dawn of science history. However, biological processes are characterized by complex interactions between cells and subcellular components that occur in 3 spatial dimensions and in time. Consequently, having a complete picture of all these events is essential to understand all the spatio-temporal dynamics of the process of interest. Unfortunately, this task is very hard to achieve with conventional microscopy. Indeed, traditional microscopy tecniques often require fixed samples, which can only provide a single snapshot of the reality and thus limiting the acqired information. In the light of this limitation, live imaging represents a valide alternative that can provide a comprehensive visualization of the entire biological process. Here, we will show three live imaging technologies that can overcome conventional microscopy limits in different flields and applications. Specifically, we will present two live-cell imaging microscopes for in vitro specimens and one for in vivo imaging. The first one is the innovative holotomographic microscope NANOLIVE that allows you to visualize cell cultures in 3D and in time-lapse without stainig or marker. This label-free technology requires no sample preparation and does not generate phototoxicity damages, allowing continuos high-frequency imaging at high resolution. The second live-cell imaging solution is called AUROX UNITY and consists in a laser-free spinning disk confocal microscope. UNITY is an all-in-one compact device equipped with LED, motorized stage and incubator, thus perfect for fluorescence live-cell imaging. Finally, the in vivo live imaging tecnology comes from Femtonics and is called FEMTO 3D ATLAS. ATLAS is the unique acousto-optic 2-photon microscope that offers ultrafast 3D live imaging in living animal models

Key Words: Live Imaging, 2-photon, Nanolive, Aurox, Femtonics

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Session III: Targeting ER stress to resolve aggresome accumulation in oculopharyngeal muscular dystrophy

Alexis Boulinguiez ¹, Jamila Dhiab ¹, Fanny Roth ¹, Ludovic Gaut ¹, Mai Thao Bui ², Teresinha Evangelista ², Norma Beatriz Romero ², Elisa Negroni ¹, Jean Lacau St Guily ¹, Vincent Mouly ¹, Gillian Butler-Browne ^1,^✉, Capucine Trollet ¹

Abstract

Oculopharyngeal muscular dystrophy (OPMD) is a rare, late-onset genetic myopathy (Trollet et al., 1993)¹ affecting mainly the eyelid and pharyngeal muscles, resulting in ptosis and dysphagia, respectively. OPMD is due to an alanine expansion in the Poly-A Binding Protein Nuclear 1 (PABPN1) gene which encodes a regulator of poly-A tail length of RNA. This mutation results in PABPN1 nuclear aggregates in OPMD myonuclei that sequester essential RNA and proteins and thus induce muscle alterations. Therefore, human OPMD muscles present a global proteostatis impairment (chaperones, ubiquitin-proteasome and autophagy-lysosomal processes) (Abu-Baker et al., 2003; Anvar et al., 2011; Raz et al., 2017).^2-4 Endoplasmic reticulum is also a key actor of cell proteostasis assuring folding of one third of the cellular proteome. Interestingly, OPMD mouse model exhibits muscle endoplasmic reticulum (ER) stress (accumulation of misfolded proteins in the ER) and restoration of the homeostatic unfolded protein response (UPR) by guanabenz acetate treatment decreases PABPN1 nuclear aggregates and improves muscle function in OPMD mice (Malerba et al., 2019).⁵ To extend these results to humans, we analyzed muscle biopsies from OPMD and healthy donors. We found that UPR genes were all upregulated in human OPMD muscles, as revealed by RNAseq analysis, strongly indicating ER stress. Consistently, GRP78 protein, the key ER chaperone, was reduced in OPMD muscles. GRP78 protein was also trapped in PABPN1 nuclear aggregates, further reducing its amount available for ER protein folding. Consequently, OPMD muscles showed an accrual of large 𝛽𝛽-sheet protein aggregates (called aggresomes) in the fibers, beside to the classical PABPN1 nuclear aggregates. Consistently, in culture, OPMD human muscle cells were more likely to develop aggresomes than healthy cells following ER stress specifically. Finally, in mouse cells overexpressing PABPN1, we found that ER stress induction leads to accumulation of aggresomes and aggravation of PABPN1 nuclear aggregates. Overall, our results demonstrated an ER protein folding disruption leading to ER stress and aggresome accumulation in human OPMD muscle that could reinforce pathogenesis of OPMD. Thus, restoration of ER folding and activation of protein aggregate clearance sounds promising therapeutic strategies against OPMD.

Key Words: Oculopharyngeal muscular dystrophy, protein aggregation, endoplasmic reticulum, proteostasis, skeletal muscle

References

1.Trollet C, Boulinguiez A, Roth F, Stojkovic T, Butler-Browne G, Evangelista T, Lacau St Guily J, Richard P. Oculopharyngeal Muscular Dystrophy (OPMD). In GeneReviews^®, Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, Amemiya A, editors. [Internet]. Seattle (WA): University of Washington, Seattle; 1993–2021. [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

New molecular network identified in Amyotrophic Lateral Sclerosis reveals microRNAs involved in the neuromuscular junction development

Raphael S Bonadio ^1,^✉, Francesco Chemello ¹, Davide Bonaldi ¹, Giulia Stomboli ¹, Caterina Peggion ², Giulia Zanetti ², Maria Lina Massimino ³, Marco Pirazzini ², Alessandro Bertoli ², Stefano Cagnin ¹

Abstract

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disorder of unknown aetiology, orphan of effective therapies (Kiernan, et al 2021)¹. Previous studies demonstrated that aberrant gene expression of myofibers can be involved in the cause of this disease (Scaricamazza et al, 2021; Fischer et al, 2004).^2,3 Thus, to understand gene expression regulation can provide new insights for more efficient treatments. We analyzed gene expression in gastrocnemius muscle of SOD1(G93A) ALS mouse model during pathology progression. We identified 669 mRNAs and 85 miRNAs differentially expressed. Gene Ontology analysis revealed that different cellular functions are altered in ALS such as: apoptotic processes, negative transcription regulation and nervous system development. We focused our attention on this last function identifying 3 miRNAs (miR-193a-3p, 193b-3p and 152-3p) that regulate NMJ function targeting Chrna1, Chrnb1, Chrnd, Chrne, Chrng, Emb, Musk and Prima1 (Fig. 1). After the in vivo modulation of these miRNAs we evidenced a reduced expression of their targets and a reduction of gastrocnemius and tibialis anterior muscle mass. In association with these observations we evidenced a fast to slow fiber shift with a reduction in the total number of NMJs (Fig. 2) and in the amplitude of evocated potentials. These results support the role of 3 miRNAs in the maintenance of NMJ function and in the regulation of skeletal muscle atrophy.

Key Words: Amyotrophic Lateral Sclerosis, miRNAs, neuromuscular junction, Acetylcholine receptor

References

1.Kiernan MC, Vucic S, Talbot K, McDermott CJ, Hardiman O, Shefner JM, Al-Chalabi A, Huynh W, Cudkowicz M, Talman P, Van den Berg LH, Dharmadasa T, Wicks P, Reilly C, Turner MR. Improving clinical trial outcomes in amyotrophic lateral sclerosis. Nat Rev Neurol. 2021. Feb;17(2):104-118. doi: 10.1038/s41582-020-00434-z. Epub 2020. Dec 18. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Proteomic profiling of the aged diaphragm from the mdx-4cv model of dystrophinopathy

Stephen Gargan ^1,^2,^✉, Paul Dowling ^1,², Dieter Swandulla ³, Kay Ohlendieck ^1,²

Abstract

X-linked Duchenne muscular dystrophy is due to mutations in the DMD gene, which causes the almost complete loss of the membrane cytoskeletal protein dystrophin. The diaphragm is severely affected in dystrophinopathy and is characterized by progressive muscle fibre degeneration, chronic inflammation and reactive myofibrosis. The major goal of this new study was to identify novel proteomic biomarker candidates of muscle weakness and respiratory deficiency in dystrophinopathy. In order to investigate proteome-wide changes in the dystrophic and aged diaphragm muscle, our laboratories have analysed 15 months old wild type mice versus age-matched mdx-4cv mice, which represent an established genetic model of Duchenne muscular dystrophy. Mass spectrometry-based proteomics was combined with systems biological analyses and verification studies using comparative immunoblotting. A variety of changes in proteins involved in the maintenance and organization of both the extracellular matrix and the intracellular cytoskeleton were identified by mass spectrometry. These new disease-related protein markers could be helpful to improve diagnostic procedures, as well as prognostic and therapy-monitoring approaches.

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Key Words: Diaphragm, Duchenne muscular dystrophy, fibrosis, mass spectrometry, proteomics

References

Ohlendieck K, Swandulla D. Complexity of skeletal muscle degeneration: multi-systems pathophysiology and organ crosstalk in dystrophinopathy. Pflugers Arch. 2021. (in press). doi: 10.1007/s00424-021-02623-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Differential dysferlin expression in human fast and slow skeletal muscle

Marija Meznaric ^1,^✉, Corrado Angelini ²

Abstract

A common form of limb girdle muscular dystrophy (LGMD) is dysferlinopathy presenting as LGMDR2/2B or distal Miyoshi myopathy, that show different clinical course. They are caused by mutations in the DYSF gene encoding the protein dysferlin, a transmembrane sarcolemmal protein which has a role in the repair of sarcolemma. Dysferlin is also a t-tubule protein. To the best of our knowledge (differential) dysferlin expression in skeletal muscle fibre types has not been studied so far. We investigated by western blotting human skeletal muscles, one proximal in the arm (biceps brachii) and the other distal in the leg (tibialis anterior) which are affected differently during the course of LGMDR2: usually first tibialis anterior (around the age of 20 years) and later on the biceps brachii, with clear involvement at the age of 45 years. Muscle specimen were collected after a few hours from sudden death or muscle biopsies of biceps brachii and tibialis anterior muscle of neuromuscular controls were analysed. Dysferlin to myosin heavy chain ratio differed in biceps brachii and tibialis anterior muscle. Preliminary results show a trend towards a higher "content" of dysferlin in fast skeletal muscle, i.e. biceps brachii, compared to slow, i.e. tibialis anterior (Figure1, Table1) which might be relevant for the different course observed in LGMDR2/2B and Miyoshi myopathy.

Table 1.

Content of dysferlin in fast skeletal muscle, i.e. biceps brachii (BB), compared to slow, i.e. tibialis anterior (TA).

Muscle	Patient (n), Gender, Age (years)	Dysferlin/Myosin
BB1 - biceps brachii	1, Male, 63	2,36
TA2 - tibialis anterior	2, Male, 60	1,17
BB3 - biceps brachii	3, Female, 48	2,26
TA4 - tibialis anterior	4, Female, 52	1,56

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Key Words: Dysferlin, fast skeletal muscle, LGMD, slow skeletal muscle, human

References

Angelini C, Nardetto L, Borsato C, Padoan R, Fanin M, Nascimbeni AC, Tasca E. The clinical course of calpainopathy (LGMD2A) and dysferlinopathy (LGMD2B). Neurol Res. 2010. Feb;32(1):41-6. doi: 10.1179/174313209X380847. [DOI] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Session IV: A Dual-AAV gene therapy strategy for Duchenne Muscular Dystrophy

Sonia Albini ^1,^✉, Laura Palmieri ¹, Nathalie Bourg-Alibert ¹, William Lostal ², Isabelle Richard ¹

Abstract

Duchenne Muscular Dystrophy (DMD) is a severe rare pediatric disease that affects the skeletal and cardiac muscles, leading to progressive muscle wasting and premature death. DMD is caused by X-linked mutations in the dystrophin gene that results in lack of dystrophin, a crucial protein involved in the biomechanical support of the muscle fibers. As a consequence of the membrane instability, DMD muscle fibers are progressively more fragile, highly susceptible to damage and fibrotic. Up to date there is no cure for DMD. Although DMD is a suitable disease for gene replacement therapy, the extent of Dystrophin transcript exceeds by far the AAVs cargo capacity and only short versions of dystrophin can be accommodated and are currently being investigating in clinical trials. However, is still not clear whether some domains of dystrophin are really dispensable for its function and stability. Our strategy consists in the generation of a larger version of dystrophin that includes additional domains required for interactions with signaling and structural proteins. To overcome the AAV limit capacity, we developed a dual system dystrophin vector, encoding for a larger dystrophin protein (Quasidystrophin) after homologous recombination of the overlapping region of the 2 dystrophin vectors. We analyzed the therapeutic efficiency of dual-AAVs gene transfer by systemic injection in DBA2-mdx mice, an aggressive DMD mouse model. Our data show high transduction efficiency in the cardiac and skeletal muscles with significant reduction in fibrosis and calcification and overall amelioration of the dystrophic phenotype using the dual-AAV vector gene transfer.

Key words: Duchenne muscular dystrophy, Dual-AAV, Dystrophin

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

D2.mdx mice undergo a transient period of left ventricular restriction prior to heart failure

Cora C Hart ^1,^✉, David W Hammers ¹, H Lee Sweeney ¹

Abstract

Duchenne muscular dystrophy (DMD) is the most frequently inherited neuromuscular disease of childhood and cardiopulmonary failure is the leading cause of death for this patient population. In this study, we used electrocardiograms (EKGs) and ultra high frequency echocardiography to longitudinally monitor the heart function of a severe mouse model of DMD, the D2.mdx mouse. By 10 months of age, D2.mdx mice have a left ventricular end diastolic volume (LV EDV) that is ~32% less than age-matched wild- type mice. This restricted LV results in a significant decrease in stroke volume (SV) and cardiac output (CO); however, parameters of systolic function, such as ejection fraction (EF) and fractional shortening (FS), are preserved. In line with this impaired relaxation, EKGs of D2.mdx mice reveal they have a prolonged QT interval, due to an increase in T wave duration and indicative of impaired repolarization. Further evidence of the diastolic dysfunction of D2.mdx hearts was found using pulsed-wave doppler of the blood flow through the mitral valve (MV), visualized using color doppler. D2.mdx mice have a reduced MV E/A ratio, and in agreement with the EKG findings, an increased isovolumic relaxation time (IVRT). In conclusion, like DMD patients and a canine model of DMD, the D2.mdx mouse displays left ventricular restriction prior to the progression to a dilated cardiomyopathy (DCM) and is a useful small animal model for the cardiomyopathy associated with DMD.

Table 1.

Pre-DCM Left Ventricular Restriction.

Echocardiographic measure	Murin		Canin		Human²
	DBA2/J	D2.mdx	Normal	GRMD	Normal	DMD
End Diastolic Volume (μL or mL)	75.77±5.99	52.10±4.15*	66.53±3.24	51.57±3.35*	64.8±11.6	41.8±7.7*
End Systolic Volume (μL or mL)	19.97±3.64	8.25±1.69*	26.87±2.68	23.51±2.43*	21.6±4.4	18.7±5.12
Stroke Volume (μL or mL)	55.81±3.07	43.85±2.98*	39.65±1.85	28.06±1.58*	43.3±10.4	29.1±5.4*
--SV Deficit (%)	--	-21.43	--	-29.23	--	-32.79
Ejection Fraction (%)	79.53±2.36	84.82±2.62	60.47±2.49	56.88±2.28	66.3±7.2	61.2±7.1

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Volumes are in μL for murine values and mL for canine and human values; SV = stroke volume; *p < 0.05 vs. control values. 2Panovský et al. 2021

Key Words: Duchenne muscular dystrophy, restrictive cardiomyopathy, mouse model

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Beneficial effects of boosting skeletal muscle metabolism by SIRT1 activator in DMD mouse model

Matteo Giovarelli ^1,^✉, Silvia Zecchini ¹, Emilio Clementi ¹, Clara De Palma ²

Abstract

Duchenne muscular dystrophy (DMD) is a severe and relentlessly progres- sive myopathy caused by out-of-frame or nonsense mutations in the X-linked DMD gene. DMD is a complex disease and multiple approaches are needed to target pathological processes, both the underlying genetic mutations and the secondary complications. Despite several therapeutic options have been developed with good results, more effective treatment options are essential and may be generated through the definition of novel therapeutic targets. A muscular metabolic dysregulation is an established DMD feature with mito- chondrial dysfunctions as one of the earliest deficits that arise from multiple cellular stressors. Among metabolic regulators, Sirtuin 1 (SIRT1) represents an intriguing candidate since it acts on different aspects of cellular metabo- lism, regulating energy homeostasis, mitochondrial biogenesis, and inflam- mation. SIRT1 overexpression represents an important counter-mechanism to alleviate the dystrophic phenotype and its pharmacological modulation could be relevant as well inDMD conditions. Consistently, SIRT1 activation by selective compound, i.e., SRT2104, has already been proven to reinforce muscular structure, mitochondrial functionality, and to reduce inflammation. SRT2104 has never been tested in muscular diseases; therefore, considering its metabolic and immunomodulatory effects, we tested SRT2104 as an at- tractive candidate for DMD treatment. Accordingly, in our preliminarydata, long-term SRT2104 administration improved muscle force and stimulated oxidative capacity. This was paralleled by reduced fibrosis and inflammatory infiltrate and increased regeneration in mdx muscle. In conclusion our results demonstrate the efficacy of SRT2104 as a new SIRT1 activator in DMD and highlight that considering DMD also as a metabolic disease and treating it as such, could provide important therapeutic strategies additional to gene therapies.

Key Words: Duchenne Muscular Dystrophy, SIRT1 activators, mitochondria

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Pathogenetic mechanism of Limb Girdle Muscular Dystrophy D2: functional characterization of Transportin 3 in cellular and animal models of disease

Roberta Costa ¹, Maria Teresa Rodia ¹, MMatteo Bergonzoni ², SSerafina Pacilio ¹, Spartaco Santi ³, Flavia Frabetti ², Giovanna Cenacchi ^1,^✉

Abstract

Limb Girdle Muscular Dystrophy D2 (LGMD D2) is caused by a heterozygous mutation in the termination codon of the TNPO3 gene. This mutation gives a protein which is 15-aminoacids longer in its C-terminal domain. TNPO3 gene encodes for TNPO3, which normally mediates the translocation to the nucleus of SR proteins, a family of splicing factors and other proteins related to RNA metabolism. The goal of this work was to investigate the pathogenetic mechanism of LGMD D2 through a dual approach, in vitro and in vivo, focusing mainly on the role of TNPO3 in the myogenic process and in possible muscle-specific molecular pathways. In vitro murine C2C12 myoblasts were treated with CRISPR/Cas9 genome editing of TNPO3. Preliminary data suggest morphological, proliferative and expression changes of genes involved in myogenic differentiation in comparison to the C2C12 control line (Fig.1). For the in vivo approach, the microinjection of mRNA encoding human wild type (WT) and mutated TNPO3 in zebrafish embryos allowed to study its effects during embryonic development. We monitored the gene expression profiles of endogenous transportin 3, of myogenic regulatory factors (MRFs) and muscle-specific proteins, suggesting morphological and gene expression changes in embryos miscroinjected with the WT form and particularly in microinjected embryos with the mutated TNPO3 form. The approaches used are a first step to understand the role of TNPO3 in muscle physiology and in the pathogenetic mechanism underlying LGMD D2 which is still unknown.

Key Words: LGMD D2, TNPO3, Zebrafish, myogenesis

References

Torella A, Fanin M, Mutarelli M, Peterle E, Del Vecchio Blanco F, Rispoli R, Savarese M, Garofalo A, Piluso G, Morandi L, Ricci G, Siciliano G, Angelini C, Nigro Vl. Next-generation sequencing identifies transportin 3 as the causative gene for LGMD1F. PLoS One. 2013;8(5):e63536. Published 2013. May 7. doi:10.1371/journal.pone.0063536. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Uncovering a novel mechanism for intramuscular fat formation

Alessandra Norris ¹, Lylybell Zhou ¹, Connor Johnson ¹, David McKellar ², Ben Cosgrove ², Daniel Kopinke ^1,^✉

Abstract

Adipose tissue can accumulate between muscle fibers, known as intramuscular fat (IMAT) and is strongly correlated with loss in muscle mass and function. IMAT infiltration is a hallmark of ageing and a diverse set of diseases such as muscular dystrophies, diabetes, and spinal cord injuries. No therapies exist currently that limit IMAT formation. We and others have shown that exogenous activation of the Hedgehog (Hh) pathway is beneficial for overall muscle regeneration and improves muscle health in a Duchenne muscular dystrophy (DMD) model (mdx mouse). We have also demonstrated that pharmacological and genetic activation of the Hh pathway blocks the formation of IMAT after an adipogenic injury and in mdx mice. To define the endogenous function of the Hh pathway during acute muscle regeneration, we evaluated which of its 3 possible ligands (Sonic [SHH], Indian [IHH] or Desert Hedgehog [DHH]) is expressed. Exploring single cell RNA sequencing data of dissociated muscle tissue pre and post injury, we found that DHH is expressed by endothelial and Schwann cells and strongly upregulated after injury. To test the functional requirement for DHH, we evaluated the regeneration of Dhh knock-out (Dhh KO) mice compared to controls. For this, 21 days post cardiotoxin (CTX) injury, we assessed IMAT and found a significant increase compared to control littermates. This suggests that the Hh pathway, activated endogenously through DHH, acts as an adipogenic break, that when lost leads to increased IMAT. To further evaluate the pathway’s physiological role, we sought to compare and manipulate Hh activity in mouse strains with differing adipogenic predisposition. When comparing IMAT 7 days after a CTX injury between CD1 and 129S1/SvlmJ mice strains, CD1 mice have higher levels of IMAT compared to 129S/1SvlmJ. To test if this difference in IMAT formation is due to differences in Hh activity, we compared the expression levels of the pathway’s downstream gene target Gli1 by RT-qPCR. Excitingly, our results demonstrate that, compared to CD1 mice, 129S1/SvlmJ mice display higher Hh activity. As ectopic Hh activation acts as a potent adipogenic brake, endogenous Hh activity levels may explain the differences in adipogenic predisposition between mouse strains. Confirming our hypothesis, inactivating endogenous Hh activation through the small molecule Vismodegib in 129S1/SvlmJ mice resulted in an increase in IMAT. Together, our data identify DHH as the key Hh ligand during muscle regeneration, and that Hh signaling, via DHH, acts as an endogenous adipogenic break. In addition, our results also indicate that endogenous differences in Hh activity may explain the differences in IMAT predisposition. Thus, the Hh pathway represents an exciting and novel therapeutic target to control IMAT formation.

Key Words: Intramuscular fat, FAPs, muscle regeneration

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Neutral Lipid Storage Diseases: a patient clinical follow-up and presentation of two novel cases

Sara Missaglia ^1,^2,^✉, Enza Torre ^1,², Daniela Tavian ^1,², Valentina Pegoraro ³, Paola Vago ⁴, Corrado Angelini ⁵

Abstract

Neutral lipid storage diseases (NLSDs) are rare inherited disorders caused by mutations in adipose triglyceride lipase (ATGL/PNPLA2) or in α-β hydrolase domain 5 (CGI-58/ABHD5). NLSD patients present defects in lipid droplets (LDs) metabolism resulting in accumulation of neutral lipids in these organelles that become larger and more numerous¹ (Fig.1). When the mutation is localized on PNPLA2 gene, the main symptom is myopathy (NLSDM)². The case report of a 29-year-old woman with NLSDM is presented. At 18 years, despite she was asymptomatic, hyperCkemia suggested NLSDM diagnosis and sequencing analysis revealed a retrotransposal insertion in PNPLA2 gene². In the following years, the patient has been experiencing increasing muscle weakness. Muscle MRI showed lipid accumulation in calves. At 27 years, she was put on MCT diet and CPK lowered³. Nevertheless, muscle weakness did not improve. In this patient, no beneficial effects were observed after treatment, probably due to complete loss of ATGL protein. When the mutation is on CGI-58 gene, the main symptom is ichthyosis (NLSDI)⁴. Here we report the genetic and clinical symptoms of two young brothers 10.5 and 2 years old, with a new ABHD5 gene mutation (p.W179Nfs22*). They presented generalized ichthyosis since birth, hepatomegaly and increased liver enzymes. The second child also had severe hypoplastic cerebellum, global developmental delay, generalized hypotonia, increased deep tendon reflexes and occasional attacks of dystonia. In conclusion, we focus the attention on NLSDs disorders, discussing the clinical follow-up of a woman affected by NLSDM and presenting two unreported cases of NLSDI.

Key Words: Neutral lipid storage diseases, Lipid droplets, myopathy, Ichthyosis, MCT treatment

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Human Brody disease and novel therapeutic approaches of its animal model cattle pseudomyotonia

Eylem Emek Akyürek ^1,^✉, Joana GP Jacinto ², Marilena Bolcato ², Elisa Bianchini ³, Doriana Sandonà ³, Arcangelo Gentile ², Roberta Sacchetto ¹

Abstract

Bovine “congenital pseudomyotonia” (PMT) is a genetic muscular disorder very similar to human Brody myopathy (Brody 1996)¹ for clinical signs. Exerciseinduced stiffness and delayed muscular relaxation following even mild physical exercise, are the major clinical symptoms. A missense mutation in the ATP2A1 gene (Drögemüller et al 2008)², encoding sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1), causes congenital PMT in cattle and Brody disease in human. Clinical symptoms genetic and biochemical findings clearly demonstrated that congenital PMT in Chianina cattle is the real analogue of Brody myopathy (Sacchetto et al 2009).³ This is not unexpected given that human diseases have recently been discovered in a variety of domestic animal species. Our research team provided evidence that in bovine PMT, SERCA1 mutation generates a protein most likely corrupted in proper folding, which was ubiquitinated and prematurely degraded by the ubiquitin-proteasome system, although it retained its catalytic properties (Bianchini et al. 2014).⁴ The treatment with proteasome inhibitors restores SERCA levels and Ca2+ homeostasis in a cellular model and in muscle fibres from PMT affected animals (ex vivo experiments) (collected in conformance with the institutional guidelines for the care and use of animals). At present, neither specific therapy nor mouse model for Brody myopathy exists. However, we have recently designed and proven in vitro a novel pharmacological approach based on the employment of protein folding correctors named CFTR (Cystic Fibrosis Transmembrane Regulator) exploited in Cystic Fibrosis. The treatment with CFTR correctors rescued the expression level of mutated SERCA1 in HEK293 cell model. These data have been confirmed in vivo by local treatments of bovine PMT muscle with the most effective in vitro CFTR corrector.

Key Words: Brody disease, Bovine pseudomyotonia, SERCA1

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Muscle clocks change with age: A potential contributor to sarcopenia?

Christopher A Wolff ^1,^2,^*, Zhiguang Huo ³, Miguel A Gutierrez ^1,², Collin M Douglas ^1,², Xiping Zhang ^1,², Karyn A Esser ^1,²

Abstract

Skeletal muscle circadian clocks regulate a daily program of gene expression that is necessary for predictive homeostasis and tissue resilience. Aging is associated with progressive decline in skeletal muscle function. To date, our lab has found age associated changes in expression of the core clock gene, PER2, in mouse muscle. However very little is known about how aging alters the circadian transcriptome. To address this gap in the field, we collected skeletal muscle from Young (6mo), Aged (18mo), and Old (27mo) male C57BL/6J mice every 4h for 48h for RNA sequencing and circadian analysis. We identified that circadian expression of the core clock genes was unchanged from Young to Aged, however, from Aged to Old there were significant changes in several genes within the repressor limb (e.g. Per1/2, and Cry1/2). Consistent with age-associated changes in core clock genes we found an age-related decline in the circadian transcriptome that was evident by 18 months, with 941 oscillating genes in the Young mice, 699 oscillating genes in the Aged mice, and 474 oscillating genes in the Old mice. Comparing across the 3 ages there was less than 10% overlap in the circadian transcriptome between any age and only 3% of genes shared across all three ages, and these shared genes were composed primarily of the core circadian clock factors. These are the first data to demonstrate a progressive age-related decline the skeletal muscle clock and the skeletal muscle circadian transcriptome. Functional cluster analysis of the transcriptomes suggest that altered circadian clock output with aging likely contributes to changes in skeletal muscle function and adaptability with age.

Key Words: Muscle clock, Circadian rhythms, Clock output

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Cancer cell-derived IL-8 and CXCL1 mediate cachexia in mice bearing human pancreatic tumors

Chandler Callaway ^1,^✉, Andrea Delitto ¹, Michael Dehyle ², Andrew D-Lugos ¹, Jose Trevino ³, Sarah Judge ¹, Andy Judge ¹

Abstract

Pancreatic Cancer Cachexia is a multifactorial wasting syndrome, where extent of muscle loss predicts survival and response to therapy in patients. Among the molecular network of factors that may be responsible for initiating pancreatic cancer cachexia are tumor-secreted factors, and in recently published work we demonstrated that human pancreatic cancer cells secrete high levels of both CXCL8 (IL-8) and CXCL1, and these factors are sufficient to induce muscle wasting. In the current study we therefore determined the requirement of pancreatic cancer cell-derived IL-8 and CXCL1 for the cachexia phenotype in mice bearing human L3.6pl pancreatic tumors. CRISPR-Cas9 technology was utilized to generate L3.6pl cells containing single or co-deletion of IL-8 and CXCL1. Genetically modified L3.6pl cells (L3.6plIL8-/-, L3.6plCXCL1-/-, L3.6plIL8-/-CXCL1-/-) or their Cas9 controls (L3.6plCas9) were injected into the pancreas of 20 week old NSG mice, and tissues harvested approximately 3 weeks later, upon L3.6plCas9 mice reaching experimental endpoint. Compared to Sham, mice with L3.6plCas9 tumors showed significant cachexia, as indicated by reduced tumor-free body mass and reductions in gonadal fat, skeletal muscle and heart mass. Although L3.6plIL8-/- and L3.6plCXCL1-/- tumors induced comparable cachexia in mice to that of L3.6plCas9 controls, mice with L3.6plIL8-/-CXCL1-/- tumors showed marked protection against cachexia, based on preservation of tumor-free body mass, fat mass and heart mass, and attenuated skeletal muscle wasting, despite comparable tumor burden. RNA-sequencing of TA muscles revealed 1293 genes differentially expressed in L3.6plCas9 mice compared to Sham, of which only 176 (13%) were similarly changed in L3.6plIL8, CXCL1-/- mice, demonstrating significant normalization of the molecular atrophy signature. Analyte profiling of serum confirmed significantly elevated levels of IL-8 and CXCL1 in L3.6-pl Cas9 mice, but not in the respective knockouts. We further found that elevations in tumor-derived (human) IL-6 and MCP-1, factors previously implicated in cachexia, were also attenuated in serum of L3.6plIL8, CXCL1-/- mice, suggesting a dependency of IL-6 and MCP-1 production and/or secretion on IL-8 and CXCL1 in pancreatic tumors. In summary our data indicate that combined, but not individual, targeting of IL-8 and CXCL1 in human pancreatic tumors significantly blocks cachexia.

Key Words: Cytokine, chemokine, atrophy, inflammation

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Succinate impairs skeletal muscle isometric and isotonic contractile function

Vinicius M Mariani ^1,^✉, Ravi Kumar ¹, Leonardo F Ferreira ¹

Abstract

Mitochondrial and metabolic disorders such as heart failure with preserved ejection fraction (HFpEF) cause skeletal muscle contractile dysfunction through mechanisms not fully understood. Abnormal mitochondrial metabolism causes cytosolic accumulation of metabolites of the tricarboxylic acid (TCA) cycle, such as succinate and its derivatives.¹ Therefore, we investigated whether succinate impairs skeletal muscle contractile function. We exposed mouse diaphragm bundles to dimethyl-succinate (0.5 to 10 mM, membrane-permeable) ex vivo for 1h to 4h at 37°C and measured contractile properties. Dimethyl-succinate (0.5 and 1 mM, 4 h) decreased diaphragm twitch force (~15%), tetanic force (~10%), and peak power (~25%; Figure 1.A). The greater decline in peak twitch force compared to maximum tetanic force allude to impairments in the excitation-contraction coupling (ECC) machinery, whereas the notable decrease in peak power implicates dysfunction in sarcomeric proteins. Myofibrillar and calcium- handling proteins are susceptible to hypersuccinylation at lysine residues, suggesting that elevated lysine succinylation contributes to the contractile impairments in conditions associated with succinate accumulation. We performed post-translation modification proteomics in diaphragm bundles exposed to dimethyl-succinate (10 mM, 1h: 75-90% decrease in force and power) and found increased lysine succinylation in nebulin (6-fold), titin (5-fold) and troponin-I (2-fold). Myosin heavy chain (fast), myosin-binding protein C (fast), troponin C, myomesin, and calsequestrin were among the proteins with lysine succinylation detected only in bundles exposed to dimethyl-succinate. Inhibition of complex II activity (dimethyl-malonate, 10 mM) had no effect on contractile function and did not prevent the loss of force and power induced by dimethyl-succinate. Exposure of permeabilized single fibers to dimethyl-succinate (10mM, 1h at 15°C) revealed a ~20% decrease in maximum isometric force in fast-twitch fibers and a ~20% decrease in submaximal force and calcium sensitivity in slow-twitch fibers. The high concentration used in single fibers accelerates mass action and elicits non-enzymatic effects² suggesting direct effects of succinate on sarcomeric proteins. Our study shows that membrane-permeable succinate causes isometric and isotonic contractile dysfunction via impairments in excitation-contraction coupling and sarcomeric protein function. Moreover, several myofibrillar proteins responsible for sarcomere structure and function emerged as being susceptible to lysine succinylation. The data suggest that cytosolic accumulation of succinate is a potential mediator of contractile dysfunction in mitochondria and metabolic disorders.

Key Words: Isotonic contraction, weakness, lysine succinylation, sarcomere, calcium handling

References

1.Chouchani ET, Pell VR, Gaude E, Aksentijević D, Sundier SY, Robb EL, Logan A, Nadtochiy SM, Ord ENJ, Smith AC, Eyassu F, Shirley R, Hu CH, Dare AJ, James AM, Rogatti S, Hartley RC, Eaton S, Costa ASH, Brookes PS, Davidson SM, Duchen MR, Saeb-Parsy K, Shattock MJ, Robinson AJ, Work LM, Frezza C, Krieg T, Murphy MP. Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature. 2014. Nov 20;515(7527):431-435. doi: 10.1038/nature13909. Epub 2014. Nov 5. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Skeletal muscle specific rescue of Bmal1 is sufficient to extend the lifespan of the Bmal1 KO mouse

Miguel A Gutierrez-Monreal ^1,^2,^✉, Christopher A Wolff ^1,², Eduardo E Rijos ^1,³, Collin Dougla s ^1,², Karyn A Esser ^1,²

Abstract

Several studies across different model organisms have indicated that age-associated changes in skeletal muscle may be a critical contributor to healthspan and lifespan of the organism. In this study we tested whether skeletal muscle specific rescue of the core clock gene, Bmal1, is sufficient to extend the lifespan of the Bmal1 knock out mouse (Bmal1 KO). The Bmal1 KO mouse exhibits 50% mortality by 37 weeks of age and features of aging have been described across multiple organ systems, including skeletal muscle. Prior studies from other labs have performed liver and brain-specific rescue of Bmal1, however neither of these approaches were sufficient to extend survival of the Bmal1 KO. We used systemic delivery with an AAV construct expressing Bmal1 behind a muscle specific promoter (CK6). We tracked a variety of physiological and behavioral parameters in the mice. We found that Bmal1 rescue in skeletal muscle of about 20-30% of control was sufficient to significantly extend lifespan assessed by Kaplan-Meier and log-rank survival tests (n=36-45 mice/group). In addition, we found that the Bmal1 KO+AAV mice exhibited improved total cage activity, motor coordination (rotarod) and measures of ex vivo muscle force. RNA-seq analysis of gastrocnemius muscle of these mice identified a significant cluster of genes related to oxidative phosphorylation, insulin signaling, and protein homeostasis that were rescued in the Bmal1 KO+AAV group. Ongoing studies are in progress to better define both muscle specific and systemic mechanism(s) through which survival is improved.

Key Words: Bmal1, survival, adeno-associated virus

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The role of muscle IGF-I after a single bout of exercise on AMPKα in mouse skeletal muscle

Chih-Hsuan Chou ^1,^✉, Elisabeth R Barton ¹

Abstract

Insulin-like growth factor I (IGF-I) contributes to glucose homeostasis and may glucose handling in skeletal muscle through activation of hybrid receptors, which contain insulin and IGF-I hemireceptors. Our previous work showed that loss of IGF-I in murine muscle can have negative effects on glucose metabolism and exercise tolerance in addition to muscle mass. AMP-activated protein kinase (AMPK) has a key role in maintaining the balance between anabolic and catabolic programs for cellular homeostasis in response to metabolic stress.Increased AMP/ATP ratio in muscle cells during repetitively contraction activates phosphorylation at AMPKαThr172, and this induces downstream signaling cascades to promote glucose uptake. Phosphorylation of AMPKαSer485/491 can inhibit AMPK activity and insulin may be one of its upstream triggers in skeletal muscle. The influence of IGF-I in glucose uptake and its interplay with AMPK activation during exercise is not well understood. The aim of this study is to determine the acute response of AMPK signaling pathway after a single bout of aerobic exercise in muscle loss or overexpression of IGF-I (Figure 1). We used two different mouse models in our study to determine the acute exercise response in muscle deficient or overexpressed IGF-I: Muscle IGF-I Deficient (MID) and mice harboring the rat Igf1a cDNA under the control of the fast myosin light chain promoter (mIgf1^+/+, MLC/mIgf). We induced Igf1 deletion with chow containing doxycycline at postnatal day 21 for 5 days in MID and the controls without floxed exon 4 of Igf1 (Igf1^fl/fl).Littermateswithout the transgene were used aswildtype controls for mIgf1^+/+ mice. We used 16-week-old MID and mIgf1^+/+ transgenic male mice, and relevant controls in our study. The mice were randomly assigned to No Exercise control (NO) or to exercise groups with three time points (0-hour, 2-hour, or 24-hour after exercise). For the exercise groups, mice ran at 20 m/min with 5% inclination for 30 minutes. Tibialis anterior muscles from each mouse were used to examine AMPKα phosphorylation. In our preliminary data, the phosphorylation of AMPKαSer485/491 was higher at rest in mIgf1^+/+ transgenic muscle compared to wild type muscle, and the trend of phosphorylation was different immediately after exercise and 2-hours later. In contrast, phospho-AMPKαSer485/491 seemed unchanged in MID mice. This suggests that high IGF-I levels may be needed to modulate the inhibitory AMPKα phosphorylation state, whereas normal or diminished IGF-I has no effect.

Key Words: Senescent skeletal muscle, IGF-I, exercise, AMPK

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Dysregulation of heme synthesis-export axis in skeletal muscle reshapes energetic metabolism and results in impaired motor performance

Miriam Mistretta ^1,^✉, Veronica Fiorito ¹, Myriam Hsu ¹, Chiara Riganti ², Giorgio Roberto Merlo ¹, Paolo Ettore Porporato ¹, Yvan Torrente ³, Emanuela Tolosano ¹

Abstract

Heme metabolism plays an essential role in the maintenance of skeletal muscle health ^{1, 2}. Feline leukemia virus subgroup C receptor 1a (FLVCR1a) is a plasma membrane heme exporter that, by removing the intracellular excess of heme, limits the feedback inhibitory effects of heme on its own production, thus sustaining heme biosynthesis. Being part of the heme synthesis-export functional axis, heme export by FLVCR1a has been reported to sustain the tricarboxylic acid cycle (TCA) flux and the electron transport chain (ETC) activity in tumors³. Here we generate skeletal muscle-specific Flvcr1a-null mice and analyze the impact of disrupted heme synthesis-export axis in this tissue. Metabolic data obtained in gastrocnemius homogenate show that, upon deletion of Flvcr1a, the activity of TCA cycle enzymes and ETC complexes is increased, along with glutaminolysis and fatty-acid beta oxidation. Conversely, the activity of glycolytic enzymes is reduced. Flvcr1a deletion also affects muscle morphology. The distribution of cross-sectional fibers’ area (CSAs) is compromised in Flvcr1a knockout mice, showing a prevalence of fibers with small area. Along with these data, increased number of PAX7+ cells was also observed. In motor behavior tests, Flvcr1a knockout mice show worse performance compared to controls, a phenotype exacerbated by aging. Collectively, these results hint that FLVCR1a is involved in the regulation of energetic metabolism in skeletal muscle, and that the heme synthesis export system is important to maintain proper muscle function.

Key Words: block of heme export, reduced fibers’ area, worse muscle performance, energetic metabolism

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The timecourse of adaptive change in gene expression across 30d of daily programmed resistance exercise in rats

Mark Viggars ^1,^2,^✉, Hazel Sutherland ¹, Hermann Lanmueller ³, Martin Schmoll ³, Manfred Bijak ³, Jonathan C Jarvis ¹

Abstract

Despite data over several decades showing that resistance exercise induces hypertrophy, the transcriptional mechanisms responsible for the process are still not fully understood. Recent meta-analyses have attempted to identify time-dependent patterns in the expression of genes that regulate the transcriptional response to resistance exercise.^1,2 The objective of this study was to perform a detailed transcriptomic analysis through RNA- sequencing of the acute response to resistance exercise (one hour after an exercise session), across a 30 day period of daily resistance training. We have developed and implemented a highly controllable resistance training model that uses implantable pulse generators in free-living rats to induce hypertrophy in the tibialis anterior by co-contraction of the dorsiflexors and plantarflexors (3). We assessed the skeletal muscle transcriptome (1-hour post exercise) after 2, 10, 20 and 30 days of our exercise training paradigm and found 5 temporal clusters across our timecourse (Self-organizing maps (SOM) 1-5, Figure 1B). SOM1 and SOM2 included known resistance-exercise responsive pathways including the ribosome, proteasome, and protein processing in the endoplasmic reticulum as well as genes associated with inflammation/macrophages. These genes were highly responsive while the muscles were growing, but their expression was blunted as muscle mass reached a new steady state. GeneMANIA network association analysis (Figure 1c) found that the genes within these clusters were centrally controlled by the transcription factor Myc, a well-known regulator of cell cycle, metabolism, growth, RNA polymerase activity and ribosomal biogenesis (4). However, the expression of many of these genes in skeletal muscle is not well understood. We propose that it will be informative to study the group of genes whose timecourse of expression is associated with the period of growth and whose response declines as the muscle adapts to a daily stimulus to achieve a new steady state.

Key Words: Transcriptomics, resistance exercise, Myc

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Peroxisomal-mitochondrial interaction impinging on muscle homeostasis

Giulia Trani ^1,^2,^*, Marco Scalabrin ^1,^2,^*, Gaia Gherardi ², Cristina Cerqua ³, Leonardo Salviati ³, Vanina Romanello ^1,²

Abstract

The skeletal muscle tissue is a primary metabolic activity site controlling almost 80% of insulin-dependent glucose uptake and storage. It is also the largest protein reservoir (75%) of our body, supplying aminoacids to support energy production in other organs during catabolic conditions. Thus, skeletal muscle is crucially important to control health and disease progression in distant tissues both for its role in balancing other organs' metabolic needs and its reserves to use in energy production. Its dysfunction is associated with poor prognosis and reduced quality of life. Peroxisomes are ubiquitous dynamic metabolic organelles that play an essential role in a variety of cellular events such as reactive oxygen species clearance and lipid metabolism. However, their role in muscle metabolism and function has largely been overlooked. To investigate the consequences of peroxisomal dysfunction in muscle we have generated a mouse model with impaired peroxisome biogenesis by disrupting specifically in skeletal muscle the Peroxisomal biogenesis factor 5 (PEX5), which serves as a shuttle receptor for the import of peroxisome matrix protein. We found the peroxisomal dysfunction in muscle causes several mitochondrial defects, and increased ROS production resulting in muscle ultrastructural alterations and reduced running performance. Thus, peroxisomal integrity is critical for skeletal muscle homeostasis.

Key Words: Peroxisomes, skeletal muscle, ROS

References

Argyriou C, D'Agostino MD, Braverman N. Peroxisome biogenesis disorders. Transl Sci Rare Dis. 2016. Nov 7;1(2):111-144. doi: 10.3233/TRD-160003. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Mitochondrial dysfunction in skeletal muscle promotes precocious sarcopenia, degeneration of multiple organs, and premature death through inflammation and metabolic changes

Caterina Tezze ^1,^2,^✉, Francesco Ivan Amendolagine ¹, Marco Sandri ^1,²

Abstract

A healthy mitochondrial network is essential for post-mitotic tissues as muscles. Mitochondria-shaping machinery is downregulated in sarcopenia and is maintained by lifelong exercise. OPA1 is a profusion protein that plays an important role in mitochondrial dynamics we have demonstrated. Interesting, OPA1 protein is significantly decreased in sedentary humans and old mice compared to young controls and, exercise is sufficient to restore the protein level. In conditional tissue-specific Opa1 ko is sufficient to induce a sarcopenic-cachectic phenotype in just 3 months of deletion and Fgf21 is a central myokine in this process. This work aims to find new specific players involved in aging sarcopenia. Particularly, that synergistically promotes the aging of the whole body and the secretome related. we are comparing different genetic mouse models to dissect the specific role of the different genes in the sarcopenia scenario. Mitochondrial dysfunction in muscle tissue is sufficient to directly drive metabolic changes and systemic inflammatory by increasing the expression and secretion of the myokines FGF21 and IL6. Despite the key role of FGF21, we have new evidence that is connecting mitochondrial dysfunction, autophagy alteration, muscular IL6, and aging. Opa1 is a "sensor" for the health of the muscle, the reduction of the protein level recapitulates the acute sarcopenia process.

The changes related to mitochondrial dysfunction are specifically correlated to the shape of these organelles. Different alteration induces some common but mainly specific metabolic and stress responses directly correlated to sarcopenia development.

Key Words: Skeletal muscle secretome, inflammaging, metabolism, sarcopenia, precoscious aging

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Identification of a novel TFEB and exercise dependent gene

Davide Steffan ^1,^2,^✉, Andrea Armani ^1,², Marco Sandri ^1,²

Abstract

While the benefits and adaptations to regular physical activity are long known, molecular networks, signalling pathways and regulatory molecules that coordinate adaptive responses to exercise are still matter of debate. Transcription Factor EB (TFEB) is a master metabolic regulator in skeletal muscle, finely tuning fuel uptake to energy production during exercise. These findings strongly support TFEB activity as crucial for mediating the beneficial effects of exercise¹. Starting from this evidence we decided to discover new uncharacterized genes that may mechanistically explain adaptive effects of TFEB on skeletal muscle. By crossing gene expression data of TFEB transgenic and trained muscles, we found a gene of unknown function belonging to the Riken cDNA collection² that we called Exe-Riken. We showed that Exe-Riken is a real protein coding gene. We also confirmed its transcriptional dependence from TFEB and exercise, with a fine tuning in different muscles; interestingly, its transcription induction does not depend on extra muscular factors. Moreover, we showed its differential localization in muscle fibers, with a positive correlation between cross sectional area and immunostaining reactivity. Oxidative muscles such as soleus did not present cytoplasmic but more nuclear Exe-Riken localization. Thanks to overexpression and NES deletion experiments, we showed that exercise is an Exe-Riken cytosol-to-nucleus shuttling stimulus. Finally, qRT-PCR experiments suggest the presence of at least two transcript variants that differ on the 3’UTR sequence. So far, all the findings emphasize Exe-Riken as a novel but compelling molecular player mediating metabolic adaptions to physical training.

graphic file with name ejtm-32-1-10440-g033.jpg

Key Words: TFEB, exercise, adaptation, RIKEN

References

1.Mansueto G, Armani A, Viscomi C, D'Orsi L, De Cegli R, Polishchuk EV, Lamperti C, Di Meo I, Romanello V, Marchet S, Saha PK, Zong H, Blaauw B, Solagna F, Tezze C, Grumati P, Bonaldo P, Pessin JE, Zeviani M, Sandri M, Ballabio A. Transcription Factor EB Controls Metabolic Flexibility during Exercise. Cell Metab. 2017. Jan 10;25(1):182-196. doi: 10.1016/j.cmet.2016.11.003. Epub 2016. Dec 20. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Understanding BMP signaling in cancer cachexia

Camilla Pezzini ^1,^2,^✉, Roberta Sartori ^1,², Adam Hagg ³, Paul Gregorevic ³, Marco Sandri ^1,²

Abstract

Cancer cachexia is a multi-factorial metabolic syndrome characterized by excessive body weight loss due to progressive fat and lean mass catabolism in spite of nutritional support,¹ occurring in more than half of cancer patients.² The frailty experienced by cachectic patients increases morbidity, reduces tolerance to treatments, complicates patient management and accounts for up to 30% of cancer deaths.^3,4 The loss of skeletal muscle mass and strength are considered as the most relevant features of cancer cachexia and predictors of poor outcomes.^5,6 Bone Morphogenetic Protein (BMP)/Smad1/5/8 pathway is a positive regulator of muscle mass repressing the transcription of MUSA1, an atrophy-associated E3 ubiquitin ligase that facilitates proteasome-dependent protein breakdown.^7,8 We demonstrated that diminished BMP (Bone and Morphogenetic Protein)/Smad1/5/8 signalling in muscles plays a critical role for the onset of cancer cachexia. Re-activation of BMP signaling in muscles is sufficient to prevent tumor-induced muscle atrophy, and to extend the lifespan in tumor bearing mice despite tumor growth. Importantly, these protective effects are prevented by knocking down Smad1/5.⁹ We aim to understand how the cancer stimulates transcriptional changes leading to BMP-Smad1/5/8 inhibition in skeletal muscles to favor cachexia onset in order to identify new potential therapeutic targets.

Key words: Cancer cachexia, gene regulation, BMP signature

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Two brothers with X-linked Charcot Marie Tooth disease and different lifestyle

Valentina Pegoraro ^1,^✉, Leonardo Salviati ², Corrado Angelini ¹

Abstract

Aim of our study was to investigate CMT1-X and its different phenotypic expression in a family with an unreported frameshift mutation in GJB1 gene encoding connexin 32. In CMT1-X family two male siblings had a different clinical phenotype we investigated genetic and clinical features. Connexin 32 is a gap junction protein that is located in paranodal regions and Schmidt-Lanterman incisures. Genetic analysis and micro RNA-206 determination was performed on DNA from 2 cases on the following genes: BSCL2, EGR2, FGD4, FIG4, GARS, GDAP1, GJB1, HSPB1, IGHMBP2, LITAF, LMNA, MFN2, MPZ, MTMR2, NEFL, PMP22, PRX, SH3TC2. Muscle specific miRNAs was also investigated in serum. A muscle biopsy (1 case) and EMG with conduction velocities were performed in both patients. Clinical features and family history were collected. One brother of 55 years had diffuse hypotrophy in lower limbs since age 28 years, he had atrophic legs bilateral pes cavus bilateralis and ataxic gait, he had difficulty grasping object with hands his micro-RNA 206 level was elevated. The younger brother of 47 years had a distal weakness, tremor in upper extremities (right more than left) and stepping gait with thin legs and abnormal conduction velocity EMG (38 m/s). He had difficulty opposing thumb and a slight deficit (4/5 MRC score) of finger extension in upper limbs. His weakness was mostly in hands and slowly evolving in lower limbs. Muscle biopsy showed neurogenic changes, MRI of the spinal cord showed in the cervical region a slight disc protrusion on C5/C6 with a rectified cervical cord., a different level of micro RNA 206 was found In first genetic analysis, no duplication of PMP22 gene was found. A second genetic analysis of a panel of genes associated with inherited peripheral neuropathy showed a frameshift mutation of connexin 32 (GJB1 gene) c.281_287del in hemizigosis not previously reported that segregated with clinical phenotype in two male brothers. Circulating miR-206 was found up-regulated in both patients. While the older sibling had evident gait difficulty and presented a chronic invalidating weakness both in upper and lower extremities the second brother was a military pilot and had minimal weakness mostly in the right hand and tremor . There was evidence from the family history of an X-linked Hereditary Sensory Motor Neuropathy that was confirmed by the mutation in connexin 32 gene. We hypothesize that in CMT X-linked there is a different expression of the connexin-32 protein in relation to its markedly reduced abundance in gap region by a semi-dominant mechanism on other connexins and consequent demyelination. Such reduction might affect not only gap function formation but also myelin and resulted in differential micro-RNA 206 expressivity observed in this family. The level of these molecules reflects different muscle mass observed.

Key Words: Charcot Marie Tooth, connexin-32, novel mutation in GJB1 gene, miR-206

References

Panosyan FB, Laura M, Rossor AM, Pisciotta C, Piscosquito G, Burns J, Li J, Yum SW, Lewis RA, Day J, Horvath R, Herrmann DN, Shy ME, Pareyson D, Reilly MM, Scherer SS; Inherited Neuropathies Consortium—Rare Diseases Clinical Research Network (INC-RDCRN). Cross-sectional analysis of a large cohort with X-linked Charcot-Marie-Tooth disease (CMTX1). Neurology. 2017. Aug 29;89(9):927-935. doi: 10.1212/WNL.00000000000 04296. Epub 2017. Aug 2. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The strength and the broadness of CFTR correctors for the treatment of sarcoglycanopathies

Martina Scano ^1,^✉, Alberto Benetollo ¹, Francesco Dalla Barba ¹, Paola Caccin ¹, Marcello Carotti ¹, Roberta Sacchetto ², Dorianna Sandonà ¹

Abstract

Sarcoglycanopathies are recessive forms of muscular dystrophies in which the disruption of the sarcoglycan complex results in sarcolemma fragility and progressive muscle degeneration. Most of the reported cases are due to missense mutations originating a folding-defective sarcoglycan (SG) eliminated by the cells’ quality control system, although potentially functional.¹ No treatment for sarcoglycanopathy is currently available. Cell and gene therapy-based approaches are under investigation, even if many challenges are present. Among others, there is the difficult of delivering the gene to skeletal muscle, which is the most abundant tissue of human body, the problem of immunogenicity of the viral vector and of the transgene (if the protein is totally absent in the patient), etc. An interesting alternative could be represented by a class of small molecules, called protein folding correctors, able to recover the mutants and avoid complex disruption. Furthermore, small molecules are easier to formulate, deliver and eventually optimize. Particularly, CFTR correctors are a subgroup of cystic fibrosis modulators able to enhance or even restore, through different mechanisms, the expression, function, and stability of type II CFTR mutants.² In α-sarcoglycanopathy, the effective rescue of different SG-mutants has been proved for a few of such molecules by using cell models and, importantly, myogenic cells from patients.^3,4 In addition, their pharmacological efficacy has been successfully tested in novel mouse models, ad hoc generated, expressing the human mutated α-SG sequence in both hind-limbs, delivered by early AAV injection. As the proof of concept of the strategy has been established for LGMDR3 both in vitro and in vivo, we checked the broadness of such approach by testing the effect of a panel of different CFTR correctors in LGMDR4 patient’s myotubes, carrying a missense mutation on the SGCB gene. The analysis revealed the increased expression of the mutated β-SG protein upon treatment with the C17 molecule, the most promising corrector in our hands. In particular, C17 induced a two-fold increase in the β-SG membrane fraction in comparison to vehicle treated cells. The myotubes surface re-localization of the β-SG protein was further proven by immunofluorescence staining. These results confirm the hypothesis that CFTR correctors could be in principle applied to all the sarcoglycanopathies cases linked to missense mutations, regardless of the SGC gene. Remarkably, these data are the proof that CFTR correctors are effective not only in cystic fibrosis, but also in sarcoglycanopathy, suggesting a great potential for the protein conformational diseases, especially for those orphan diseases where no current therapeutics are available.

Key Words: Sarcoglycanopathies, CFTR correctors, therapeutic approaches

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

IGF-I from satellite cells is critical for skeletal muscle growth and regeneration

Hui Jean Kok ^1,^✉, Elisabeth R Barton ¹

Abstract

Satellite cells are an absolute requirement for postnatal muscle growth and muscle regeneration. Insulin-like Growth Factor-I (IGF-I) is well known to promote satellite cell function by expanding satellite cell proliferation and accelerating differentiation. We previously established that muscle fiber provides an important endogenous source of IGF-I for anabolic actions on the skeletal muscle;¹ however, whether satellite cells require IGF-I from other cell types remains unclear.² Much speculation determined that IGF-I diffuses into the satellite cell niche for its actions, although no direct evidence had been shown. Thus, we sought to determine the critical sources of IGF-I for muscle growth and regeneration. We generated novel mouse models with inducible tissue- and cell-specific ablation of IGF-I: SID (Satellite cell IGF-I Deficient), MID (Muscle IGF-I Deficient), SMID (Satellite cell and Muscle IGF-I Deficient), to test the hypothesis that satellite cells require an autocrine source of IGF-I for muscle growth and regeneration. Mice lacking the floxed exon 4 of Igf1 (Igf1fl/fl) alleles but retaining the Cre drivers and Rosa reporters served as controls (CTRL) for doxycycline (DOX) and tamoxifen (TAM) treatment effects. Male pups at postnatal day 3 (P3) were treated with TAM/VEH(3) and/or DOX for 5 consecutive days to induce Igf1 deletion, and hindlimb muscles were harvested at P28. SID and SMID pups that received TAM were 22% and 34% smaller in body weight when compared to VEH treated littermates. The absolute mass of major hindlimb muscles (TA, soleus, EDL, Gastrocnemius, and Quadriceps) for SID and SMID mice were significantly smaller than VEH treated littermates, suggesting the importance of IGF-I from satellite cells for muscle development. To determine if IGF-I from satellite cells is crucial for muscle regeneration, we treated 20-24-week-old mice (n=4/sex/timepoint/genotype) with TAM and/or DOX prior to unilateral Cardiotoxin (CTX) injection into the TA(3), while the contralateral TA served as an internal control. Muscles were harvested at days 3, 5, 7, 11, and 14 post-CTX. TA masses from all groups at days 3-7 post-CTX were significantly lower (p<0.05) compared to contralateral non-damaged muscles. All groups were able to recover to non-damaged mass levels by day 14 post-CTX except SID animals. There were no differences when comparing CTRL and MID muscle fiber areas, instead, SID muscle fibers were smaller when compared to CTRL muscles, suggesting that satellite cell IGF-I are crucial for muscle regeneration. Surprisingly, SMID mice were able to recover to CTRL levels in fiber area, indicating a potential compensatory IGF-I supply from non-muscle sources. Next, in-situ functional testing(4) was performed comparing damaged vs. non-damaged TAs. At day 14 post-injury, normalized forces were lower in damaged TAs of CTRL, MID, and SMID animals when compared to contralateral non-damaged TA; however, there was no difference in force for SID animals, suggesting the importance of muscle-specific IGF-I for muscle function post-injury. In conclusion, IGF-I generated by satellite cells is critical for efficient skeletal muscle regeneration, while both satellite cells and myofibers provide integral sources of IGF-I for muscle development.

Key Words: Satellite cells, IGF-I, muscle regeneration, postnatal muscle growth

References

1.Vassilakos G, Lei H, Yang Y, Puglise J, Matheny M, Durzynska J, Ozery M, Bennett K, Spradlin R, Bonanno H, Park S, Ahima RS, Barton ER. Deletion of muscle IGF-I transiently impairs growth and progressively disrupts glucose homeostasis in male mice. FASEB J. 2019. Jan;33(1):181-194. doi: 10.1096/fj.201800459R. Epub 2018. Jun 22. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Kok HJ, Barton ER. Actions and interactions of IGF-I and MMPs during muscle regeneration. Semin Cell Dev Biol. 2021. Nov;119:11-22. doi: 10.1016/j.semcdb.2021.04.018. Epub 2021. May 5. [DOI] [PMC free article] [PubMed] [Google Scholar]
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4.Spinazzola JM, Smith TC, Liu M, Luna EJ, Barton ER. Gamma-sarcoglycan is required for the response of archvillin to mechanical stimulation in skeletal muscle. Hum Mol Genet. 2015. May 1;24(9):2470-81. doi: 10.1093/hmg/ddv008. Epub 2015. Jan 20. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION V: Energy balance and skeletal muscle in microgravity

Filippo Giorgio Di Girolamo ¹, Giovanni Valentini ¹, Roberta Situlin ¹, Nicola Fiotti ¹, Elisa Carrubba ¹, Gabriele Mascetti ¹, Gianni Biolo ^1,^✉

Abstract

Energy balance maintenance is crucial to preserve astronaut’s wellness during longduration-spaceflight. Nutritional intervention can counteract the detrimental effect of microgravity on body composition and metabolism. Methods and procedures: NutrISS is an Italian Space Agency biomedical experiment. This proof-of-concept study aim to monitor the body composition of astronauts and to provide nutritional advice during long-term mission. The study has been executed on the ISS and sponsored by ESA. The payloads selected for both missions (i.e. “BEYOND” and “COSMIC KISS”) result from a public-call funded and coordinated by ASI, in the frame of its national mission of promoting and fostering the culture of space across the Country and providing access to the ISS as a laboratory in space to the Italian research community. The Italian astronaut Luca Parmitano already completed the protocol during his 6-month spaceflight. While Mathias Maurer is now starting the firsts measurements. Each astronaut underwent to baseline data collections, i.e. body composition, anthropometric evaluation and energy and metabolic assessment carried out by the scientific team. Body composition has been assessed by a BioImpedance Analyser device (Akern) modified by Kayser Italia. To maintain the near-neutral energy balance, the NutISS team monitored monthly the astronaut body mass during flight. Results are now classified for privacy reasons. The study was extended since collected data improved physical performance and quality of life of the astronaut during spaceflight and optimize the astronaut recovery- phases on Earth after landing. Obtained experimental data could have an important impact both on astronaut quality of life and clinical management of malnourished and/or obese immobilized patients, therefore improving the quality of human bedridden patients on Earth.

graphic file with name ejtm-32-1-10440-g038.jpg

Key Words: Space flight, energy balance, body composition, muscle atrophy

Acknowledgements

Funding by ASI, NUTRISS and MARS-PRE Projects

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Peripheral impairments of oxidative metabolism during exercise following inactivity

Bruno Grassi ^1,^✉, Giovanni Baldassarre ¹, Lucrezia Zuccarelli ¹

Abstract

Oxidative metabolism is responsible for most of ATP re-synthesis during work, leisure or sport activities lasting longer than 1-2 minutes. A substantial impairment of oxidative metabolism during exercise is known to occur following inactivity. Whereas the “central” (mainly cardiovascular) factors contributing to this impairment have been thoroughly investigated, “peripheral” impairments (related to microvascular and endothelial functions, intramuscular O2 delivery-O2 uptake coupling, peripheral O2 diffusion, skeletal muscle oxidative metabolism, mitochondrial respiration) have received relatively less attention. We recently investigated several central and peripheral factors potentially responsible for an impairment of oxidative metabolism during exercise in 10 young males undergoing 10 days of horizontal bed rest (BR) (Zuccarelli et al. 2021). As expected, peak pulmonary O2 uptake (V̇O2peak) and peak cardiac output were significantly lower after (POST) vs. before (PRE) BR (44.4 ± 7.2 mL kg-1 min-1 vs. 40.3 ± 6.1, P=0.001; 25.2 ± 5.5 L min-1 vs. 19.0 ± 3.2, P=0.003, respectively). Microvascular/endothelial function was evaluated by measuring the blood flow increase in the common femoral artery, by Eco-Doppler, during 1-min passive leg movements (PLM). A significant impairment was observed following BR: the area under the blood flow vs. time curve during PLM was smaller (P=0.03) in POST (274 ± 233 mL) vs. PRE (427 ± 291). The intramuscular matching between O2 delivery and O2 uptake was evaluated by measuring the muscle deoxygenation “overshoot”, by near-infrared spectroscopy (NIRS), during a rest-to-exercise transition. Also for this variable a significant impairment was observed following BR, with a significantly greater overshoot in POST vs. PRE (P=0.02). Resting vastus lateralis muscle V̇O2, estimated by calculating the slope of muscle deoxygenation (determined by NIRS) during a transient ischemia of the limb, was 27% lower (P=0.006) in POST vs. PRE. Other variables related to skeletal muscle oxidative metabolism were unaffected (or even slightly improved) following BR. The kinetics of recovery of vastus lateralis muscle V̇O2 (determined by NIRS and the repeated-occlusions method [Zuccarelli et al. 2020]) were unaffected by BR: time-constants were 21.7 ± 5.7 s in PRE vs. 22.2 ± 5.9 in POST (P=0.79). Skeletal muscle citrate synthase activity, taken as an estimate of muscle mass, was not different (P=0.11) in POST (131 ± 16 nmol.min-1.mg-1) vs. PRE (138 ± 19). Mitochondrial respiration was evaluated ex vivo by high-resolution respirometry in isolated vastus lateralis muscle fibers obtained by biopsy. Maximal ADP-stimulated mitochondrial respiration (JO2max) (66 ± 18 pmol.s-1.mg-1 [POST] vs. 72 ± 14 [PRE], P=0.41) was not affected by BR. The sensitivity of mitochondrial respiration to submaximal [ADP] was evaluated by calculating the [ADP] corresponding to 50% of JO2max: values were significantly lower (P=0.046) in POST (409 ± 398 μM) vs. PRE (1270 ± 1015), demonstrating an increased sensitivity to submaximal [ADP]. In conclusion, peripheral limitations to oxidative metabolism after a 10-day BR were “upstream” of mitochondrial function, at the level of microvascular O2 delivery/endothelial function, and at the intramuscular matching between O2 delivery and O2 uptake. On the other hand, mitochondrial content and mitochondrial respiration were unaffected or even improved (enhanced mitochondrial sensitivity to submaximal [ADP]). The decreased resting muscle V̇O2 following BR could represent an adaptive phenomenon in response to simulated microgravity–inactivity, attributable to the fact that muscle catabolic processes within muscles are less expensive, in terms of energy, than anabolic ones. These concepts, besides being of interest from a basic science point of view, may be relevant for patients with pathological conditions characterized by relatively short periods of profound inactivity, and could affect the definition of countermeasures or rehabilitative interventions.

Key Words: Microgravity, inactivity, skeletal muscle oxidative metabolism, microvascular/endothelial function, mitochondrial respiration

Acknowledgements

Funding by ASI, MARS-PRE Project, DC-VUM-2017-006; PRIN Project 2017CBF8NJ; WP6, Project ALT FRAILTY, Active Ageing, UNIUD.

References

Zuccarelli L, do Nascimento Salvador PC, Del Torto A, Fiorentino R, Grassi B. Skeletal muscle V̇o2 kinetics by the NIRS repeated occlusions method during the recovery from cycle ergometer exercise. J Appl Physiol (1985). 2020. Mar 1;128(3):534-544. doi: 10.1152/japplphysiol.00580.2019. Epub 2020. Jan 23. [DOI] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Metabolic dysfunction and exercise preconditioning in disuse muscle atrophy

Lorenza Brocca ¹, Maria Antonietta Pellegrino ¹, Roberto Bottinelli ^1,^✉

Abstract

Disuse induced loss of muscle mass is a frequent phenomenon and a very relevant clinical problem. It exposes subjects to falls and fractures and favour further deconditioning. It is related to metabolic alterations, such as insulin resistance, and to low grade chronic inflammation which are among the major risk factors of chronic diseases. It worsens the prognosis of many chronic diseases 1, which benefit from exercise training.

We studied structural, functional and proteomic adaptations of skeletal muscle and the underlying cellular and molecular mechanisms induced by spaceflight in humans and by simulated microgravity both in humans, bed rest (BR) and unilateral lower limb suspension (ULLS), and in mice, hindlimb unloading (HU). As expected, in both human and mice models we found muscle atrophy and a slow to fast myosin isoform shift. Moreover, a downregulation of most myofibrillar proteins was observed by 2D proteomic and of myosin by quantitative one-dimensional electrophoresis. To address the mechanisms of such adaptations, we studied intracellular signalling pathways controlling muscle mass and metabolism. In human bed rest and mice HU, we observed: upregulation of NRF2, a transcription factor sensing reactive oxygen species (ROS) in the cell; adaptations of major ROS buffering systems (e.g., SOD, catalase, peroxiredoxins, Hsp); protein oxidation. Such findings were consistent with a potential role of redox imbalance in causing muscle wasting as suggested by several previous findings. To test such hypothesis, we administered trolox, a potent antioxidant, to HU mice. Importantly, NRF2 was not up-regulated and no protein oxidation was found, indicating that redox imbalance was prevented by trolox. However, expressions of MuRF-1 and atrogin-1, major ubiquitin ligases of the ubiquitin proteasome system, were still up-regulated and muscle atrophy was not prevented by trolox. Therefore, redox imbalance was not likely to cause muscle atrophy, at least in mice. Importantly, in human BR and ULLS and in mice HU, proteomic analysis indicated downregulation of both aerobic and anaerobic metabolic enzymes, i.e., a general derangement of energy metabolism. In human BR and mice HU, PGC1α, an activator of transcription controlling mitochondrial dynamics and biogenesis and inhibiting the ubiquitin proteasome system, was downregulated and expression of proteins involved in mitochondrial dynamics was altered. On such ground, we hypothesized that a metabolic program of muscle atrophy could be activated in disuse. To test such hypothesis, we subjected transgenic mice selectively overexpressing PGC1α in muscle to HU. In both soleus and gastrocnemius muscles of such mice, no upregulation of atrogenes expression was observed following HU and muscle atrophy was almost completely prevented confirming a major role of a metabolic program in disuse muscle atrophy. Exercise is an attractive tool to prevent muscle wasting as it is known to cause mitochondrial adaptations and to enhance PCG1α expression. Few, but encouraging evidences on the use of exercise as a tool to prevent disuse atrophy were published. The mechanism of exercise-induced protection on muscle atrophy appears not clearly defined yet. We will present novel data on the mechanisms underlying prevention of muscle atrophy by exercise preconditioning.

Acknowledgements

Funding by ASI, MARS-PRE Project, PRIN Project 2017CBF8NJ.

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Alterations in the behaviour of individual motor units with inactivity

Giacomo Valli ¹, Fabio Sarto ¹, Andrea Casolo ¹, Martino Franchi ¹, Marco Narici ¹, Giuseppe De Vito ^1,^✉

Abstract

A reduction in skeletal muscle activation (disuse), regardless the cause (i.e.: physical inactivity, disease and microgravity), can induce profound functional transformations affecting the metabolic, and neuromuscular properties of the the muscle tissue. It is also well known, both from animal and human studies, that although the main alteration caused by disuse is represented by muscle atrophy, there is evidence that similarly the quality of the muscle is altered, as demonstrated by the fact that the loss of muscle strength is considerably greater than that of muscle size.¹ The reduction of muscle quality can be ascribed to a complex interaction of various factors affecting neuromuscular transmission,¹ muscle architecture,² and contractile apparatus properties.³ Short‐term disuse is a common condition experienced by those individuals, that are bedridden because of an injury, surgery and/or chronic medical conditions. In the present study we focused our attention on the functional skeletal muscle alterations induced by short term disuse, consisting of 10 days of unilateral lower limb suspension (ULLS, walking with crutches), and the effect of a retraining period on the contractile properties of neuromuscular system investigated at the level of the single motor units (MUs). To this purpose, the electrical activity of the muscle vastus lateralis of 11 young males (22.1 ± 2.9 years) was recorded with high-density electromyography (HD-EMG), using a grid of 64 electrodes (5 columns × 13 rows; gold-coated; diameter of 1 mm; inter-electrode distance of 8 mm), during isometric trapezoidal contractions executed at 10, 25 and 50% of maximum voluntary contraction (MVC) and characterized by 3 phases: a linear increase (recruitment phase) in force at 5% MVC/s, a 20 s constant force at target (plateau phase) and a linear decrease (de-recruitment phase) in force at 5% MVC/s. These measures were performed at baseline (BLS0), and repeated immediately after the suspension period (LS10) and after 21 days of retraining (RT1). As expected, MVC decreased at LS10 (from 816.18 ± 97.22 to 573.20 ± 104.69 N; p<0.001) as did the ability of the participants to maintain the constancy (steadiness) of the isometric contraction, during the plateau phase, with a reduction observed at 25% MVC. After the HD-EMG decomposition and the removal of duplicated MUs, a total of 1422 unique MUs were identified. Moreover, we observed a reduction in absolute MUs recruitment and de-recruitment thresholds at all intensity of contractions. The MUs discharge rate decreased at 10 and 25% MVC, at all the points of evaluation (e.g., recruitment, derecruitment and plateau), but not at 50%. The coefficient of variation of MUs interspike interval (COVisi), a marker of irregular firing pattern, increased during the steady state phase at 25% MVC. After the 21 days of retraining, MVC, recruitment threshold and discharge rate recovered to baseline levels while the COVisi and steadiness improved compared to baseline. In conclusion, these results highlight that 10 days of ULLS can induce significant alterations of the skeletal muscle function at the level of the single MUs and that 21 days of resistance training can restore these impairments.

Key Words: Aging, ords: Motor unit, discharge rate, conduction velocity, disuse

Acknowledgements

Funding by ASI, MARS-PRE Project, DC-VUM-2017-006 is acknowledged

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION VI: NMJ aging: Contributions of motor neurons and synaptic Schwann cells

Gregorio Valdez ^1,^✉

Abstract

The cellular and molecular changes that contribute to aging of the neuromuscular junction (NMJ) remain either debated or unknown despite extensive research efforts over several decades. For instance, there are contradictory reports regarding the effect of aging on motor neurons and perisynaptic Schwann cells (PSCs). Additionally, there is a paucity of knowledge about molecular pathways dysregulated in aged motor neurons and PSCs. In this talk, I will present compelling evidence that aging does not result in the loss of motor neurons. I will instead show that motor neurons undergo subcellular and molecular changes during aging. These include loss of synaptic inputs and altered expression of genes with potential roles in synaptic maintenance and repair. I will also present data showing that PSCs accrue a myriad of age-related cellular and molecular features but do not decrease in number with advancing age. I will end by describing studies needed to further advance our understanding of aging NMJs, and its contribution to sarcopenia.

Key Words: Aging, neuromuscular junction, motor neurons, perisynaptic Schwann cells, synapse

References

Maxwell N, Castro RW, Sutherland NM, Vaughan KL, Szarowicz MD, de Cabo R, Mattison JA, Valdez G. α-motor are spared from aging while their synaptic inputs degenerate in monkeys and mice. Aging Cell. doi: 10.1111/acel.12726. Accepted 11 Dec 2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Changes of neuromuscular innervation during aging in healthy males

Sandrine Marchand ¹, Joanne Vallée ¹, José Morais ³, Charlotte H Pion ^2,^5,⁶, Marc Bélanger ^2,⁵, Mylène Aubertin-Leheudre ^2,^5,⁶, Richard Robitaille ^1,^4,^✉

Abstract

The progressive loss of muscle mass (sarcopenia) leads to serious consequences such as a loss of mobility and autonomy.¹ Deterioration of the neuromuscular junction (NMJ) is an emerging factor in the development of sarcopenia. The JNM is a tripartite structure, composed of the presynaptic terminal, the postsynaptic element and by glial cells which are the perisynaptic Schwann cells (PSCs). PSCs regulate the maintenance, remodeling, and repair of JNMs in mouse models,² functions that are altered in aging purportedly to facilitate the re- innervation of the denervated NMJs.^3,4 However, not only is human NMJ poorly characterized, but PSCs have never been investigated despite the essential role attributed to them in mice. Here we report the characteristic of NMJs at young and older active males. NMJ-enriched biopsies were performed using the BeeNMJ method5. In young individuals, the NMJ innervation was robust and showed little variability as no denervation was observed and only a small percentage of partially innervated NMJs was observed. In contrast, in older individuals, we observed more variability between individuals, showing a significantly larger percentage of partial innervation. We will discuss the relationship between the innervation status and the physiological performances of the individuals

Key Words: Human neuromuscular junction, perisynaptic Schwann cells, aging

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Mechanisms of exacerbated denervation in aging muscle

Russell Hepple ^1,^✉

Abstract

Denervation of skeletal muscle fibers is amongst the most influential processes in aging skeletal muscles. It contributes to various aging muscle phenotypes, including muscle atrophy and fiber type shift. Notably, skeletal muscle denervation with aging can be exacerbated in various conditions, such as chronic obstructive pulmonary disease (COPD)¹ and physical frailty,² leading to an acceleration of muscle atrophy, weakness, and mobility impairment. COPD is most often precipitated by years of heavy smoking and interestingly, previous studies have shown that smoking can yield marked fiber type shift even in the absence of lung disease, consistent with the established impact of denervation. Furthermore, smoking accelerates muscle atrophy with aging,³ and has recently been shown to alter neuromuscular junction morphology.¹ Physical frailty is another condition associated with exacerbated denervation and more severe muscle atrophy with aging, and recent studies link this with an elevated blood level of metabolites of tryptophan, such as kynurenine.⁴ Interestingly, motoneurons in culture exposed to kynurenine exhibit axonal degeneration,⁴ which could be a contributing mechanism to the exacerbated atrophy and muscle dysfunction in the frail elderly. In seeking a mechanistic explanation for the neuromuscular junction alterations seen with chronic tobacco smoke and kynurenine, we note that both cigarette smoke and kynurenine stimulate the aryl hydrocarbon receptor (AHR). The AHR is a ligand-activated transcription factor that upregulates detoxifying pathways which include cytochrome P450 enzymes (e.g., Cyp1A1) in response to a wide variety of endogenous and exogenous chemicals, and whereas short-term AHR activation can be adaptive, chronic activation can lead to toxicity via mitochondrial mechanisms. Notably, we find that knock-in of a constitutively active mutant of the AHR in skeletal muscle also causes neuromuscular junction alterations,⁵ suggesting chronic AHR activation may contribute to the exacerbated denervation seen in COPD and physical frailty. Future studies that manipulate AHR activation (e.g., via AHR inhibition or knockout) will be valuable in identifying whether AHR-targeting therapies may be beneficial in attenuating exacerbated denervation in aging muscle.

Key Words: Aging, skeletal muscle, denervation, frailty, aryl hydrocarbon receptor

References

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5.Thome T, Miguez K, Willms AJ, Burke SK, Chandran V, de Souza AR, Fitzgerald LF, Baglole C, Anagnostou ME, Bourbeau J, Jagoe RT, Morais JA, Goddard Y, Taivassalo T, Ryan TE, Hepple RT. Chronic aryl hydrocarbon receptor activity phenocopies smoking-induced skeletal muscle impairment. J Cachexia Sarcopenia Muscle. 2022. Feb;13(1):589-604. doi: 10.1002/jcsm.12826. Epub 2021. Nov 1. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Mosole’s evidence of transforming muscle fibers coexpressing fast and slow myosin heavy chains in slow type muscle-groupings of life-long active seniors

Ugo Carraro ^1,²

Abstract

Ugo Carraro, ugo.carraro@unipd.it I like to use the words of Dario Coletti, taken from a review published in this Issue of EJTM 32(1), 2022.¹ “With aging, type 2 fast fibers preferentially undergo denervation and are reinnervated by slow-twitch motor neurons. They spread forming new neuro-muscular junctions with the denervated fibers: the result is an increased proportion of slow fibers that group together since they are associated in the same motor unit. Grouping and fiber type shifting are indeed major histological features of aging skeletal muscle. Exercise has been proposed as an intervention for age-related sarcopenia due to its numerous beneficial effects on muscle mechanical and biochemical features. In 2013, a precursor study in humans was published in the European Journal of Translation Myology (formerly known as Basic and Applied Myology), highlighting the occurrence of reinnervation in the musculature of aged, exercise-trained individuals as compared to matching control. This paper, entitled Reinnervation of Vastus lateralis is increased significantly in seniors (70-years old) with a lifelong history of high-level exercise, is now being reprinted as an «Ejtm Seminal Paper».^2,3 In this short review we discuss those results in the light of the most recent advances confirming the occurrence of exercise-mediated reinnervation, ultimately preserving muscle structure and function in elderly people who exercise.”¹ The figure 1 is taken from the Mosole’s paper of 2014.⁴

Key Words: Aging, coexpression of fast and slow myosin heavy chains, denervation and reinnervation, fiber-type grouping, Human skeletal muscle, recreational sport activity

References

1.Coletti C, Acosta GF, Keslacy S, Coletti D. Exercise-mediated reinnervation of skeletal muscle in elderly people: An update. Eur J Transl Myol. 2022; 32(1): 10416, doi: 10.4081/ejtm.2022.10416. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Mosole S, Rossini K, Kern H, Löfler S, Simone Fruhmann H, Vogelauer M, Burggraf S, Grim-Stieger M, Cvečka J, Hamar D, Sedliak M, Šarabon N, Pond A, Biral D, Carraro U, Zampieri S. Reinnervation of Vastus lateralis is increased significantly in seniors (70-years old) with a lifelong history of high-level exercise. Eur J Transl Myol Basic Appl Myol. 2013;23(4):205–10. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Mosole S, Carraro U, Kern H, Loefler S, Zampieri S. Use it or Lose It: Tonic Activity of Slow Motoneurons Promotes Their Survival and Preferentially Increases Slow Fiber-Type Groupings in Muscles of Old Lifelong Recreational Sportsmen. Eur J Transl Myol. 2016. Nov 25;26(4):5972. doi: 10.4081/ejtm.2016.5972. eCollection 2016. Sep 15. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Mosole S, Carraro U, Kern H, Loefler S, Fruhmann H, Vogelauer M, Burggraf S, Mayr W, Krenn M, Paternostro-Sluga T, Hamar D, Cvecka J, Sedliak M, Tirpakova V, Sarabon N, Musarò A, Sandri M, Protasi F, Nori A, Pond A, Zampieri S. Long-term high-level exercise promotes muscle reinnervation with age. J Neuropathol Exp Neurol. 2014. Apr;73(4):284-94. doi: 10.1097/NEN.0000000000000032. [DOI] [PubMed] [Google Scholar]
5.Carraro U, Kern H, Gava P, Hofer C, Loefler S, Gargiulo P, Edmunds K, Árnadóttir ÍD, Zampieri S, Ravara B, Gava F, Nori A, Gobbo V, Masiero S, Marcante A, Baba A, Piccione F, Schils S, Pond A, Mosole S. Recovery from muscle weakness by exercise and FES: lessons from Masters, active or sedentary seniors and SCI patients. Aging Clin Exp Res. 2017. Aug;29(4):579-590. doi: 10.1007/s40520-016-0619-1. Epub 2016. Sep 3. [DOI] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION VII: Slow and Fast Muscle Fiber Functional Dynamics with Lifelong Exercise

Scott Trappe ^1,^✉

Abstract

The exercise boom of the late 1960’s and early 1970’s resulted in a generation of individuals that performed structured endurance exercise ranging from recreational to competitive endeavors during their entire adult life. These unique individuals, now >70y old, have cardiovascular and skeletal muscle health profiles that are comparable to individuals that are 30-40 years younger resulting in a substantially lower risk of mortality and larger physiological reserve above the frailty threshold.¹ One aspect of skeletal muscle health that is particularly challenging as we age is the reduction in size and number of the fast muscle fibers,^2,3 which are critical for muscle power.^4,5 However, the degree to which lifelong exercise may provide benefits for fast muscle fiber health are relatively unexplored. This presentation will profile the physiological benefits of lifelong exercise from whole body to single fiber level in men and women highlighting adaptations at the myocellular level.^1,6

Key Words: Aging performance ords: Masters athletes, aging, myocellular, contractile function

References

1.Gries KJ, Raue U, Perkins RK, Lavin KM, Overstreet BS, D'Acquisto LJ, Graham B, Finch WH, Kaminsky LA, Trappe TA, Trappe S. Cardiovascular and skeletal muscle health with lifelong exercise. J Appl Physiol (1985). 2018. Nov 1;125(5):1636-1645. doi: 10.1152/japplphysiol.00174.2018. Epub 2018. Aug 30. [DOI] [PMC free article] [PubMed] [Google Scholar]
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6.Grosicki GJ, Gries KJ, Minchev K, Raue U, Chambers TL, Begue G, Finch H, Graham B, Trappe TA, Trappe S. Single muscle fibre contractile characteristics with lifelong endurance exercise. J Physiol. 2021. Jul;599(14):3549-3565. doi: 10.1113/JP281666. Epub 2021. Jun 15 [DOI] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Motor unit adaptations in Masters Athletes

Matthew Piasecki ^1,^✉

Abstract

Several human studies have demonstrated an age-related reduction in the number of motor units (MU), yet the ability of a MU to undergo remodelling and to expand to rescue denervated muscle fibres is also a defining feature of their plasticity. Remodelling of MUs involves two coordinated processes; i) axonal sprouting and new branching growth from adjacent surviving neurons, and ii) the formation of key structures around the neuromuscular junction (NMJ) to resume muscle-nerve communication. Exercise in older age has demonstrated benefits in multiple physiological systems including skeletal muscle and Masters Athletes may be viewed as an effective model to study the effects of ageing dissociated from the effects of reduced activity. Several lines of evidence also suggest exercise into older age may also extend to peripheral MU remodelling. There is, however, a lack of research in humans due to methodological limitations which are easily surmountable in animal models. As such, the ability to accurately quantify remodelling and relate it to clinically relevant neuromuscular function has proven difficult. This talk will address the neuromuscular function and MU structure and function of elite Masters Athletes competing in endurance and power events, in comparison to their less-active counterparts, and crucially, to frail older adults.

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Insights to mechanisms of healthy muscle aging in octogenarian Track & Field Athletes

Russell T Hepple ^1,^✉, Tanja Taivassalo ¹

Abstract

Although advanced age is typically associated with poor physical function that can adversely affect mobility and quality of life, some individuals retain high function well into their advanced years and can provide clues to mechanisms of healthy muscle aging. With this in mind, we have been conducting extensive studies of competitive octogenarian athletes. Herein, we present data on 15 world class track and field masters athletes (MA; 7 female), 8 of whom were the current world record holders for their age and discipline at the time of study, in comparison to a group of 14 age- and sex-matched healthy non-athlete individuals (NA; 6 female). As expected, the MA group had superior physical function on a variety of clinical function tests (e.g., 4 m fast walking speed, cardiopulmonary exercise testing to VO2peak, isokinetic knee extensor strength), and this was associated with higher thigh cross-sectional area, higher motor unit number estimates, smaller accumulation of very small muscle fibers, less denervation, fewer oxidatively compromised muscle fibers, and higher mtDNA copy number. Muscle proteomics analysis revealed that MA muscle was characterized by over-representation of proteins involved in mitochondrial biology (nuclear and mitochondrial DNA encoded proteins, mitochondrial Sirtuins, mitochondrial dynamics, mitochondrial cristae remodeling, mitochondrial proteostasis), and an under-representation of proteins involved with nuclear pore complexes and the spliceosome. Interestingly, when comparing our MA to previous studies examining exercise-responsive proteins,¹ and age-responsive proteins,² although there was substantial overlap between proteins over-represented in MA and those elevated in subjects with higher physical activity, there were 301 proteins that were uniquely over-represented in MA that had not previously been identified as responsive to exercise. As such, our results suggest that a high level of physical activity is likely not the only factor explaining the superior function in MA and provides exciting insights to novel mechanisms for promoting healthy muscle aging.

Key Words: Aging, skeletal muscle, mitochondria, exercise, masters athlete

References

1.Ubaida-Mohien C., et al. , Physical Activity Associated Proteomics of Skeletal Muscle: Being Physically Active in Daily Life May Protect Skeletal Muscle From Aging. Front Physiol, 2019. 10: p. 312. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Ubaida-Mohien C.et al., Discovery proteomics in aging human skeletal muscle finds change in spliceosome, immunity, proteostasis and mitochondria. Elife, 2019. 8. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The Venice Marathon 2007-2019 as a model for analyses of Master Athletes

Giovanna Albertin ^1,^2,^✉, Laura Astolfi ¹, Marco Falda ¹, Diego Guidolin ¹, Ugo Carraro ^3,⁴

Abstract

The number of marathon events increased during the last 30 years, and in particular increased those of the Master Athletes.^1,2 There are evidences that long-distance running could provide considerable health benefits for older runners, specifically risk reduction of cardiovascular diseases, cancer, diabetes, depression, and falls.^3,4 In this study we have analyzed data from several editions of the Venice marathon, a famous Italian race that attracts people from every corner of the world. The data analysis was done on age category distribution (left panel), nation (right panel) and performance in 13 editions (2007-2019) evaluating the performance of all the athletes vs. the top 10% fastest athletes (TOP 10%). The analysis has shown a steady increase in female participation, in particular for 40-50 years old, and a substantial reduction of performance in the younger category of 30-35 years old, both male and female, from 2003 to 2019. The intra-category differences of performances have shown that the trends in male and female were similar and a specific steeper decline in performance was identified at age 55 for the women and at 50 for the men with specific differences between the set of all athletes and the TOP10% one. The model of the Venice marathon can therefore be useful to analyze the gender participation and in particular the human model of successful aging .^5-7

Key Words: Aging performance decay, Masters, gender differences

References

1.Cheuvront SN, Carter R, Deruisseau KC, Moffatt RJ. Running performance differences between men and women:an update. Sports Med. 2005;35(12):1017-24. doi: 10.2165/00007256-200535120-00002. [DOI] [PubMed] [Google Scholar]
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4.Gries KJ, Raue U, Perkins RK, Lavin KM, Overstreet BS, D'Acquisto LJ, Graham B, Finch WH, Kaminsky LA, Trappe TA, Trappe S. Cardiovascular and skeletal muscle health with lifelong exercise. J Appl Physiol (1985). 2018. Nov 1;125(5):1636-1645. doi: 10.1152/japplphysiol.00174.2018. Epub 2018. Aug 30. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Gava P, Kern H, Carraro U. Age-associated power decline from running, jumping, and throwing male Masters World Records. Exp Aging Res. 2015;41:115-35. doi: 10.1080/0361073X.2015.1001648.. [DOI] [PubMed] [Google Scholar]
6.Carraro U, Kern H, Gava P, Hofer C, Loefler S, Gargiulo P, Edmunds K, Árnadóttir ÍD, Zampieri S, Ravara B, Gava F, Nori A, Gobbo V, Masiero S, Marcante A, Baba A, Piccione F, Schils S, Pond A, Mosole S. Recovery from muscle weakness by exercise and FES: lessons from Masters, active or sedentary seniors and SCI patients. Aging Clin Exp Res. 2017. Aug;29(4):579-590. doi: 10.1007/s40520-016-0619-1. Epub 2016. Sep 3. [DOI] [PubMed] [Google Scholar]
7.Gava P, Ravara B. Master World Records show minor gender differences of performance decline with aging. Eur J Transl Myol. 2019. Aug 2;29(3):8327. doi: 10.4081/ejtm.2019.8327. eCollection 2019. Aug 2 [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Masters studies in Padua to honor the legacy of Paolo Gava

Paolo Gava ¹, Barbara Ravara ¹, Ugo Carraro ^1,^✉

Abstract

Aging behaviors differ in females and males. Females are weaker, but survive longer. Paolo Gava described the gender decay of muscle performance by comparing the world records of Masters, who compete in age groups of 5 years from 35 to 100 or older. After normalizing - that is, taking the all-time world record of young sports athletes to 100% - the women's and men's world records are lists of 16 data showing declining trends in muscle performance as aging, indicating only minimal gender differences in the process. All trend lines tend to zero at around 110 years. The decline of short running disciplines is very similar. It is smooth from 30 to 50 years, it is almost linear from 50 to 70 years, then the decay is progressively steeper. Since gender hormones appear to have little influence on the decay of aging performance, other key bioregulators, such as those of cellular energy metabolism, appear to be more relevant.

Key Words: Aging performance decay, Masters world records, gender differences

References

Gava P, Kern H, Carraro U. Age-associated power decline from running, jumping, and throwing male Masters World Records. Exp Aging Res. 2015;41:115-35. doi: 10.1080/0361073X.2015. 1001648. [DOI] [PubMed] [Google Scholar]
World Master Atheltics - http://www.worldmasters-athletics.org/ [Google Scholar]
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Carraro U, Kern H, Gava P, Hofer C, Loefler S, Gargiulo P, Edmunds K, Árnadóttir ÍD, Zampieri S, Ravara B, Gava F, Nori A, Gobbo V, Masiero S, Marcante A, Baba A, Piccione F, Schils S, Pond A, Mosole S. Recovery from muscle weakness by exercise and FES: lessons from Masters, active or sedentary seniors and SCI patients. Aging Clin Exp Res. 2017. Aug;29(4):579-590. doi: 10.1007/s40520-016-0619-1. Epub 2016. Sep 3. [DOI] [PubMed] [Google Scholar]
Gava P, Ravara B. Master World Records show minor gender differences of performance decline with aging. Eur J Transl Myol. 2019. Aug 2;29(3):8327. doi: 10.4081/ejtm.2019.8327. eCollection 2019. Aug 2. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Session VIII: Fiber Strains and Strain Tensor Mapping of Medial Gastrocnemius at Sub-Maximal Isometric Contraction at Different Ankle Angles

Usha Sinha ^1,^✉

Abstract

Prior studies of isometric contraction in calf muscle using velocity encoded phase contrast (VE-PC) imaging have revealed several interesting features in the medial gastrocnemius including strain heterogeneity along and across fibers, gear ratios decreasing with unloading, and constant in-plane areas during isometric contraction suggesting little or no deformation in the out-plane direction ^1,2. In addition to analyzing fiber strains, spatially localized strain and strain rate (SR) tensors can be computed from velocity encoded phase contrast images that have the potential to identify changes in tissue deformation along and perpendicular to the muscle fiber. Strain tensor analysis has revealed anisotropy of deformation in the cross-section of the muscle fiber, deviation of the principal strain direction from the muscle fiber orientation, heterogeneity of strains along the length of the muscle and across the muscle fiber and in study of age-related effects, maximum shear strain emerging as the most significant predictor of force loss with age ³. Earlier studies identified fiber direction from fascicle directions on water suppressed MR images (fascicles appear bright due to fat infiltration at the fascicles).

Fig. 1. — Shows the principal negative and positive strain values during the contraction peak for (L-R) N, D and P ankle positions.

Table 1.

Fiber strains for the three ankle positions: N(eutral), D(orsiflexed), P(lantarflexed) and two %MVC.

graphic file with name ejtm-32-1-10440-t001.jpg

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However, identification of fascicles in young subjects is difficult since the fascicles are relatively thin and do not have sufficient fat infiltration. We used the average of the lead DTI eigenvector in each region to extract the direction of fibers. Fiber strains, length and angle changes were calculated for three-foot positions and at two % MVC at each position. This study calculates regional fiber strains, and strain and strain rate (SR) tensor in the medial gastrocnemius muscle from 2D velocity encoded phase contrast (VE-PC) images acquired under isometric contraction at three-foot positions (representing different muscle fiber pennation angles/length) at two % MVC’s at each position. The plantarflexed foot position had the highest normalized fiber strain while the dorsiflexed position had the lowest normalized fiber strains (to force and to torque) (Table 1). In the strain and strain rate tensor analysis, plantarflexed foot position had the highest normalized strain and strain rate (to force) and strains decreased with %MVC’s compared to the neutral and dorsiflexed foot positions (Figure 1). Strain in the fiber cross-section showed radial expansion in the in-plane and radial contraction in the out-plane direction; radial deformation was lowest in the plantarflexed position.

Key Words: Dynamic muscle MRI, fiber architecture and function, fiber strain, strain tensor mapping

References

1.Shin DD, Hodgson JA, Edgerton VR, Sinha S. In vivo intramuscular fascicle-aponeuroses dynamics of the human medial gastrocnemius during plantarflexion and dorsiflexion of the foot. J Appl Physiol (1985) 2009;107:1276-84. [DOI] [PMC free article] [PubMed] [Google Scholar]
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3.Sinha U, Malis V, Csapo R, Moghadasi A, Kinugasa R, Sinha S. Age-related differences in strain rate tensor of the medial gastrocnemius muscle during passive plantarflexion and active isometric contraction using velocity encoded MR imaging: potential index of lateral force transmission. Magn Reson Med. 2015;73:1852-63. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Calf Muscle 3D Strain Imaging and Initial Results on Correlation with Histology

Shantanu Sinha ^1,^✉

Abstract

Prior studies of isometric contraction in calf muscle using velocity encoded phase contrast (VE-PC) imaging have used either single slice imaging to extract 2D strain and strain rate tensors^1,2 or sequentially acquired single slice images which were concatenated to form a volume to extract 3D strain and strain rate tensors³. The advantage of 2D VE-PC is that it is fast but requires one to acquire the images in the plane of the fibers to leverage the fact that muscles exhibit planar deformation; a 3D acquisition circumvents the need for this positioning. A volume acquisition VE-PC sequence is usually too long for subjects to maintain consistent contractions through the scan duration. In this study, we present (i) sequentially acquired anatomical slices to extract 3D strain tensor and projections on the myofibrillar aggregate in a cohort of young and old subjects along with correlation of the strain indices to histology and (ii) initial results using a highly accelerated 4D Compressed Sensing flow sequence⁴ to image the much smaller motion seen in muscle tissue. Significant age -related differences were identified in principal 3D strain (E) and strain rate (SR) indices (from sequential 2D slices) including in shear strain in the medial gastrocnemius (MG) and in the soleus (SOL). The tensor projections in the muscle fiber direction and cross-section (Figure 1) did not show any age-related differences but several principal and fiber-aligned strain indices showed correlation with collagen 3 from histology. The second part of the study establishes the feasibility of 3D volume acquisition with 3 directional velocity-encoding for calf muscle dynamic imaging to monitor sub-maximal isometric contractions using a 4D Compressed Sensing Flow imaging sequence originally developed for blood flow imaging (Figure 2).

Fig. 2. — Sagittal images of the calf muscle through the dynamic cycle (50 temporal phases) showing colormaps of the displacement in the x-, y- and z-axis derived from the 4D CS Flow imaging sequence.

Key Words: Aging muscle, dynamic MRI, compressed sensing, 3D strain imaging, fiber aligned strains

References

1.Sinha U, Malis V, Csapo R, Moghadasi A, Kinugasa R, Sinha S. Age-related differences in strain rate tensor of the medial gastrocnemius muscle during passive plantarflexion and active isometric contraction using velocity encoded MR imaging: potential index of lateral force transmission. Magn Reson Med. 2015;73:1852-63. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Malis V, Sinha U, Csapo R, Narici M, Sinha S. Relationship of changes in strain rate indices estimated from velocity-encoded MR imaging to loss of muscle force following disuse atrophy. Magn Reson Med. 2018. Feb;79(2):912-922. doi: 10.1002/mrm.26759. Epub 2017. May 30. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Our Discovery of new intracellular junctions: The calcium entry units (CEUs)

Simona Boncompagni ^1,^✉

Abstract

In 2017, Boncompagni, Michelucci et al. demonstrated that during exercise the sarcotubular system of extensor digitorum longus (EDL) fibers undergoes a profound remodeling that leads to the assembly of new junctions between T-tubule extensions at the I band and sarcoplasmic reticulum (SR) stacks. As these junctions contain colocalized STIM1 and Orai1 and enhance store-operated Ca2+ entry (SOCE), they have been named Ca2+ entry units (CEUs). In addition, it has been more recently shown that (1) CEUs disassemble following recovery, with T-tubules retraction from the I band faster than SR stacks disassembly, and (2) lack of calsequestrin-1 (CASQ1) induces a constitutive assembly of CEUs, resulting in enhanced SOCE that counteracts the SR Ca2+ depletion. We have now analyzed (1) CEUs during postnatal maturation (at 2 wk of age) and (2) whether CEUs form in slow-twitch fibers (soleus). (a) Compared with adult (4 mo) EDL fibers of resting WT mice, at 2 wk of age we found a greater longitudinal disposition of T-tubules associated to SR membranes forming junctions virtually identical to CEUs in adult EDLs of exercised WT mice, which promote increased STIM1/Orai1-mediated SOCE. (b) We also compared structure and function of soleus (which also express the cardiac isoform CASQ2) from WT mice and from mice lacking either CASQ1 (CASQ1-null) or CASQ1/2 (dCASQ-null). In soleus from both genotypes, CEUs are constitutively assembled although they appear structurally smaller than those described previously in exercised WT or CASQ1-null EDLs. A detailed EM quantitative analysis revealed that CEUs were more abundant in dCASQ-null than CASQ1-null mice. The amount of CEUs strictly correlated with the ability of soleus fibers to recover extracellular Ca2+ via SOCE to support contractility during high-frequency stimulation. Molecular analysis of Western blots, showing that Orai1 expression was enhanced following ablation of CASQ, support these data.

Key Words: Sarcotubular system, CASQ1-null, Ca²⁺ entry units, CEUs

References

Boncompagni S, Michelucci A, Pietrangelo L, Dirksen RT, Protasi F. Exercise-dependent formation of new junctions that promote STIM1-Orai1 assembly in skeletal muscle. Sci Rep. 2017. Oct 27;7(1):14286. doi: 10.1038/s41598-017-14134-0. Erratum in: Sci Rep. 2018. Nov 27;8(1): 17463. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Michelucci A, Boncompagni S, Pietrangelo L, Takano T, Protasi F, Dirksen RT. Pre-assembled Ca2+ entry units and constitutively active Ca2+ entry in skeletal muscle of calsequestrin-1 knockout mice. J Gen Physiol. 2020. Oct 5;152(10):e202012617. doi: 10.1085/jgp.202012617. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Looking at the proteome landscape in single muscle fibers

Marcus Krüger ^1,^✉

Abstract

Skeletal muscles consist of heterogeneous collections of fibres with different metabolic profiles. Due to the different neuronal innervation and fibre composition, each muscle fulfils specific functions and reacts differently to stimuli and pertubations. So far, myosin heavy chain molecules (MyHC) are currently the best marker proteins for characterization of individual fiber types, and several proteome profiling studies have helped to dissect the molecular signature of whole muscles and individual fibers. To precisely quantify fiber type-specific protein patterns, we developed a mass spectrometric workflow to measure skeletal muscle fiber type-specific proteomes. To bypass the limited protein levels in individual fibres, we have designed a proteomics high-throughput fibre typing method (ProFiT) that enables MyHC profiling in individual fibres. Aliquots of protein extracts from separated muscle fibers were subjected to capillary LC-MS gradients to profile MyHC isoforms in a 96-well format. Muscle fibers with the same MyHC protein expression were pooled and subjected to proteomic, pulsed-SILAC, and phosphoproteomic analysis. Our fiber type-specific quantitative proteome analysis confirmed the distribution of fiber types in the soleus muscle, substantiates metabolic adaptions in oxidative and glycolytic fibers, and highlighted significant differences between the proteomes of type IIb fibers from different muscle groups, including a differential expression of desmin and actinin-3. A detailed map of Lys-6 incorporation rates in muscle fibres revealed protein turnover dependent on fibre type, and labelling of mitochondrial respiratory chain complexes revealed a wide range of Lys-6 incorporation rates dependent on the localisation of subunits within different respiratory complexes. Overall, the ProFiT approach provides a versatile tool to rapidly characterize muscle fibers and obtain fiber-specific proteomes for different muscle groups

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

PCM1 labelling reveals myonuclear and nuclear dynamics in skeletal muscle across species

Mark Viggars ^1,², Daniel J Owens ^1,³, Claire HStewart ¹, Catherine Coirault ³, Abigail L Mackey ^4,⁵, Jonathan C Jarvis ^1,^✉

Abstract

Myonuclei transcriptionally regulate muscle fibres and are key regulators of muscle plasticity. Their cellular location and quantity are important when characterising phenotypes of myopathies, the effect of treatments and to understand the roles of satellite cells in muscle adaptation and muscle ‘memory’.¹ We aimed to determine whether PCM1 is, as claimed,² a reliable specific marker of myonuclei in-vitro and in-vivo. Single isolated myofibres and transverse sections from mouse, rat and humans were studied from several models including Wild-type and Lamin A/C mutant mice after functional overload,³ resistance training in rats,⁴ and damage and recovery in humans following forced eccentric contractions.⁵ Fibres were immuno-labelled for PCM1, Pax7 and DNA. C2C12 myoblasts and human primary muscle cells were also studied in vitro to investigate changes in PCM1 localisation during differentiation and fusion into myotubes. PCM1 labelled the nuclear envelope of myonuclei in mature myofibres and in newly formed myotubes but also labelled centrosomes in proliferating myogenic precursors which may or may not fuse to join the myofibre syncytium. It also labelled non-myogenic nuclei near the sarcolemma especially in regenerating areas of the LMNA+/ΔK32 mouse and damaged human muscle. Such nuclei were found in satellite cells, macrophages, and other interstitial cells. PCM1 is therefore not completely specific to myonuclei, and especially in damaged or regenerating muscle the impact of false-positive identification of interstitial cells on myonuclei counts would tend to cause overestimation of myonuclei per fiber compared with classical counting based on myonuclear positioning relative to the sarcolemma. PCM1 may further prove useful as a marker of satellite cell dynamics due to the distinct change in localisation during differentiation, revealing satellite cells in their quiescent (PCM1-), proliferating (PCM1+ centrosome), and pre-fusion states (PCM1+ nuclear envelope).

Key Words: PCM1, hypertrophy, regeneration, myonuclei

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The HERG K⁺ Channel Increases Intracellular Calcium Concentration in Myotubes by Modulation of IP3 Signaling

Amber L Pond ^1,^✉, Emily LaVigne ², Omar Khader ¹, Jennifer Koran ³, Gregory H Hockerman ²

Abstract

The HERG K⁺ channel is upregulated in skeletal muscle atrophying in response to disuse¹, cancer¹, and denervation². Using C2C12 myotubes transduced with HERG-encoded virus, we have shown that over-expression of the HERG channel increases basal intracellular calcium concentration and calpain activity³. However, the mechanism by which HERG modulates intracellular calcium levels is not known. To explore this further, we increased intracellular calcium levels by depolarization with 100 mM KCl and used Fura2 dyes and immunoblot to show that HERG does not alter myotube calcium levels by affecting L-type calcium channels (i.e., Cav1.1). Instead, using the SERCA blocking agent thapsigargin with our Fura2 assay, we discovered that the HERG-mediated increase in calcium occurs through modulation of intracellular calcium stores.⁴ Therefore, we hypothesized that HERG may be modulating the phospholipase C (PLC)-PIP2-IP3 pathway. To investigate this, we produced an increase in intracellular calcium using bethanechol to activate muscarinic receptors, which activate the PLC-PIP2-IP3 pathway. Using single cell calcium imaging, we reveal that, relative to myotubes transduced with an appropriate control virus, bethanechol treatment of HERG-expressing myotubes produces a significant 2-fold increase (p<0.05) in intracellular calcium levels. The data suggest that HERG may indeed modulate the PLC-PIP2-IP3 pathway. Interestingly, however, this HERG-mediated increase in bethanechol-induced calcium levels is not a result of increased PLC activity as demonstrated by IP1-Gq HRTF kit assays, which show no increase in the concentration of IP1 (an IP3 degradation product) in HERG-expressing myotubes relative to controls. However, single cell calcium imaging reveals that the HERG-mediated increase in calcium is inhibited by treatment with the IP3 receptor antagonist Xestospongin-C. The data suggest that HERG enhances calcium release through IP3 receptor activation without affecting PLC activity.

Key Words: HERG, IP3 signaling, intracellular calcium, myotubes

References

1.Wang X, Hockerman GH, Green HW, 3rd, Babbs CF, Mohammad SI, Gerrard D, Latour MA, London B, Hannon KM, Pond AL. Merg1a K+ channel induces skeletal muscle atrophy by activating the ubiquitin proteasome pathway. FASEB J. 2006. Jul;20(9):1531-3. doi: 10.1096/fj.05-5350fje. Epub 2006. May 24 [DOI] [PubMed] [Google Scholar]
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3.Whitmore C, Pratt EPS, Anderson L, Bradley K, Latour SM, Hashmi MN, Urazaev AK, Weilbaecher R, Davie JK, Wang WH, Hockerman GH, Pond AL. The ERG1a potassium channel increases basal intracellular calcium concentration and calpain activity in skeletal muscle cells. Skelet Muscle. 2020. Jan 16;10(1):1. doi: 10.1186/s13395-019-0220-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Cytosolic calcium as intracellular signal: local and average concentrations and their variations in relation to release from SR

Lorenzo Marcucci ^1,^✉, Marta Canato ¹, Leonardo Nogara ¹, Anna Raffaello ¹, Simona Boncompagni ², Feliciano Protasi ³, Carlo Reggiani ¹

Abstract

Calcium signaling is the most important pathway which regulates muscle contraction and relaxation. The knowledge of its spatial and temporal distribution among the major compartments in the muscle cells, namely cytosol, sarcoplasmic reticulum, but also mitochondria and T tubular extracellular space, is crucial to drive our understanding in physiological and pathological behavior in muscle mechanics. Despite a general agreement in the fluxes of calcium in skeletal muscle at the qualitative level, there are several open questions in the literature regarding a quantitative assessment of its concentration both in average and temporally/spatially resolved. We approach this problem through a three-dimensional diffusional model of calcium fluxes inside a half sarcomere, based on experimental data on FDB in WT mice.¹ We first estimate the citosolic [Ca2+] comparing the signal of our Fura-2 indicator during a twitch and during a 60Hz stimulation, accounting for the kinetic term through the Schneider equation,² with the simulated ratio of occupation of the two Ca-binding sites of the Troponin in the model, and the experimental tension developed by intact muscle fibers. Based on this data, we used the model to assess the relative amount of calcium which enter the mitochondria, which enter from T tubules through SOCE and which exit from the cell through PMCA and NCX. We extended our model to study the calcium fluxes in the parvalbumin knock-out mice, considering two different sets of experimental data recently published.^3,4 In particular we estimated the possible fate of the calcium released from the sarcoplasmic reticulum in the absence of the major cytosolic buffer and showed the contribution of the export to extracellular space and the uptake into mitochondrial matrix to stabilize the cytosolic concentration.

Key Words: Cytosolic calcium, intracellular signal, local and average concentrations, variations in relation to release from SR

References

1.Marcucci L, Canato M, Protasi F, Stienen GJM, Reggiani C. A 3D diffusional-compartmental model of the calcium dynamics in cytosol, sarcoplasmic reticulum and mitochondria of murine skeletal muscle fibers. PLoS One. 2018. Jul 26;13(7):e0201050. doi: 10.1371/journal.pone.020 1050. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Klein MG, Simon BJ, Szucs G, Schneider MF. Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators. Biophys J. 1988. Jun;53(6):971-88. doi: 10.1016/S0006-3495(88)83178-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Butera G, Vecellio Reane D, Canato M, Pietrangelo L, Boncompagni S, Protasi F, Rizzuto R, Reggiani C, Raffaello A. Parvalbumin affects skeletal muscle trophism through modulation of mitochondrial calcium uptake. Cell Rep. 2021. May 4;35(5):109087. doi: 10.1016/j.celrep.2021.109087. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION IX: Gait analysis interplay with non-motor mental symptoms in Parkinson’s disease

Michela Russo ¹, Marianna Amboni ^2,³, Antonio Volzone ², Gianluca Ricciardelli ⁴, Giuseppe Cesarelli ^5,⁶, Alfonso Maria Ponsiglione ¹, Paolo Barone ², Maria Romano ¹, Carlo Ricciardi ^1,^6,^✉

Abstract

Parkinson’s Disease (PD) is a neurodegenerative disease which involves both motor and non-motor symptoms.¹ Non-motor mental symptoms are very common among patients with PD since the earliest stage. They include mood and affective symptoms like apathy, anhedonia, anxiet and depression, cognitive dysfunction, and psychotic symptoms such as hallucinosis and delusions. In this context, gait analysis allows to detect quantitative gait variables able to distinguish patients affected by non-motor mental symptoms from patients without these symptoms. In this research, 68 PD patients were grouped in subjects with and without non-motor mental symptoms based on MDS-UPDRS I (item 1-6). All patients performed gait analysis according to the Davis protocol,² both in single and dual-task. Spatial and temporal parameters describing gait were extracted, as already done in previous researches.³ The results of statistical analysis showed significant different gait patterns in patients with non-motor mental symptoms as compared with those without. Single-task variables showed that 9 out of 16 spatiotemporal features were statistically significant for the univariate statistical analysis (p< 0.05). In addition, a statistically significant difference was found in stance phase (p=0.032), swing phase (p=0.042) and cycle length (p=0.03) of the dual task. In conclusion, PD patients with non-motor mental symptoms versus PD patients without this cluster of symptoms showed a worse gait pattern in both single and dual task, thus supporting the notion of a more widespread disease involving neuro-transmitters systems other than dopamine in patients with neuropsychiatric symptoms.

Key Words: Parkinson’s Disease, non-motor mental symptoms, gait analysis

References

1.Pearce JM. Aetiology and natural history of Parkinson's disease. Br Med J. 1978. Dec 16;2(6153):1664-6. doi: 10.1136/bmj.2.6153.1664-a. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Davis RB, Ounpuu S, Tyburski D, Gage J.R. A gait analysis data collection and reduction technique –Hum Mov Sci. 1991; 10 (5, Oct) 575-587. [Google Scholar]
3.Amboni M, Ricciardi C, Picillo M, De Santis C, Ricciardelli G, Abate F, Tepedino MF, D'Addio G, Cesarelli G, Volpe G, Calabrese MC, Cesarelli M, Barone P. Gait analysis may distinguish progressive supranuclear palsy and Parkinson disease since the earliest stages. Sci Rep. 2021. Apr 29;11(1):9297. doi: 10.1038/s41598-021-88877-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

A Novel Knee Bone and Cartilage Osteoarthritis Index Extracted from a Patient-Specific Image Feature Analysis

Marco Recenti ^1,^✉, Romain Aubonnet ¹, Deborah Jacobs ¹, Lorena Guerrini ^1,², Federica K Ciliberti ^1,³, Jogelina Ramos ¹, Magnus K Gislason ¹, Olafur Sigurjonsson ¹, Mariella Tsirilaki ⁴, Halldor Jr Jonsson ⁴, Paolo Gargiulo ^1,⁴

Abstract

Knee Osteoarthritis is one of the most common form of arthritis and affects a significant percentage of the world population.¹ Multiple indexes created to classify the cartilage status are present in the medical literature and are mainly based on physicians’ image analysis and subjected to different and personal interpretations.^2,3 In the frame of the European Project RESTORE a database of MRI and CT-scan images of degenerative, traumatic, and healthy patients is developed. Approximately 100 objective features are extracted from both 2D, and 3D bone and cartilage images. Machine Learning is then performed to select the most important features which will define a novel patient-specific index able to classify the knee osteoarthritis status with high accuracy.

Key Words: Knee Osteoarthritis, machine learning, medical imaging, knee cartilage

References

1.Zhang Y, Jordan JM. Epidemiology of osteoarthritis. Clin Geriatr Med. 2010. Aug;26(3):355-69. doi: 10.1016/j.cger.2010.03.001. Erratum in: Clin Geriatr Med. 2013. May;29(2):ix. [DOI] [PMC free article] [PubMed] [Google Scholar]
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3.Ahlbäck S. Osteoarthrosis of the knee. A radiographic investigation. Acta Radiol Diagn (Stockh). 1968:Suppl 277:7-72. [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Postural Control Assessment through Visual Induced Motion Sickness and a Moving Force Platform

Andrea Colacino ^1,², Romain Aubonnet ¹, Deborah Jacobs ¹, Hannes Petersen ^3,⁴, Paolo Gargiulo ^1,^5,^✉, Marco Recenti ¹

Abstract

In this research a novel and unique Motion Sickness moving platform is used to study different postural control reactions.^1,2 The platform simulates a rough sea environment through virtual reality which visualize waves that coordinate the platform movements. Together with EEG, EMG, and hearth rate sensors a force platform is installed under the subject feet during the experiment. This research is focused on the extraction of multiple features from the force platform to study the postural movements of the different subjects before, during and after the sea simulation. These features are then selected using a machine learning approach focusing on the ones which allows to distinguish between motion sick and not sick subjects with higher accuracy. Then the different postural behaviors during the simulation are confronted between different classes of participants like elderly and young, sporty, and not and others. Finally, the selected features are correlated with the EMG data to study the relations between postural and muscle reaction to the induced motion sickness environment.

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Key Words: Motion Sickness, postural control, machine learning, force platform, virtual reality

References

1.Recenti M, Ricciardi C, Aubonnet R, Picone I, Jacob D, Svansson HÁR, Agnarsdóttir S, Karlsson GH, Baeringsdóttir V, Petersen H, Gargiulo P. Toward Predicting Motion Sickness Using Virtual Reality and a Moving Platform Assessing Brain, Muscles, and Heart Signals. Front Bioeng Biotechnol. 2021. Apr 1;9:635661. doi: 10.3389/fbioe.2021.635661. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Barollo F, Frioriksdottir R, Edmunds KJ, Karlsson GH, Svansson HA, Hassan M, Fratini A, Petersen H, Gargiulo P. Postural Control Adaptation and Habituation During Vibratory Proprioceptive Stimulation: An HD-EEG Investigation of Cortical Recruitment and Kinematics. IEEE Trans Neural Syst Rehabil Eng. 2020. Jun;28(6):1381-1388. doi: 10.1109/TNSRE.2020.2988585. Epub 2020. Apr 17. [DOI] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Multi-scale Bone Remodeling Prediction in Patients Undergoing Total Hip Arthroplasty

Maria Laura Gatto ^1,^*, Donatella Malta ^3,^2,^*, Paolo Mengucci ¹, Halldor Jonsson Jr ^4,⁵, Francesco Tortorella ³, Carlo Mengucci, Paolo Gargiulo ^2,^4,^*

Abstract

Total hip arthroplasty (THA) is the most widely used surgery in case of severe damage of hip joints. However, the implant produces significant variations of stress distribution in the operated femur, determining the host bone remodeling, which, in turn, influences the bone quality of THA patient. The aim of the study is to create a prediction model, which allows determining significant parameters for THA femur quality during the remodeling, starting from patient clinical data, diagnostic x-ray computed tomography (XCT) and 3D high resolution imaging. Patient clinical data as age, gender, body mass index, type of operation (unilateral or bilateral), prosthesis (cemented or uncemented) and diagnosed pathologies connected to bone tissue, are correlated to multi-scale parameters of femur. Information at macro level is obtained by the analysis of diagnostic X-ray computed tomography (d-XCT) scans of patient femurs. Data are segmented to collect the densitometry of regions of orthopedic interest (gruen zones). The densitometry of rectus femoris muscle is also considered, due to the fact that remains roughly recognizable also when it is degenerated. Micro and nano femur morphometric parameters are obtained by high resolution 3D imaging on femur biopsies, achieving information on bone microarchitecture and ultrastructure. After a learning, based on above-mentioned THA patients’ data at 24 hours, 1 years and 6 years after the implant, the prediction model can predict the bone quality and the better-regenerated zones. The prediction model of THA femur quality works also as support of further clinical decisions on post-surgery treatments and rehabilitation, by providing a projection on patient mobility.

Key Words: THA, Artificial Intelligence, bone remodeling

References

Ciliberti FK, Guerrini L, Gunnarsson AE, Recenti M, Jacob D, Cangiano V, Tesfahunegn YA, Islind AS, Tortorella F, Tsirilaki M, Jónsson H Jr, Gargiulo P, Aubonnet R. CT- and MRI-Based 3D Reconstruction of Knee Joint to Assess Cartilage and Bone. Diagnostics (Basel). 2022. Jan 22;12(2):279. doi: 10.3390/diagnostics12020279. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Neuromuscular control in the neck muscles in patients suffering from whiplash associated disorders and traumatic brain injury

Magnús Kjartan Gíslason ^1,^✉, Sandra Ýr Dagnýjardóttir ¹, Kristjana Sigurðardóttir ¹, Paolo Gargiulo ¹, Jóna Marín Ólafsdóttir ¹

Abstract

Neck pain is an increasing healthcare problem throughout the developed world and is the fourth leading cause of disability globally. It can be difficult to diagnose and treat effectively because of the complex pathophysiology and variable presentation between patients. Misdiagnosed and improperly treated neck pain can develop into chronic conditions that last for years after initial onset. Despite a rise in the incidence of neck pain (21% globally between 2005 and 2015),¹ there is no standardized methodology to objectively define the underlying pathology that is the cause of the pain. The Icelandic startup company, NeckCare, had developed a system, measuring the kinematic variables of the head and neck.² The three main pillars of clinical assessment are: i) Proprioception: Measures the deviation in degrees from the initial neutral position of the head after a blindfolded subject has rotated the head to the left/right/flexion/extension and back to find again the initial position of the head; ii) Neuromuscular control (The Butterfly test): Measures the movement control of the head. The subject controls a cross on the screen and the objective is to chase a moving target. The outcome measures are average distance away from the target, the percentage of time spent on the target and the smoothness of the movement. The Butterfly trajectories are divided into three difficulty levels that come up in random order and the patient will only see a single point moving and not the overall trajectory. The trajectories along the measurements for the two groups can be seen in Fig. 1; iii) Range of motion: Measures the overall range of motion in each plane. The outcome measures are in degrees. Measurements were taken of a control group and patients suffering from whiplash associated disorders (WAD). The measurements are carried out so that the subject was sitting in a chair 90cm in front a computer monitor that was adjusted so that the middle of the screen was at eye level. A movement sensor was placed on the head that connected via Bluetooth to a computer. The movement sensor used an IMU sensor which calculates the 3D Euler angles of the head and projects them onto the monitor for a visual feedback. The results demonstrated that there was a significant difference between the neuromuscular control of the control group and the whiplash group in the Butterfly test. The whiplash group was on average further away from the moving target and spent less time in the vicinity of it than the control group. The whiplash group had less range of motion in all planes and poorer proprioceptive control. These tests can help to identify the muscular impairment for patients suffering from whiplash associated disorders and can be used in the rehabilitation of these patients to activate the deep neck flexor muscles rather than using the larger superficial muscles of the neck.

Key Words: Neck muscles, Neuromuscular control, Rehabilitation, Head kinematics

References

1.Global Burden of Disease Study 2013. Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet. 2015. Aug 22;386(9995):743-800. doi: 10.1016/S0140-6736(15)60692-4. Epub 2015. Jun 7. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Prospects for translational research on outcome measures in musculoskeletal rehabilitation: the search for critical success factors

Vincent Grote ^1,^✉, Michael J Fischer ^1,²

Abstract

Several factors influence the course of convalescence during rehabilitation. Outcome measures and endpoints are often systematically associated with influences other than treatment. This leads to large variability and confounds the observed outcomes: “Garbage In, Garbage Out”, whereby the data quality determines the quality of the results. Therefore, it is becoming increasingly important to find the right input and critical success factors to describe medical quality outcomes in musculoskeletal rehabilitation, where translational myology can make important contributions. We present clinical results for relevant moderating variables from rehabilitation practice and the performance score stratification (T2D). Associations between clinician-reported and patient-reported outcome measures are low to moderate. Overall, musculoskeletal rehabilitation in pre-post designs leads to an outcome with a large effect size. Women, the elderly, and patients with obesity and low physical activity often have poorer baseline scores (t1); therefore, they sometimes appear to respond better to interventions in an inpatient setting. Because the improvement depends on the patient's baseline status (t1), the simple formula T2D = t2 + (t2 - t1) better reflects outcomes, taking into account the functional status at the beginning of rehabilitation (Figure 1). We believe that the T2D concept is highly relevant for many studies and allows better clinical discrimination. T2D may be an appropriate external selection and validation criterion for new opportunities and applications, such as artificial intelligence for the diagnosis and management of myopathology.

Key Words: Rehabilitation, outcomes, Critical Success Factors, confounder, baseline status, translational research, performance score

References

Zdravkovic A, Grote V, Pirchl M, Stockinger M, Crevenna R, Fischer MJ. Comparison of patient- and clinician-reported outcome measures in lower back rehabilitation: introducing a new integrated performance measure (t2D). Qual Life Res. 2022. Jan;31(1):303-315. doi: 10.1007/s11136-021-02905-2. Epub 2021. Jun 15. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Project “Rehabilitation and Health”: URL: www.researchgate.net/project/Rehabilitation-and-Health. [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION X: Centre of Active Ageing (Austria): current status

Helmut Kern

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

AMB-REMOB - study protocol of an early outpatient rehabilitation program

Stefan Loefler

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Exercise intervention in elderly: a novel system within the Centre of Active Aging in Bratislava

Ján Cvečka

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The potential of eccentric training in older adults

Nejc Šarabon ^1,^✉

Abstract

The interest in potential of eccentric training in older adults seems to be increasing in the recent years. Two very recent systematic reviewers focused on the effect of eccentric training in healthy older adults (Kulkarni et al., 2021; Molinari et al., 2019). Molinari et al. (2019) included five studies with muscle strength as outcomes, and reported similar effects of eccentric and concentric exercises, with the data slightly favoring the former. Kulkarni et al. (2021) examined 10 studies and reported that eccentric exercises can be as effective as conventional exercises in older adults for improving functional performance. We performed a broader review and included 18 studies that compared eccentric and concentric exercise interventions. Our analyses suggest that eccentric exercise is actually superior to concentric exercise in terms of the effect on functional performance. Specifically, larger improvements after eccentric compared to concentric exercise were seen for timed up and go test, 2-min stepping test and 30-s sit-stand test (moderate effect sizes). There was also a tendency for eccentric exercise to induce larger improvements in muscle strength and size, but the overall effects were not statistically significant. Together with previous reviews, our analyses suggest that eccentric training may evoke superior effects to conventional/concentric training, wherein functional performance outcomes were most illustrative.

Key Words: Flywheel training, eccentric exercise, elderly, functional outcomes

References

Kulkarni D, Gregory S, Evans M. Effectiveness of eccentric-biased exercise interventions in reducing the incidence of falls and improving functional performance in older adults: a systematic review. Eur Geriatr Med. 2021. Oct 11:1–14. doi: 10.1007/s41999-021-00571-8. Epub ahead of print. [DOI] [PMC free article] [PubMed] [Google Scholar]
Molinari T, Steffens T, Roncada C, Rodrigues R, Dias CP. Effects of Eccentric-Focused Versus Conventional Training on Lower Limb Muscular Strength in Older People: A Systematic Review With Meta-Analysis. J Aging Phys Act. 2019. Dec 1;27(4):823-830. doi: 10.1123/japa.2018-0294. [DOI] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Mimicking disuse and rehabiliation in a mouse model

Feliciano Protasi

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The effects of exercise on skeletal muscle atrophy and denervation in ageing and disuse

Sandra Zampieri

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION XI: Heatwaves in a warming climate: overview and impacts

Piero di Carlo ^1,^✉

Abstract

One of the effects of the global warming is the increase of heatwaves in terms of intensity, frequency, and duration. While global warming is a worldwide concern, heatwaves is a local issue depending on land use change, urbanization, orography, and meteorological conditions.^1,2 Even the local specificity of heatwave, in almost all regions a measure of cumulative heat shows significant increases since the 1950s, mainly driven by heatwave days.³ Understanding past decades heatwave trends and future projection have strong implications to know the impact on human health, on ecosystems and on infrastructure planning.⁴ Here we will show how temperature is changing at different scales from global to local,⁵ focusing on the formation and evolution of urban heat islands. The analysis will cover the last 200 years of data and projections of future trends. Finally, will be discuss the impacts of heatwaves on ecosystems and human health pointing out drivers, mitigation, and adaptation strategies.

graphic file with name ejtm-32-1-10440-g058.jpg

Key Words: Climate Change, heatwaves, radiative forcing

References

1.Stone B Jr, Vargo J, Liu P, Habeeb D, DeLucia A, Trail M, Hu Y, Russell A. Avoided heat-related mortality through climate adaptation strategies in three US cities. PLoS One. 2014. Jun 25;9(6):e100852. doi: 10.1371/journal.pone.0100 852. [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Ding T, Qian W, Yan Z. Changes in hot days and heat waves in China during 1961–2007. Int J Climatol. 2010;30: 1452–1462. [Google Scholar]
3.Perkins-Kirkpatrick SE, Lewis SC. Increasing trends in regional heatwaves. Nat Commun. 2020. Jul 3;11(1):3357. doi: 10.1038/s41467-020-16970-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Philip SP, et al. Cities must protect people from extreme heat. Nature. 2021. Jul;595(7867):331-332. doi: 10.1038/d41586-021-01903-1. [DOI] [PubMed] [Google Scholar]
5.Aruffo E, Di Carlo P. Homogenization of instrumental time series of air temperature in Central Italy (1930−2015), Clim Res. 2019;77: 193–204. doi: 10.3354/cr01552 [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Environmental and Exertional Heat Stroke: the role of skeletal muscle

Feliciano Protasi ^1,^✉

Abstract

Climatic changes are increasing the incidence of heat waves, periods in which the temperature raise above the average temperatures for three or more days. Heat waves are very dangerous for human health, as it is now clear that the mortality rate increases dramatically during those days.¹ Even if several factors may contribute to sudden death in high environmental temperatures, the most common cause of death attributable to heat is dehydration, heat cramps and exhaustion, and hyperthermia, i.e. in one word heat stroke (HS).^2,3 The cellular and molecular mechanisms underlying HS are multiple and still debated; as a result: a) a cure for acute treatment of HS is still missing; b) patients are usually treated only with supportive measures (cooling and hydration); and finally c) clear guidelines for proper lifestyle habits before and during heat waves are not well defined. We have collected compelling evidence that HS shares common symptoms with malignant hyperthermia (MH) susceptibility (Figure), a life-threatening syndrome caused by mutations in proteins deputed to Ca²⁺ handling in muscle and triggered by administration of halogenated anaesthetics. There is general agreement that the main mechanism triggering MH crises is an abnormal SR Ca²⁺ leak through mutated RYR1, causing uncontrolled contraction and rhabdomyolysis. However, other factors must be taken in consideration: A) Ca²⁺ leak from the SR triggers a feed-forward mechanism leading to overproduction of reactive species of oxygen and nitrogen (ROS and RNS) by mitochondria, which in turns causes further Ca²⁺ release through RYR1-nitrosylation.⁴ B) MH responses do not occur in the absence of extracellular Ca²⁺ (5) suggesting that Ca²⁺ influx from the extracellular space is an essential component to be considered. The Central Hypotheses guiding our recent work are: a) skeletal muscle plays a central role in HS; b) lifestyle habits may affect proper muscle function, hence cause either increased or diminished risk of the normal population to HS in challenging environmental conditions.

Key Words: Heat-wave, malignant hyperthermia (MH), Environmental and Exertional Heat Stroke (EHS)

References

1.Robine JM, Cheung SL, Le Roy S, Van Oyen H, Griffiths C, Michel JP, Herrmann FR. Death toll exceeded 70,000 in Europe during the summer of 2003. C R Biol. 2008;331(2):171-8. doi: 10.1016/j.crvi.2007.12.001. Epub 2007. Dec 31. [DOI] [PubMed] [Google Scholar]
2.Bouchama A, Knochel JP. Heat stroke. N Engl J Med. 2002;346(25):1978-88. doi: 10.1056/NEJMra011089. [DOI] [PubMed] [Google Scholar]
3.Yeo TP. Heat stroke: a comprehensive review. AACN Clin Issues. 2004;15(2):280-93. doi: 10.1097/00044067-200404000-00013. [DOI] [PubMed] [Google Scholar]
4.Durham WJ, Aracena-Parks P, Long C, Rossi AE, Goonasekera SA, Boncompagni S, Galvan DL, Gilman CP, Baker MR, Shirokova N, Protasi F, Dirksen R, Hamilton SL. RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice. Cell. 2008;133(1):53-65. doi: 10.1016/j.cell.2008.02.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Fletcher JE, Huggins FJ, Rosenberg H. The importance of calcium ions for in vitro malignant hyperthermia testing. Can J Anaesth. 1990;;37(6):695-8. doi: 10.1007/BF03006495. [DOI] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

High-fat diet increases the risk of environmental heatstroke in mice

Matteo Serano ^1,^✉, Antonio Michelucci ¹, Giorgia Rastelli ¹, Cecilia Paolini ¹, Laura Pietrangelo ¹, Flavia Alessandra Guarnier ², Feliciano Protasi ¹

Abstract

Heat-stroke (HS) is a life-threatening response to heat characterized by an abnormal increase in body temperature (>40°C) that causes dysfunction of organs, central nervous system and may end in death.¹ Environmental heat-stroke (EHS), often triggered by a hot and humid environment, is caused by excessive heat production in muscle, which in turn is the result of abnormal Ca2+ leak from the sarcoplasmic reticulum (SR) and oxidative stress.² See Figure for a Mechanistic Model. As high fat diet is known to increase oxidative stress,³ the objective of the present study was to investigate the effects of high-fat diet in the heat-stroke susceptibility of c57bl/6 wild type (WT) mice of 4 months of age (adult). Our results show that, in comparison with mice fed with a control diet, mice after 3 months of high-fat: a) increased heat generation and energy expenditure (assessed by indirect calorimetry) during heat stress; b) elevated oxidative stress in both EDL and Soleus muscles; and c) enhanced sensitivity to caffeine and temperature of isolated EDL and Soleus muscles during in vitro contracture test (IVCT, the gold standard procedure to test in-vitro EHS susceptibility). Our data suggest that high-fat diet predispose mice to EHS, possibly as a result of increased oxidative stress and excessive release of Ca2+ from SR. This study may have important implications for guidelines regarding food intake during periods of intense environmental heat.

graphic file with name ejtm-32-1-10440-g060.jpg

Key Words: High-fat diet, heat-stroke, oxidative stress

References

1.Bouchama A, Knochel JP. Heat stroke. N Engl J Med. 2002. Jun 20;346(25):1978-88. doi: 10.1056/NEJMra011089. [DOI] [PubMed] [Google Scholar]
2.Durham WJ, Aracena-Parks P, Long C, Rossi AE, Goonasekera SA, Boncompagni S, Galvan DL, Gilman CP, Baker MR, Shirokova N, Protasi F, Dirksen R, Hamilton SL. RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice. Cell. 2008. Apr 4;133(1):53-65. doi: 10.1016/j.cell.2008.02.042 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Matsuzawa-Nagata N, Takamura T, Ando H, Nakamura S, Kurita S, Misu H, Ota T, Yokoyama M, Honda M, Miyamoto K, Kaneko S. Increased oxidative stress precedes the onset of high-fat diet-induced insulin resistance and obesity. Metabolism. 2008. Aug;57(8):1071-7. doi: 10.1016/j.metabol.2008.03.010 [DOI] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Exertional Heat Stroke: the possible role of external Ca²⁺

Barbara Girolami ^1,^✉, Matteo Serano ¹, Antonio Michelucci ¹, Laura Pietrangelo ¹, Feliciano Protasi ¹

Abstract

Exertional/Environmental Heat Strokes (EHSs) are hyperthermic crises triggered by strenuous physical exercise and/or exposure to environmental heat, which are caused by an altered intracellular Ca²⁺ homeostasis in muscle.^1,2We recently demonstrated that a single bout of exercise on treadmill leads to formation of Calcium Entry Units (CEUs), intracellular junctions that promote interaction between STIM1 and Orai1, the two proteins that mediate Store-Operated Ca²⁺ Entry (SOCE). SOCE is a mechanism that is activated during muscle fatigue and that allows recovery of extracellular Ca²⁺ during prolonged activity.^3,4 The hypothesis underlying this project is that assembly of CEUs during prolonged exercise may predispose to EHS, when exercise is performed in challenging environmental conditions. To test this hypothesis, we used 4 months old WT mice that were: a) first, divided in 3 experimental groups: control, trained-1m (1 month of voluntary running in wheel cages), and exercised-1h (1 hour of incremental treadmill run); and b) second, subjected to an incremental treadmill run of 45 min at 34°C and 40% humidity. We then: i) measured the internal temperature of mice, which was higher in the pre-exercised groups (trained-1m: 38.9°C ± 0.33; exercised-1h: 38.7°C ± 0.40) compared to control (37.9°C ± 0.17); ii) applied an ex-vivo exertional stress protocol to isolated EDL muscles (tetanic stimulation performed at 30°C) and verified that samples from trained-1m and exercised-1h mice generated a tension significantly greater than control; iii) Analyzed CEUs by electron microscopy (EM) and verified that EDL muscles of exercised-1h ad trained-1m mice contained a greater number of elements forming CEUs. The data collected suggest that assembly of Calcium Entry Units during exercise could predispose to EHS when exercise is performed in challenging environmental conditions.

graphic file with name ejtm-32-1-10440-g060a.jpg

Key Words: Calcium entry unit (CEU), heat-stroke, store-operated Ca²⁺ entry (SOCE)

References

1.Bouchama A, Knochel JP. Heat stroke. N Engl J Med. 2002. Jun 20;346(25):1978-88. doi: 10.1056/NEJMra011089. [DOI] [PubMed] [Google Scholar]
2.Durham WJ, Aracena-Parks P, Long C, Rossi AE, Goonasekera SA, Boncompagni S, Galvan DL, Gilman CP, Baker MR, Shirokova N, Protasi F, Dirksen R, Hamilton SL. RyR1 S-nitrosylation underlies environmental heat stroke and sudden death in Y522S RyR1 knockin mice. Cell. 2008. Apr 4;133(1):53-65. doi: 10.1016/j.cell.2008.02.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Boncompagni S, Michelucci A, Pietrangelo L, Dirksen RT, Protasi F. Exercise-dependent formation of new junctions that promote STIM1-Orai1 assembly in skeletal muscle. Sci Rep. 2017. Oct 27;7(1):14286. doi: 10.1038/s41598-017-14134-0. Erratum in: Sci Rep. 2018. Nov 27;8(1): 17463. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Protasi F, Pietrangelo L, Boncompagni S. Calcium entry units (CEUs): perspectives in skeletal muscle function and disease. J Muscle Res Cell Motil. 2021. Jun;42(2):233-249. doi: 10.1007/s10974-020-09586-3. Epub 2020. Aug 18. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION XII: Integration of Motion, Forces, and the Central Nervous System

Alessandro Del Vecchio ^1,^✉, M Oßwald ¹, R Simpetru ¹, M Cakici ¹, D Braun ¹, D O Souza ¹

Abstract

Movement is encoded by spinal motor units. The motor unit is formed by a motoneuron, and the muscle fibres innervated by its axon. The motoneuron receives efferent and afferent synaptic inputs that modulate the force output of the muscle. There is very little knowledge on the changes in motor unit behaviour during natural dynamic movements consisting of changes in muscle length and neural drive. Moreover, the associations between kinematics, kinetics and motor unit dynamics are largely unknown. Here we aimed to explore these associations with a linear and nonlinear method. With the linear method, we decomposed high-density EMG signals into constituent motor unit action potentials during dynamic and isometric hand movements. With the nonlinear method, we built a framework with digital cameras that recorded the movements of the hand as well as recording isometric forces with an instrumented circular object. Deep neural networks were trained to understand the exact position of the hand from the digital cameras as well as from the high-density EMG signals. The movements consisted of more than 20 degrees of freedom, ranging from individual and combined finger flexion/extensions, and different hand and wrist gestures at different speeds (0.5 and 1.5 Hz). Four hundred EMG channels recorded the activity of the forearm muscles. The non-linear method was able to identify with 99.6% accuracy the exact movement of the fingers. The decomposition of the electromyogram revealed unique motor unit firing patterns that were significantly different than the control during isometric contractions. The delays between the onset of neural activity and movement kinematics were similar across all fingers and decreased with the speed of force contraction suggesting that the central nervous system compensates the muscle dynamics with unique control strategies for each finger. Because the hand digits were flexed and extended at various speeds, our interface shows that the dynamical output of the hand is fully explained by the neural strategies and that the musculotendinous unit has only a passive role for the rate of speed production.

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

On the role of proprioception in the sense of force

Stéphane Baudry ^1,^✉, Alp Eşrefoğlu ¹, Mélanie Henry ¹

Abstract

Successful performance of motor tasks requires the adequate modulation of force output, which depends, in part, on the integration of information from various mechanoreceptors located in muscles, muscle-tendon interfaces, and joints. These signals form the basis for the perception of self-movement and body position – termed proprioception – which is defined as the conscious or unconscious perception of the position of different parts of the body in space. Along with the kinaesthetic sense (the senses of position and movement), the sense of force is another aspect of proprioception. The sense of force represents the ability to correctly perceive and reproduce a given level of force. However, it is still debated whether the sense of force really relies on proprioceptive inputs. This may depend on the experimental approach used for its assessment. Indeed, it can be evaluated by asking the participants to match with one limb the force produced by the contralateral limb (two-arm matching task), or by asking to reproduce a force with the same limb (force reproduction task). It has been suggested that the two-arm matching task does not require proprioceptive feedback but rather a centrally generated signal. In contrast, the force reproduction task is suggested to mainly rely on proprioceptive inputs, including signals from muscle spindles and Golgi tendon organs, in addition to sensory inputs from skin mechanoreceptors. By manipulating proprioceptive and visual inputs within different phases of the force reproduction task, we provide relevant elements supporting the role of proprioceptive signal from muscle and tendons sensors during the force reproduction task. Furthermore, we show a decrease in the sense of force with ageing, which could reflect age-related decline in sensorimotor integration of proprioceptive and somaesthetic inputs.

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The role of age on neuromuscular performance decay induced by a maximal intensity sprint session in a group of competitive athletes

Leonardo Cesanelli ^1,^*, Danguole Satkunskiene ^2,^*, Nerijus Eimantas ², Angelo Iovane ³, Giuseppe Messina ³

Abstract

Age-related changes in the neuromuscular system functions may affect profoundly high-level athletes’ performance across their career, and potentially determining its length. Therefore, the present study aimed to explore the role of age on neuromuscular functions in a group of competitive athletes, and in particular, to analyse the fatiguing effect of a maximal intensity sprint session (MISS). Thirty-one competitive endurance athletes completed a knee extensors and flexors’ maximal-voluntary-isometric-contraction (MVC) test (3x5s, 60s recovery) before and after a maximal intensity sprint session (MISS) consisting of 4x15s Wingate-tests. The data have been stratified considering three age categories (18-28, n=11, 29-38; n=10; 39-43, n=10). Non-significant differences in neuromuscular performance markers emerged between the three age groups at baseline (p>.05). Overall, both quadricep and hamstring muscles early and late RTD dropped significantly more than the MVT (p<.05). Age had a significant effect on early RTD with older athletes exhibiting greater RTD drop compared to younger athletes (p<.05). A significant effect of age emerged also for the changes in surface electromyography (sEMG) variables, in which the frequency spectrum variables (MDF and MNF) dropped significantly more than the sEMG amplitude (RMS) (p<.05). In addition, age was largely significantly correlated with early RTD drop. On the contrary, no significant effects of age emerged for the MVT and late RTD drop (p>.05). The dynamics of changes in neuromuscular performance markers after a MISS suggested that getting older, competitive athletes may potentially experience greater lost in early explosive strength compared to maximal or late explosive strength, due to neuromuscular fatigue.

Key Words: Ageing, physical performance, maximal strength, explosive strength

References

Thompson BJ, Ryan ED, Herda TJ, Costa PB, Herda AA, Cramer JT. Age-related changes in the rate of muscle activation and rapid force characteristics. Age (Dordr). 2014. Apr;36(2):839-49. doi: 10.1007/s11357-013-9605-0. Epub 2013. Dec 12 [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Adaptive Force in Patients with Long-COVID

Frank N Bittmann ^1,^✉, Laura V Schaefer ¹

Abstract

After 6 month 68% of COVID-19 survivors, and still 49% after 12 months, show one or more long-COVID symptoms.¹ The symptoms persist regardless of COVID-19 severity.² The public attention to Long-COVID contributed to shed light on a similar but neglected syndrome. Chronic fatigue syndromes after acute infections (e.g., Ebstein-Barr virus) are known since decades, but mostly are without adequate recognition from medical professionals.³ Based on established diagnostics, there is still no satisfying approach to understand the phenomena of post-infectious syndromes and, consequently, to apply purposeful therapies. In view of the immense and increasing number of Long-COVID patients, diagnostical approaches are urgently needed. The most common symptoms of post-infectious diseases are fatigue and muscle weakness.¹ In clinical practice, a considerably reduced ability to resist external forces is regularly seen in those patients. Therefore, it is questioned whether the newly inaugurated Adaptive Force (AF) could be a promising approach to measure post-infectious states. The AF reflects the capacity of the neuromuscular system to adapt adequately to external increasing forces in an isometric holding manner. Healthy persons show a high maximal isometric AF (AFisomax (N)) assessed during manual muscle test (MMT) objectified by a handheld device.^3,4 Thereby AFisomax is close to the maximal AF (AFmax (N)) reached in the same measurement, reflecting a proper adaptability of the neuromuscular system. AF pilot measurements of Long-COVID patients gathered in clinical routine (no clinical study) will be presented. The AF of the hip and elbow flexors of one side was assessed manually (MMT) each three times (alternating) by well-skilled examiners objectified by the handheld device. The maximal holding capacity (AFisomax) and the total maximal AF (AFmax) were evaluated (Fig. 1) as well as the ratio of AFisomax/AFmax (%). Long-COVID patients show a substantial and significantly lower AFisomax and AFisomax/AFmax compared to healthy subjects. After successful treatment the holding capacity is immediately improved and the ratio is significantly higher compared to the initial measurements. This suggests the holding capacity is essentially lower in post-infectious conditions and could possibly be a sensitive biomarker for post-infection fatigue. These preliminary data need to be confirmed by future clinical studies. In case of verification it will be used to optimize individual diagnostics and to apply purposeful treatments.

Key Words: Adaptive Force, holding isometric muscle function, long-COVID, post-COVID

References

1.Huang L, Yao Q, Gu X, Wang Q, Ren L, Wang Y, Hu P, Guo L, Liu M, Xu J, Zhang X, Qu Y, Fan Y, Li X, Li C, Yu T, Xia J, Wei M, Chen L, Li Y, Xiao F, Liu D, Wang J, Wang X, Cao B. 1-year outcomes in hospital survivors with COVID-19: a longitudinal cohort study. Lancet. 2021. Aug 28;398(10302):747-758. doi: 10.1016/S0140-6736(21)01755-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
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3.Schaefer LV, Dech S, Bittmann FN. Adaptive Force and emotionally related imaginations - preliminary results suggest a reduction of the maximal holding capacity as reaction to disgusting food imagination. Heliyon. 2021. Aug 19;7(8):e07827. doi: 10.1016/j.heliyon.2021.e07827. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Schaefer LV, Dech S, Aehle M, Bittmann FN. Disgusting odours affect the characteristics of the Adaptive Force in contrast to neutral and pleasant odours. Sci Rep. 2021. Aug 12;11(1):16410. doi: 10.1038/s41598-021-95759-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION XIII: How far can electrical stimuli recruit specific neurons: mechanisms, realistic options and limits

Winfried Mayr ^1,^✉, Jose Luis Vargas Luna ¹, Dietmar Rafolt ¹

Abstract

Electrical stimuli play an important role in many clinical diagnostic and therapeutic methods as well as in experimental and clinical studies. Key for understanding elicited physiological mechanisms is understanding of interaction of the induced artificial electrical fields with electrically excitable neural and muscular structures. Adjustable variables in stimulation setups are size and position of electrodes,¹ shape of stimuli, sequence of their delivery and variation of their size and rate. Goals are selective activation of neurons or, in case those are denervated, muscle fibers, and safe delivery of currents, without harm to skin and other tissues. Coupling of the technical systems to the physiological structures is made via contacting electrode surface, at the skin or embedded in soft tissue, with a transition from a technical electron conductor to a physiological ion conductor. Careful provisions are necessary for keeping associated electrolytic processes safe and avoiding tissue damage risks, acutely and in long-term perspective. Functional coupling happens at the electrically excitable membranes, which form the envelope of nerve or muscle fibers. At such membranes, induced electrical fields can elicit traveling action potentials, starting at a trigger point, where a discharge condition is reached, in addition, there are also control options, where the induced field influences resting potentials and increases or lowers excitability for reaching a discharge threshold.² Basic principles of fiber recruitment are the size dependence, the bigger the fiber in diameter, the lower the discharge threshold, and distance dependent, threshold increases with distance between electrode and neuron.^3,4 In the mixed peripheral nerve, preferred recruitment of groups of large proprioceptive afferent neurons is reached with lowest stimulation intensity, followed by co-recruitment of cutaneous afferents, efferent neurons of type 2 motor units and then type 1 motor units, and finally smaller afferent pain fibers, when stimulation intensity is increased (Fig. 1). It is important to consider this as limitation, when physiological responses are interpreted, as recruitment has strong impact in central interneuron processing of afferent input signals as well as in motor control and metabolic processes in activated muscles. In addition to stimulus size dependent recruitment, frequency of trains of stimuli plays a major role in both central neural processing and peripheral activation of muscles ⁴.

Key Words: Functional Electrical Stimulation, neuron recruitment, fiber size, threshold

References

1.Krenn MJ, Vargas Luna JL, Mayr W, Stokic DS. Bipolar transcutaneous spinal stimulation evokes short-latency reflex responses in human lower limbs alike standard unipolar electrode configuration. J Neurophysiol. 2020. Oct 1;124(4):1072-1082. [DOI] [PubMed] [Google Scholar]
2.Vargas Luna JL, Krenn M, Löfler S, Kern H, Cortés R JA, Mayr W. Comparison of Twitch Responses During Current- or Voltage-Controlled Transcutaneous Neuromuscular Electrical Stimulation. Artif Organs. 2015. Oct;39(10):868-75. [DOI] [PubMed] [Google Scholar]
3.Rattay F. (1990). Electrical Nerve Stimulation: Theory, Experiments, and Applications. Vienna: Springer. [Google Scholar]
4.Vargas Luna JL, Brown J, Krenn MJ, McKay B, Mayr W, Rothwell JC, Dimitrijevic MR. Neurophysiology of epidurally evoked spinal cord reflexes in clinically motor-complete posttraumatic spinal cord injury. Exp Brain Res. 2021. Aug;239(8):2605-2620. [DOI] [PMC free article] [PubMed] [Google Scholar]

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The effect of direct muscle stimulation on denervated gluteal muscles and tissue composition in people with chronic spinal cord injury – preliminary results

Marie Alberty ¹, Ines Bersch ^1,^✉

Abstract

Electrical stimulation of denervated muscles with long pulses to convert connective and adipose tissue into contractile muscle tissue has been investigated in persons with chronic spinal cord injury.^1–3 A European funded project (RISE) has demonstrated that a stimulation period of two years led to significant changes in muscle growth and reduction in adipose tissue in the lower limb muscles.⁴ It is still unclear after what time the transformation process from connective and fatty tissue to contractile muscle tissue begins in chronic paraplegia with damage to the lower motor neuron. Likewise, the process of this conversion and how it occurs remains unclear. In other words, is there an immediate visible increase in muscle mass or does connective and adipose tissue break down first and when do these changes in tissue composition start to occur? Kern and Carraro have visualized and emphasized on the importance of life-long stimulation in people with lower motor neuron lesion in order to maintain proper blood circulation and to produce a so-called "cushioning effect" in people with spinal cord injury (SCI) to prevent them from skin injuries.⁵ The aim of the present study is to investigate the effect of electrical stimulation of chronic denervated gluteal muscles in the first six months after stimulation onset. Changes in muscle as well as adipose tissue thickness were assessed over the stimulation period of six months using standardized magnetic resonance imaging (MRI) measurements of the gluteal area on the projected line of the sacroiliac joint (SIJ), the acetabulum and the coccyx. The study participants (four participants with AIS A and one with AIS C) stimulated the denervated gluteal muscles five times a week during a period of six months. The stimulation was composed of a warm-up phase and a training phase lasting a total of 33 minutes per session. The electrical stimulation was administered with the Stimulette den2x (Schuhfried GmbH Vienna, Austria) with the sponge electrodes being placed on the buttocks to generate a horizontally oriented electrical field to cover the entire gluteal area.

Key Words: Denervation, Electrical stimulation, SCI, Tissue composition, Magnetic resonance imaging

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Spinal cord stimulation review and recent progress

Thordur Helgason ^1,^✉, Arndís Þóra Þórisdóttir ¹, Linda Björk Halldórsdóttir ¹, Sara Kristinsdóttir ¹, Gígja Magnúsdóttir ¹, Brynja Ingólfsdóttir ¹, Páll E Ingvarsson ¹

Abstract

Epidural stimulation of the spinal cord (eSCS) has for several decades been used successfully for the treatment of pain and spasticity. It has also been used to facilitate voluntary control of walking by individuals with partial lesion. Newly (Feb. 2022) walking in complete paraplegic individuals has been demonstrated using eSCS. eSCS has the advantage compared to tSCS that the electrodes are placed near to the stimulation target and can activate these with less coactivation of other muscles. This though given that the placement of the electrodes can be done during an operation and that they are stable in place. Using leads with an electrode array makes it possible to adjust for misplacement and capsuling through connective tissue to some limit. tSCS has, following the experience with eSCS, been used with similar results for spasticity treatment and facilitation of voluntary controlled walking in spinal cord injured (SCI) individuals. This is achieved using common gel electrodes on the surface of the skin above T11 – T12. Being further away from the targeted cells the specificity of tSCS through localisation of the current field is lower than by eSCS but sensory fibres in the posterior roots have a larger diameter than lower motor neuron fibres and can be targeted with lower current intensity without activating the latter. In resent years progress has been made in evaluating spasticity although there is a lack of consensus in the definition of spasticity. Also, our group has developed a new parameter for quantifying spasticity based on Wartenberg pendulum test targeting the excitability of the stretch reflex. It is defined as the time span from 50% of the maximum velocity of the first leg swing, determined from the pendulum test, to the activation of the muscle contraction in the quadriceps, determined from the EMG. The result is a parameter reflecting changes in excitability of the stretch reflex. Spinal cord stimulation effect on the brains or the spine plasticity has not been in focus of research so far. Our group has developed a methodology to evaluate EEG, EMG and joint angle measurements made during several common neurological and physio therapeutical tests as well as SCS in order to analyse the reaction of brain and muscles to these tests. Pipeline for brain network analysis and time-frequency analysis of the neurological signals has been developed. This enables studies of changes in the brain due to different condition of the subject.

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Patellar reflex test, transcutaneous spinal cord stimulation (tSCS), H-reflex analysis, and brain activity analysis in healthy, spinal cord injury and brain disorders

Sara Kristinsdóttir ¹, Arndís Þóra Þórisdóttir ¹, Linda Björk Halldórsdóttir ¹, Gígja Magnúsdóttir ¹, Brynja Ingólfsdóttir ¹, Páll E Ingvarsson ¹, Thordur Helgason ^1,^✉

Abstract

Epidural spinal cord stimulation (eSCS) has shown to be an effective way for neuromodulation in the spinal cord. Treatment of pain with eSCS has a long time been a clinical routine. Also, has the treatment of spasticity in SCI been proven to be effective although it has not found its way into a daily clinical application, mostly due to the fact that it is an invasive treatment. In recent years several groups, including ours, have shown that tSCS can be used to treat spasticity and facilitate walking in SCI in a similar way as eSCS. This is not the case for brain insulted individuals. It is believed that the tSCS affects the spinal cord neural network with inhibiting signals partly replacing the brain’s function. This process is poorly understood. Also, the interaction of the spinal cord with the brain or the effect of tSCS on the spine and brain has not been the focus of research. Plastic processes in the spinal cord and in the brain as a result of SCI are known to some extent but not the effect of tSCS on the same organs. The neural structures influencing the activity on the lower motor neuron are the target of the treatment of spasticity in the lower extremities. These effects and the structures are to be studied in this work through the monosynaptic reflex and tSCS effects mainly through EMG of the thigh and the connection to the brain through EEG. In this project a measurement system for recording of H-reflex and m-wave as well as EEG and joint angles during patellar reflex test and tSCS is constructed and tested. Especially exact time measurements (Δt ≤ 0,5 ms) between events are targeted. Additionally, EEG, EMG and joint angles registrations during three standard tests are evaluated. They are 10 m walk, stand up and go and five times stand up (if movement ability is there). This is done by healthy, SCI and brain insulted individuals. These tests are standard for evaluating movement ability in individuals with lesions in the central nervous system (CNS). A tested and evaluated measurement system enabling exact time measurements between events for research on the monosynaptic reflex arc including H-reflex and m-wave. Maps and statistics on individual variability between healthy subjects as well as SCI and brain insulted. Also, a tested and evaluated measurement system for research on EEG, EMG, and joint angle signals. Recordings, processing, and evaluation of brain potentials evoked by patellar tendon test and tSCS and statistical analysis on brain activity between groups.

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Skeletal muscle tissue restoration using functionalized biomaterials

Serafina Pacilio ¹, Roberta Costa ¹, Valentina Papa ¹, Maria Teresa Rodia ¹, Carlo Gotti ², Giorgia Pagnotta ², Maria Letizia Focarete ^2,^✉, Giovanna Cenacchi ¹

Abstract

Skeletal muscle (SKM) loss is considered to be largely irreversible and therapeutic options to restore muscle mass have been limited. Frequent reasons for SKM loss are injuries, surgery, aging, metabolic diseases, as diabetes, and inherited genetic diseases. SKM has good regenerative capacity due to the presence of resident stem cells, the satellite cells, located between the cell membrane and the basal lamina, however in vitro studies demonstrated their poor ability to migrate and to self-renewal in situ: that means their intrinsic proliferative capacity is lost in normal or suitable cell culture. New strategies are required to overcome these limitations; recently, various studies have focused on recreating the niche-like environment combining biomaterials (tissue engineering) and cells (cell-based therapy). Tissue engineering (TE), in particular, has been recognized as a promising tool and a new therapeutic approach for the renewal of SKM tissue. Biomaterials for SKM TE designed in this work are able to persist long enough to support functional SKM tissue formation, while degrading gradually. The device developed is a biocompatible scaffold composed of aligned nanofibers obtained through the electrospinning technique. The alignment provides the anisotropy of SKM tissue mimicking its natural morphology. Moreover, the scaffold acts as carrier for cell vitality, proliferation and myogenic differentiation. Nanofibrous scaffold endows SKM tissue morphogenesis into parallel-oriented myotubes, promoting early cell cycle exit and contributing to an early myoblast differentiation and fusion into myotubes in comparison to conventional monolayer myoblast culture.

Key Words: Skeletal muscle, Myogenesis, Tissue engineering, Regenerative Medicine

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Establishment of models for mechanical and oxidative stress based on tissue-engineered skeletal muscle

Janine Tomasch ^1,^✉, Babette Maleiner ¹, Adelheid Weidinger ¹, Andrey Kozlov ¹, Heinz Redl ¹, Andreas Teuschl ¹

Abstract

Models adequately mimicking skeletal muscle injuries and diseases are important tools to understand and study mechanisms underlying initiation and progression of diseases as well as tissue regeneration and remodeling after injuries.¹ 3D tissue models help increase significance of generated results due to their similarity to in vivo conditions compared to conventional cell culture without the obvious drawbacks of animal tests. Herein, we present how an established tissue engineering approach to create biomimetic skeletal muscle-like tissue can be used to model pathologies. Healthy tissue constructs created through mechanical stimulation of murine myoblasts with a bioreactor,² and subsequently subjected to different types of stress present the basis of this study. Stress was induced either by 1.) mechanical overstimulation with high frequency or high tensile stress with the same bioreactor;² 2.) complete deprivation of mechanical stimulation or 3.) by exposure to oxidative stress with another bioreactor system based on reactive oxygen species generated through hydrostatic pressure.³ Mechanical overstimulation resulted in an immediate stress response (Fig. 1) as well as prolonged downstream signaling activity. Furthermore, high frequency overstimulation led to increased gene expression of markers of proliferation and early- and mid-stage myogenesis, and changed morphology of myotubes (Fig. 2). Deprivation of mechanical stimulation for seven days led to severely impaired integrity of myotubes on a morphological level (Fig. 2), but also to downregulation of signaling pathways involved in stress response and myogenic development. Oxidative stress induced decreased viability (Fig. 3) and loss of structural integrity of myotubes. Thus, we conclude that the first steps towards the establishment of a biomimetic tissue-engineered model for skeletal muscle pathologies were successful. Nevertheless, further refinement and investigation of the mechanisms involved are still needed.

Figure 3. — Live (green) / dead (red) staining of biomimetic skeletal muscle-like tissue after oxidative stress.

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION XIV: Topical aspects of neuro-rehabilitation

Sergey A Rachin ¹, Andrey P Rachin ^2,^✉

Abstract

The goal of neuro-rehabilitation is to create optimal conditions for the patient’s active engagement in domestic and social life, a return to active social and domestic activities and, ultimately, to improve the quality of life of both the patient himself and his/her relatives. The main tasks of neuro-rehabilitation are the impact on the restoration of the patient's vital functions using physical and mental methods, the impact on his/her body with the help of drug therapy to improve and normalize metabolic processes, as well as the development of an adaptation system for the changes caused by the pathological process. It is important to take into account theoretical aspects of the organization of physiological and pathological movements, as well as the formation of short- and long-term compensatory processes of damaged structures and the impaired functions of the body systems in the course of neuro-rehabilitation. The formation of compensatory mechanisms is greatly influenced by signaling from the external and internal environment of the body, i.e. feedback.¹ A reliable brain function is ensured by dynamic inter-neuronal activity, resulting in morphologically redundant connections. Neuro-rehabilitation is also based on the systemic organization of the brain functions, the reserve capacity of the brain in reorganizing its structures under the conditions of the pathological process. This reorganization causes the patient's motor retraining, which leads to the restoration or compensation of the impaired functions. When conducting neurorehabilitation, it is necessary to strive for a true restoration of the disturbed functions of the patient's body. This level of HP seems to be the highest and lies in the fact that the functions of the body completely return or are as close as possible to the original state. The main mechanism ensuring a true functional recovery is the disinhibition of inactivated nerve elements by means of techniques aimed at stimulating them.² With brain damage, recovery occurs due to intact functional systems. This process is ensured by the plasticity of the nervous system and the anatomical connections between its departments. The plasticity of the brain lies in the ability of the nervous tissue to change its structural and functional organization under the influence of external and internal factors. It is due to the following mechanisms: the functioning of the previously inactive connections; sprouting of the fibers of the remaining neurons (renewed of growth of the affected axon, changes in fiber branching, the area and density of the dendritic spines); formation of new synapses; synaptic remodeling (changes in the configuration and properties of synapses); reorganization of neuronal circuits; extrasynaptic transmission of excitation; a change in the structure of astrocytes, an increase in the number of contacts between synapses and astrocytes. During the medical and social rehabilitation, one must bear in mind that severe disabling neurological diseases cause permanent disability and social maladjustment. Thus, regarding the main aspects and principles of neuro-rehabilitation, it is important to understand the complexity, aims and problem orientation of this process, which is primarily aimed at increasing the degree of recovery of neurological functions of patients after a stroke, their level of social adaptation and, ultimately, improving the quality of life of patients and their relatives. ³

Key Words: Neuro-rehabilitation, stroke, neuroplasticity

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Head and neck functional analysis: the Functional Anatomy Research Center experience

Claudia Dellavia ^1,^✉

Abstract

The physiological functions of the human body represent the expression of biological phenomena easily observable, but hardly quantifiable in their entirety as they result from the dynamic interaction of multiple factors. Each phenomenon may present in different ways through a different combination of the same parameters that characterize it at different times. Therefore, the evaluation of a biological phenomenon implies the possibility of measuring only ONE or more aspects, and not the whole complexity. The same stimulus can in fact elicit different responses in the same individual in relation to time and environment. It is essential to associate the measurement of the phenomenon with a series of numbers that allow its interpretation, temporal evolution and comparison with other phenomena. In the scientific field, the numerical description of biological functions needs reliable instruments that provide accurate and repeatable values of one or more characterizing parameters. The response to the stimulus is not solely determined: the input-output relationship can lead to the same result by exploiting different paths. Hence, despite the knowledge of the input and the output, the biological phenomenon is not necessarily known. Each individual owns a specific adaptability to the different stimuli that undergoes fluctuations over time. When an input exceeds the adaptability threshold, unexpected and previously undetectable problems may be generated. In the clinical dental field, it is necessary to quantify the function and/ or dysfunction and the need of intervention to restore the functional alteration, by means of objective measures and not only of patient subjective symptoms. It is crucial to set instrumental tests with dedicated protocols that allow to measure the stability of the occlusal relationships both in static and dynamic conditions and to uniquely study the experimentally-induced variations. The evaluation should integrate the functional aspects with the morphological ones at micro and macrostructural levels. The research activity of the Functional Anatomy Laboratory of the stomatognathic apparatus (Functional Anatomy Research Center- FARC- University of Milan) has been developed aiming at defining standardized indices that measure the overall activity of the chewing muscles. Strength, recruitment and coordination of muscles are gathered in order to identify the impact of various occlusal conditions on the masticatory synergism and possible effects on other districts, primarily on the neck. The FARC 30-years-experience applied innovative non-invasive technologies (electro magnetic and electromechanical digitizers, optical morphometric methods, surface electromyographs, dynamometers, etc.) for the creation of protocols that clinician could use to measure the chewing function in each individual patient in a simple way, testing its fluctuations over time.

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Key Words: Physiological functions, scientific instrumentation, biological measures, masticatory muscles activity

References

Ferrario VF, Sforza C, Tartaglia GM, Dellavia C. Immediate effect of a stabilization splint on masticatory muscle activity in temporomandibular disorder patients. J Oral Rehabil. 2002;29:810-815. [DOI] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Masticatory muscles guided orthodontic treatments

Giacomo Begnoni ^1,^✉

Abstract

The purpose of orthodontic treatment is to guide the development of the upper and lower jaw during childhood and adolescence to establish proper eruption and arrangement of teeth in the alveolar bone in balance with oral functions.¹ Centripetal forces are exerted on the dental arches by the perioral muscles and centrifugal forces by the tongue. These structures exert slight pressures at rest for prolonged periods of time and are therefore fundamental in determining the dental position. Alteration in these forces influence the orientation of the skeletal structures thus resulting in malocclusion.² The development of the masticatory system is mostly determined by hereditary and environmental factors. In this context the activity of masticatory and perioral muscles is determinant not only in the setting of malocclusion but also in the definition of the treatment plan and in the long-term retention phase.³ During childhood the highest risk to develop malocclusion is linked to alterations in the normal pattern of oral functions (atypical swallowing, oral breathing, bad oral habits) with a consequent modification of the skeletal relations between the two jaws. In this period orthopedic and myofunctional treatment are pursued to restore a correct balance in the jaw’s growth.⁴ In adolescence patients presenting skeletal class II malocclusion (most frequent malocclusion in Europe) are mostly treated by using functional appliance to correct the discrepancy between the upper and lower jaw (increased overjet). To do so the removable appliance stress out the soft tissue and muscles fibers to allow a forward repositioning of the mandible.⁵ In adults’ patients the lack of residual growth affects the treatment toward a surgical procedure or a camouflage treatment. Even in this period, muscles activity represents a determinant element in the treatment planning and in the long-term stability.⁶

Key Words: Orthodontics, malocclusion, muscles activity, myofunctional therapy

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Dental rehabilitation from a muscular point of view

Elena P Ivanova ¹, Riccardo Rosati ^2,^✉

Abstract

Dental occlusion changes may be needed for several reasons. Osseous and dental static and dynamic relationships alterations often result from severe tooth wear, crowding, loss of masticatory units and oncologic surgical resections that require reconstruction of dental and/or skeletal occlusal relationships. Any dental work that changes the occlusal surfaces and/or the tooth position could potentially modify oral proprioception. Kogawa., et al.¹ demonstrate how the interdental perception threshold in healthy subjects is less than two hundredths of a millimeter. Neuromuscular system could be forced to develop functional adaptations to dental treatments involving occlusal surfaces that change oral proprioception. It has been clearly demonstrated how dental afferents play a role in the masticatory muscles recruitment. Occlusal modifications could change masticatory muscles global contraction intensity but also their functional relationship.² Moreover muscle imbalances may originate from several factors including interference in the working or balancing side and loss of posterior vertical support.³ Indeed, it has been demonstrated how proprioceptive dental alterations due to iatrogenic occlusal disturbances (200 μm thick) may require also neck muscles functional adaptations.⁴ The relationship between abnormal muscle recruitment and symptoms such as pain or signs of dysfunction like movement limitation does not appear linear, underlining that a great number of subjects has a wide functional adaptability.⁵ The absence of clinical symptoms (mainly pain) following an occlusal intervention does not correspond directly to a procedure free from imperfections and/or anomalies. The nervous tissue can adapt the muscles rectruitment to new oral conditions (without causing symptoms such as pain),⁶ “masking” changes in other structures, for example, teeth, bones and joints. Occlusal proprioception requiring (asymptomatic) muscle adaptations could causes changes in the distribution of the occlusal forces causing the following major complications: 1) Functional. The adaptation capability is not constant between individuals (some patients may develop symptoms as a result of altered occlusion) and over time; 2) Biological. Mechanical forces play a role on bone biology; the bone apposition and resorption mechanisms are managed by chemical processes initiated by mechanical stimuli. It has been demonstrated how the bone modifies in order to support functional loading needs; 3) Mechanical. The prosthetic (crowns and implants) reliability is the result of the stress developed in artificial products. In this context the use of masticatory function instrumental evaluations (before and after therapies that modify dental occlusion) are suggested to support the clinician to quantify the effect of occlusal changes on the oral biology. In fact, the biomechanical studies show that each muscle has its own specific action vectors (or more than one) and that changes in the muscle forces distribution cause alterations of the mechanical stresses on the hard structures.⁷ In conclusion, a reliable dental treatment include masticatory functional evaluation to ensure greater reliability and durability of homogeneously stressed oral rehabilitations.

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Back muscles training and balance therapy in rehabilitation of patients with osteoporotic vertebral fractures

Ekaterina V Makarova ^1,^✉, Larisa A Marchenkova ², Mikhail A Eryomushkin ³, Elena M Styazkina ³, Ekaterina I Chesnikova ³

Abstract

Medical rehabilitation of patients with osteoporotic vertebral fractures (VF) remains an insufficiently developed topic and requires additional research. Aim of the study was to assess the efficiency of back muscles training and balance therapy in rehabilitation of patients with osteoporotic vertebral fractures. Prospective, interventional, open-label, controlled study in two parallel groups, performed in inpatient department settings at "National Medical Research Center of Rehabilitation and Balneology" during 2018. The study involved 120 patients (11 men and 109 women) aged 40-80 (mean age 65.4±9.1 years) who were admitted for medical rehabilitation for systemic OP and VF. The rehabilitation program in the main group included: 1) Mechanotherapy on the Back-Therapy-Center Dr. Wolf complex with biofeedback (Germany); 2) Balance therapy on a double unstable COBS platform, with biofeedback (Germany); 3) Hydrokinesiotherapy in a pool; 4) Gymnastic exercises (Gorinevskaya-Dreving method). Results. The use of the three-week program of physical rehabilitation using mechanotherapy, balance therapy and special complexes of physiotherapy exercises in the gym and in the pool in patients with osteoporotic VF significantly increases the strength of the muscle corset, helps to eliminate the existing muscle deficit in TE and TF and results in a more physiological distribution of the strength ratio between TE and TF. The rehabilitation program improves the function of static and dynamic balance, both with closed and open eyes, which can be observed in the return of the center of gravity to a physiological position and in improved reaction speed to changes in body position. Usage of mechanotherapeutic methods in rehabilitation of patients with osteoporotic VF is effective for basic motor function improvement and disability reduction.

Key Words: Rehabilitation, balance therapy, osteoporosis, active aging, physical therapy, muscle strength

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION XV: Fascia lata alterations in Hip Osteoarthritis

Ilaria Fantoni ¹, Carmelo Pirri ², Carlo Biz ¹, Caterina Fede ², Raffaele De Caro ², Carla Stecco ^2,^✉

Abstract

Osteoarthritis (OA) is a degenerative pathology of the joint,¹ characterized by progressive damage to articular cartilage, which currently represents an important cause of disability,² with an estimated prevalence ranging from 1 to 10% in the general population.^3,4 Despite several mechanisms having been proposed to explain its onset and progression, the aetiopathogenesis of osteoarthritis is still doubtful. This study hypothesizes an association between fascia lata alterations and hip OA; in particular, changes in the production of collagen and HA could result in altered fascial structure and behaviour in OA patients. The structure and composition of fascia lata in healthy subjects and in patients with hip OA were compared with the aim to evaluate any differences in the amount of Collagen type I (COL I), Collagen type III (COL III), and Hyaluronan (HA). Ten samples from healthy subjects and 11 samples from OA patients were collected. COL I was significantly more abundant in the OA group (p = 0.0015), with a median percentage positivity of 75.2 % (IQR 13.11), while representing only 67 % (IQR: 8.71) in control cases. COL III, with median values of 9.5 % (IQR 3.63) (OA group) and 17.10 % (IQR 11) (control cases), respectively, showed significant reduction in OA patients (p = 0.002). HA showed a median value of 10.01 % (IQR 8.11) in OA patients, denoting significant decrease (p < 0.0001) with respect to the control group median of 39.31 % (IQR 5.62). The observed differences suggest a relationship between fascial pathology and hip OA: the increase in COL I in OA patients, along with the reduction of COL III and HA, could lead to fascial stiffening, which could alter fascial mechanics and be linked to the development and symptoms of OA.

Key Words: Fascia, hip osteoarthritis, hyaluronan, collagen, stiffness, myofascial pain

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Connections between hyaluronan properties and fascial health

Caterina Fede ^1,^✉, Ilaria Fortunati ², Carmelo Pirri ¹, Lucia Petrelli ¹, Camilla Ferrante ², Raffaele De Caro ¹, Carla Stecco ¹

Abstract

Hyaluronan (HA) is the most abundant polysaccharide of the extracellular matrix of connective tissues and the key element that underlies the fascial gliding, lubricating the loose connective tissue between the densely packed collagen layers, between the deep fascia and muscle, and within the muscle itself¹. It plays also an important role in cell proliferation and mobility, inflammation and angiogenesis, and it is involved in various diseases such as cancer and diabetes². The mean amount of HA in muscular deep fasciae changes according to the various gliding functions to the anatomical site: we found about 43 μg/g of HA in aponeurotic fasciae, which are free to glide over the muscles; there was a drastic decrease (about 6 μg/g) in epimysial fasciae, completely adherent to the underlying muscles, and an increase in the retinacula surrounding joints (90.4 μg/g)³. Many physical, mechanical, hormonal and pharmacological factors can influence the production of the various fibrous and glycosaminoglycans components of the fascial extracellular matrix: we found that human fascial fibroblasts are able to produce HA-rich vesicles in vitro within a few hours of cannabinoid CB2 receptor agonist treatment, leading to greater tissue fluidity⁴. In the same way, extracorporeal shock-waves also produce a change in the extracellular matrix and an increase in the HA production, with a reduction in tissue stiffness. Furthermore, viscosity of HA is increased by changes in body temperature (cold) and immobilization. These data can have many clinical implications. For example, an increase in temperature, even of only 2 degrees centigrade, caused by a thermal care, sauna or a deep massage, brings a progressive break-up of the three-dimensional superstructure of HA chains, with a consequent decrease in viscosity. At the contrary, cold environment and immobility can increase viscosity of the loose connective tissue, leading to less flexibility of the fasciae and more friction among muscle bundles. Understanding the physicochemical characteristics of HA can help to understand which are the behaviors and the targeted treatments for a healthy fascia.

Key Words: Hyaluronan, fascia, viscosity, gliding, stiffness

References

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2.Fede C, Pirri C, Fan C, Petrelli L, Guidolin D, De Caro R, Stecco C. A Closer Look at the Cellular and Molecular Components of the Deep/Muscular Fasciae. Int J Mol Sci. 2021. Jan 30;22(3):1411. doi: 10.3390/ijms22031411 [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Rehabilitation of peripheral nerve disorders by the use of physical agents. A multiperspective literature evaluation

Daniele Coraci ^1,^✉, Stefano Masiero ¹

Abstract

A large number of diseases can affect the peripheral nervous system, with different etiologies and different relative needed managements. The proper diagnosis is necessary for the correct indication of treatment bases on drugs and surgery. Besides these, rehabilitation plays a fundamental role to get the amelioration of functions and quality of life. Among the several approaches, physical agents represent an effective group of conservative methods, which may positively influence nerve recovery. These agents are based on different types of energies (mechanical, electrical, magnetic, thermal) with peculiar indications and targets aimed at pain relief, tissue restoration, neuromuscular stimulation, anti-inflammatory action. We performed a multiperspective literature evaluation to present the state of art about the use of physical agents in the rehabilitation of nerve injuries. We collected data about the major studied diseases and the relative effects, the most common outcome measures, the most involved countries in the world about this topic and the modality of the description of the physical agents. We searched in PubMed the terms “physical agents rehabilitation AND peripheral nerv*”, finding 131 papers. Our literature review showed the potential benefits of the physical agents even if further studies should be needed for confirmation.

Key Words: Rehabilitation, peripheral nerve, physical agents, literature review

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Extracororeal Shock Wave Therapy (ESWT) in muscular pathologies

Lucrezia Tognolo ^1,^✉

Abstract

Extracorporeal Shock Wave Therapy (ESWT) is considered part of the Regenerative Therapy, that represents one of the most challenging branches of rehabilitation field and, more generally, in modern medicine. Basic science studies demonstrated the effectiveness of the ESWT in stimulating biological activities, by the mechanotransduction of the acoustic signal into a biological response, that involves both intra-cell and cell–matrix interactions. According to the Consensus Statement elaborated by ISMST (International Society for Medical Shockwave Treatment) in 2016 and subscribed by SITOD (Italian Society for Shockwave Therapy), some muscular pathologies are among the indications based on clinical experience. Indeed, despite the few studies in the literature, ESWs are widely used in clinical practice in many acquired muscle diseases. The rationale for the use of this treatment is related to its well-known anti-inflammatory, analgesic, anti- fibrotic and myorelaxant action. Moreover, ESWT could act on Muscular Satellite Cells, myogenic progenitors, by stimulating them to proliferate, and eventually differentiate through fusion with each other or to damaged fibers to reconstitute fiber integrity and functionality. This hypothesized mechanism may play an important role in helping muscle regeneration, if ESWT is properly inserted in a multimodal rehabilitative approach including therapeutic exercise. However, there is still a lot of confusion about the different types of shock waves (focal, radial), the different generators (electrohydraulic, electromagnetic, piezoelectric), the parameters and treatment protocols. Therefore, this presentation aims to provide an update on the efficacy of extracorporeal shockwave treatment for muscular acquired pathologies by focusing on the evidence.

Key Words: Extracorporeal shock wave therapy (ESWT), muscle, evidence, regenerative medicine

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Can home-based rehabilitation be effective to counteract skeletal muscle atrophy and to ameliorate physical functioning of elderly patients?

Maria Chiara Maccarone ^1,^✉, Ugo Carraro ², Stefano Masiero ³

Abstract

Elderly subjects with musculoskeletal and neurological diseases spend less time training on a regular basis, worsening their mobility concerns and undergoing muscular atrophy. As a result of the pandemic, access to regular physical activity has reduced even in healthy older individuals, due to mobility restrictions, quarantine, and lockdown. It might be beneficial to provide all these individuals, both affected by chronic disorders and healthy, with rehabilitation program to be admistered at home or even in bed (Full-Body In-Bed Gym). Home-based exercise programs are increasingly being employed in order to improve physical functioning of elderly patients with orthopedic (e.g., hip and knee osteoarthritis) and neurological disorders (e.g., Parkinson’s disease, spinal cord injury, etc.). Through a comprehensive review of the literature, we investigated what is the evidence of the efficacy of home-based exercise programs for different subjects and what are the most appropriate modalities to perform these programs. The evidence in literature seems to be promising: home-based treatment seems to be effective, safe and usable by elderly patients suffering from different diseases as well as healthy ones. In the future, home-based exercise programs may represent a valuable option to counteract muscle atrophy and reduced motor function in the elderly, taking also advantage of modern devices such as those employed in telerehabilitation.

Key Words: Full-Body In-Bed Gym, Telerehabilitation, Muscle atrophy

References

Gava P, Kern H, Carraro U. Age-associated power decline from running, jumping, and throwing male masters world records. Exp Aging Res. 2015;41(2):115-35. doi: 10.1080/0361073X.2015.1001648. [DOI] [PubMed] [Google Scholar]
Carraro U, Marcante A, Ravara B, Albertin G, Maccarone MC, Piccione F, Kern H, Masiero S. Skeletal muscle weakness in older adults home-restricted due to COVID-19 pandemic: a role for full-body in-bed gym and functional electrical stimulation. Aging Clin Exp Res. 2021. Jul;33(7):2053-2059. doi: 10.1007/s40520-021-01885-0. Epub 2021. May 28 [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Early rehabilitation of ischaemic stroke with medicinal acupuncture: A clinical study

Olga M Oleynikova ¹, Oganes L Badalyan ¹, Georgiy G Avakyan ¹, Kirill V Terentev ^2,^✉, Natalia P Sanina ²

Abstract

Purpose of this study was to assess the effect of a course of homeopathic preparations using acupuncture points on the severity of motor impairment against the background of early rehabilitation of the ischaemic stroke (IS). More than 60 patients with different localisation of ischaemic stroke (vertebro-basilar and carotid territories) were treated. All the patients were treated according to the standards of medical care for ischaemic stroke. The degree of impairment was assessed using National Institutes of Health Stroke Scale (NIHSS), which included impairment of consciousness, degree of paresis (0 - no paresis, 4 - no active movements), sensory disturbance and speech disorders manifested in the form of aphasia and dysarthria. The control group consisted of 10 patients with ischaemic stroke. All the patients underwent a computer-assisted tomography of the brain to rule out the hemorrhagic nature of the stroke. Treatment was given during the acute period of IS within 1 to 3 days after onset. Exclusion criteria: hemorrhagic and mixed strokes, thrombolysis, history of cancer. The basic therapy of IS was carried out according to the standards. HEEL homeopathic medicines with anti-inflammatory, metabolic and antioxidant effects were used, which were administered on the common action points of the Yang meridians of the paretic limbs. The control group received acupuncture according to the same scheme on the paretic limbs. The treatment duration was 10 sessions; the observation lasted for 21 days, according to the standards of hospitalization for stroke patients. The degree of functional impairment of patients treated with medicinal acupuncture and the patients of the control group assessed by the National Institutes of Health Stroke Scale (NIHSS), (Fig. 1,2). The outcomes of the medicinal acupuncture treatment: positive effect in the form of a reduction in the degree of paresis (Fig. 3,4) were observed in patients, who received medicinal acupuncture. The lowest treatment outcome was observed in patients with IS in the vertebro-basilar system. Medicinal acupuncture can be used in early rehabilitation for patients with mild and moderate IS, since its possible mechanism is the enhancement of neuroplasticity in the conditions of brain damage. The use of homeopathic medicines can increase the positive effect compared with acupuncture. The most promising “candidates” for this treatment are the patients with IS in the carotid territory.

Key Words: ischaemic stroke, medicinal acupuncture

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Can aqua exercises in fresh water improve the gait stereotype function in patients with a neurological disease?

Irina A Grishechkina ^1,^✉, Andrey A Lobanov ¹, Sergei V ¹, Andronov ¹, Gleb N Barashkov ¹, Andrey I Popov ¹, Elena P Ivanova ¹

Abstract

An open descriptive study was carried out, which involved 12 patients (7 men, 5 women) aged between 40 and 62 years, with disorders of maintaining the vertical posture, which correspond to the functional diagnosis coded according to ICF “Gait Stereotype Function” b770.1 – minor disorders (5–24%). The test group (n = 12), in addition to basic therapy and exercises with a physiotherapy instructor, did aqua exercises in fresh water for two weeks (30 minutes, 6 days a week). Evaluation technique: the study before and after the rehabilitation course was carried out using “Habilect” video analysis system. To assess the significance of differences between the groups of qualitative variables χ2 test was used. To assess the significance of differences of quantitative variables of the two test groups Wilcoxon rank sum test was used. The significance of differences is considered to be established at p<0, 05. The study calculated the amplitudes of deviations of the body and the head along Х, Y, Z axes before and after the exercises, as well as deviations of the body motion vector before and after the exercises. While calculating the increase of deviation (deviations of the main body axes from the reference value) using Wilcoxon rank sum test, statistically significant deviations along the X-axis were found (an increase by 306,5%, р=0,0504), along the Z-axis (an increase by 112,68%, р=0,0225) and the Body Angle parameter (an increase by 1973,86% р=0,0323). While calculating the increase of deviations of the head axis from the reference value using Wilcoxon rank sum test, statistically significant deviations along the X-axis were found (an increase by 163,04%, р=0,0280), along the Y-axis (an increase by 85,71%, р=0,0199) and Z-parameter (an increase by 173,34% р=0,0292) Thus, aqua exercises, due to a decrease of the deviations amplitude along the three axes (Z, Y, X), contribute to the adjustment of disorders of maintaining the vertical posture, a statistically significant decrease in the amplitude of head and neck movements.

Key Words: Movement, exercise therapy, gait, posture

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Trauma of peripheral innervation impairs content of epidermal Langerhans cells and skin immunoreactions

George Aurelian Toader et al

Eur J Transl Myol. 2022 Mar 10;32(1):10440.

SESSION XV: Sanatorium and spa resort treatment of patients who have recovered from COVID-19

Anatoly D Fesyun ^1,^✉, Maxim Yu Yakovlev ^1,²

Abstract

In 2019, a new coronavirus infection, subsequently identified as a pandemic, broke out around the world. On 11 February, 2020, the World Health Organization gave the infection caused by the new coronavirus an official name - COVID-19. On 11 February, 2020, the International Committee on Taxonomy of Viruses (ICTV) gave the infectious agent its official name - SARS-CoV-2. In its turn, a comprehensive rehabilitation treatment combined with open-air stays, terrainkur (terrain therapy), diet therapy and balneotherapy has demonstrated its effectiveness for a multitude of diseases and pathological conditions.¹ There is no reason to doubt that for COVID-19 survivors, too, the possibilities of recovery in sanatorium and spa resort environment will be successfully realized. Although the effects of the SARS-CoV-2 virus on the human organism are, in a sense, unique, the pathological processes that occur undergo the "traditional" stages of regression. The procedures offered by sanatoriums for the treatment of diseases of the respiratory, nervous and cardiovascular systems usually have broad indications and can be offered for recovery to patients who have had COVID-19. The provision of spa treatment and rehabilitation activities is crucial for the effective completion of the recovery process and rehabilitation after COVID-19. Rehabilitation in a sanatorium and spa resort environment enables a comprehensive and effective recovery process for the patient, and for the healthcare system it is an opportunity to relieve the burden on specialised in-patient rehabilitation facilities and polyclinic structures. A study of pathogenesis of pathological processes in COVID-19, morpho-functional and metabolic changes shows that the regression of multi-organ lesions caused by SARS-CoV-2: fibrosis and granulation changes in lungs, respiratory failure, extensive ischemia and micro-thrombosis, myocardial and brain lesions, will require a long period for recovery.^2,3 However, it is possible to optimize this process through sanatorium and spa resort treatment. When the patient arrives at the sanatorium-and-spa institution, his or her condition should be stabilized. The main objectives of spa treatment are to provide general strengthening therapy, symptomatic treatment, prevention of disability and improvement of the quality of life. When organizing sanatorium and spa treatment for patients who have had COVID-19, there are two main components: 1. A spa location characterized by natural healing factors; 2. Areas and methods of sanatorium and spa treatment, in which a nutritional therapy plays an essential role. Particularly, in the case of rehabilitation of patients after a severe and extremely severe course of the disease, who have a weight deficit. In conclusion it should be noted that the sanatorium and spa resort treatment of patients after COVID-19 is defined by the following main areas and methods used for the recovery of this category of patients: 1. Nutritional therapy; 2. Active lifestyle; 3. Natural healing factors; 4. Physiotherapy; 5. Terrainkur; 6. Massage; 7. Aerosol therapy; 8. Photohaemotherapy; 9. Electro-neurostimulation of the lymphatic drainage; 10. Low-frequency magnetic therapy; 11. SMC therapy; 12. Interference therapy; 13. Pulsed low frequency electrostatic field or electrostatic massage; 14. Decimetre-wave band electromagnetic radiation; 15. Vibrotherapy; 16. Laser therapy; 17. Oxygen-barotherapy; 18. Ultrasonic therapy; 19. General magnetotherapy; 20. Carbon dioxide baths; 21. Transcranial electrotherapy; 22. Psychological correction and Psychotherapy; 23. Biofeedback (biofeedback therapy).⁴ All of the above-mentioned methods of treatment are effectively used both individually and as part of comprehensive sanatorium and spa resort treatment and medical rehabilitation programmes.

Key Words: Coronavirus infection, rehabilitation treatment, sanatorium and spa resort

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Focal Muscle Vibration and Action Observation: a novel approach for muscle strengthening

Stefania Dalise ¹, Valentina Azzollini ^2,^✉, Noemi Fragapane ², Vincenzo Spina ¹, Carmelo Chisari ²

Abstract

Focal vibration (FV), when applied with specific intensities and frequencies on belly muscle, can improve muscle strengthening inducing peripheral and, in the long term, central modifications. Therefore, this approach is useful when subjects need a strengthening treatment after o during prolonged immobility or disabling diseases that prevent them from performing traditional protocols. Similarly, action observation (AO) and "motor imagery" techniques are used to intensify the effect of rehabilitation on muscle strength and to reduce recovery time. The synergy effect of the two approaches was not demonstrated. The aim of this study is to assess the effectiveness of the integration of a strengthening protocol based on FV associated with an AO paradigm. The study involved healthy subjects, who were randomly assigned to a specific treatment: a group underwent 100 Hz FV treatment on the right femoral quadriceps and a second group underwent the same protocol associated with an AO paradigm of the muscle training (AO+FV) for two weeks, five days/week. Before (T0), after the first week (T1), at the end of treatment (T2), and after a week from the end (T3) for the follow-up, we assessed the maximum voluntary contraction (MVC) expressed at knee extension bilaterally with dynamometer and at T0, T2 and T3 we assessed changes in cortical excitability by transcranial magnetic stimulation (Resting Motor Threshold - RMT; Silent Period - SP). Preliminary data seems to show that MVC increases bilaterally in both groups at T1 and T2. A major increase is evident in the AO+FV group, this increase is maintained even at T3. RTM is reduced at T3 in AO + FV. The SP is reduced only in group AO + FV at T2 and T3. In conclusion, AO seems to be able to enhance the effect of muscle reinforcement induced by FV on maximum strength. This effect seems to be mediated by peripheral and central modifications. More evidence is needed to support treatment effectiveness, which could be a valuable addition to existing protocols as well as an alternative treatment when the subject is not able to perform traditional reinforcement protocols.

Key Words: Parkinson’s Disease, postural deviation, Pisa Syndrome, trunk dystonia

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

A combined treatment protocol for postural instability in Pisa Syndrome

Martina Cafalli ¹, Elisa Doro ¹, Fulvio Vercillo ¹, Stefania Dalise ², Carmelo Chisari ^1,^✉

Abstract

Postural disorders such as Pisa Syndrome (PS) are frequently observed in Parkinson’s Disease (PD). PS is characterized by postural instability that affects both balance and gait pattern, leading to progressive loss of autonomy, increase in fall risk and deterioration of quality of life. To date, no consensus has been reached on its pathogenesis, nor on the definition of a specific standard therapeutic protocol. Taking into consideration a central etiopathogenetic hypothesis, characterized by dystonic activation of trunk muscles, in particular the external oblique muscle (EOM), we evaluated the efficacy of an intensive rehabilitation training combined with botulinum toxin type A (BoNT-A) treatment. Patients with PD and PS, defined as a reversible lateral trunk flexion >10°, were selected. All patients were on chronic antiparkinsonian therapy with dopaminergic drugs, stable on their daily during the 4 weeks previous the study. Exclusion criteria were presence of other neurological diseases, clinically significant psychiatric disturbances, and orthopedic spine abnormalities. Before treatment (T0), all patients underwent the following evaluations:- 3D Gait Analysis (GA); - Needle EMG of the EOM: if hyperactivation was detected, concomitant BoNT-A inoculation was Performed - Berg Balance Scale (BBS) (Bronstein, 2013); - Clinical-Functional Scales: Walking Test (10mWT, TUG, 6MWT), Berg Balance Scale (BBS), Parkinson Disease Questionnaire (PDQ-8), Fall Risk scale (FRS), Falls Efficacy Scale (FES-I). Numerical Rating Scale (NRS); - Truncal flexion angle (TFA) measurement with “Image J” system. One week after T0, patients underwent a three-week specific high intensity rehabilitation program (trunk muscles stretching, balance and postural control) structured in 5 session per week. At the end of treatment (T1) initial evaluations were repeated. Nine patients were enrolled and completed the protocol (age 71± 4,1 y; M: F=5:4). The comparison of the T0 and T1 data displayed a significant reduction of TFA (T0= 11,45 ± 4,74; T1= 7,71 ± 6,21; p= 0,027), NRS (T0= 6 ± 1,8; T1= 1,77 ± 1,48; P= 0,007) and BBS (T0= 48,88 ±4,85; T1= 52,11 ± 3,29; p= 0,018). 3D Gait. Analysis showed an improvement in the time-space parameters after the treatment, specifically in stride duration (T0= 1,36 ± 0,21; T1 = 1,25 ± 0,20; p= 0,03) and cadence (T0= 90,18 ± 14,90; T1= 97,66 ± 15,15; p= 0,05). In conclusion, the results seem to confirm the importance of EOM in PS. Our protocol was well tolerated and showed to be successful in reducing the postural abnormality and posture related pain, as suggested by the significative variation of TFA and the major improvement seen in clinical-functional scales .

Key Words: Fascia, hip osteoarthritis, hyaluronan, collagen, stiffness, myofascial pain

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Improvement of the expression of upper limb muscle strength and balance in a group of patients with multiple sclerosis, through a proprioceptive rehabilitation protocol combined with the application of photon emission devices

Patrizia Proia ^1,^2,^✉, Federica Rizzo ^1,², Alessandra Amato ^1,², Francesco Fischetti ³, Diego Genua ^1,², Vincenzo Francavilla ⁴, Angelo Iovane ¹, Giuseppe Messina ^1,²

Abstract

In multiple sclerosis (MS) patients, symptoms such as: fatigue, lack of physical energy, spasticity, difficulty in performing movements, and motor coordination disorders, and with tremors, dizziness and postural instability are among the most common complications. Cattaneo et al. (2007) studied the effects of stability training on MS patients and described it as an effective intervention in reducing falls risk, improving stability, and strength. Therefore, the present study aimed to confirm early observations by the application of a proprioceptive rehabilitation program targeting balance and strength on MS patients. 13 subjects with MS, 5 male and 8 female, volunteered in the study. A MAP hand dynamometer was used to determine the handgrip strength, as the maximum isometric grip (MIG), recognized as an important health indicator for determining musculoskeletal function, as well as weakness and disability. Sensor Medica® systems allowed us to carry out baropodometric and stabilometric measurements using the associated program (Freestep® by Sensor Medica®). The device consists in a pressure platform with resistive sensors with conductive rubber. The rehabilitation protocol included: A) 10 minutes of Motomed; B) 10 minutes of Human Tecar proprioceptive path; C) 15 minutes in total of physical exercises; D) 15 minutes of massage therapy of the whole spine All patients wore the photon emission devices TAOPATCH by TAO technologies, according to a protocol used in a previous study (Amato et al., 2021). Testing procedures were carried out before and after the rehabilitation protocol. The paired sample t-test revealed statistically significant improvements for the baropodometric measures (p<0.05) associated with the intervention. In particular, plantar surface pressure, forefoot and hindfoot pressure improved significantly, as well as the maximal and mean foot pressures and the forefoot and hindfoot loads (figure 1a). In addition, the intervention induced a statistically significant improvement in the right (p = 0.023) and left (p = 0.021) handgrip strength (figure 1b). The combination of an adequate and specific rehabilitation protocol combined with the application of the photon emission devices Taopatch, emerged as a valid protocol to improve handgrip strength of the upper limbs and the expression of the body weight on the ground in contrast with the force of gravity. Thus, determining improvements in a cluster of parameters severely decompensated in MS patientsIn multiple sclerosis (MS) patients, symptoms such as: fatigue, lack of physical energy, spasticity, difficulty in performing movements, and motor coordination disorders, and with tremors, dizziness and postural instability are among the most common complications. Cattaneo et al. (2007) studied the effects of stability training on MS patients and described it as an effective intervention in reducing falls risk, improving stability, and strength. Therefore, the present study aimed to confirm early observations by the application of a In multiple sclerosis (MS) patients, symptoms such as: fatigue, lack of physical energy, spasticity, difficulty in performing movements, and motor coordination disorders, and with tremors, dizziness and postural instability are among the most common complications. Cattaneo et al. (2007) studied the effects of stability training on MS patients and described it as an effective intervention in reducing falls risk, improving stability, and strength. Therefore, the present study aimed to confirm early observations by the application of a proprioceptive rehabilitation program targeting balance and strength on MS patients. 13 subjects with MS, 5 male and 8 female, volunteered in the study. A MAP hand dynamometer was used to determine the handgrip strength, as the maximum isometric grip (MIG), recognized as an important health indicator for determining musculoskeletal function, as well as weakness and disability. Sensor Medica® systems allowed us to carry out baropodometric and stabilometric measurements using the associated program (Freestep® by Sensor Medica®). The device consists in a pressure platform with resistive sensors with conductive rubber. The rehabilitation protocol included: A) 10 minutes of Motomed; B) 10 minutes of Human Tecar proprioceptive path; C) 15 minutes in total of physical exercises; D) 15 minutes of massage therapy of the whole spine All patients wore the photon emission devices TAOPATCH by TAO technologies, according to a protocol used in a previous study (Amato et al., 2021). Testing procedures were carried out before and after the rehabilitation protocol. The paired sample t-test revealed statistically significant improvements for the baropodometric measures (p<0.05) associated with the intervention. In particular, plantar surface pressure, forefoot and hindfoot pressure improved significantly, as well as the maximal and mean foot pressures and the forefoot and hindfoot loads (figure 1a). In addition, the intervention induced a statistically significant improvement in the right (p = 0.023) and left (p = 0.021) handgrip strength (figure 1b). The combination of an adequate and specific rehabilitation protocol combined with the application of the photon emission devices Taopatch, emerged as a valid protocol to improve handgrip strength of the upper limbs and the expression of the body weight on the ground in contrast with the force of gravity. Thus, determining improvements in a cluster of parameters severely decompensated in MS patientsproprioceptive rehabilitation program targeting balance and strength on MS patients. 13 subjects with MS, 5 male and 8 female, volunteered in the study. A MAP hand dynamometer was used to determine the handgrip strength, as the maximum isometric grip (MIG), recognized as an important health indicator for determining musculoskeletal function, as well as weakness and disability. Sensor Medica® systems allowed us to carry out baropodometric and stabilometric measurements using the associated program (Freestep® by Sensor Medica®). The device consists in a pressure platform with resistive sensors with conductive rubber. The rehabilitation protocol included: A) 10 minutes of Motomed; B) 10 minutes of Human Tecar proprioceptive path; C) 15 minutes in total of physical exercises; D) 15 minutes of massage therapy of the whole spine. All patients wore the photon emission devices TAOPATCH by TAO technologies, according to a protocol used in a previous study (Amato et al., 2021). Testing procedures were carried out before and after the rehabilitation protocol. The paired sample t-test revealed statistically significant improvements for the baropodometric measures (p<0.05) associated with the intervention. In particular, plantar surface pressure, forefoot and hindfoot pressure improved significantly, as well as the maximal and mean foot pressures and the forefoot and hindfoot loads (figure 1a). In addition, the intervention induced a statistically significant improvement in the right (p = 0.023) and left (p = 0.021) handgrip strength (figure 1b). The combination of an adequate and specific rehabilitation protocol combined with the application of the photon emission devices Taopatch, emerged as a valid protocol to improve handgrip strength of the upper limbs and the expression of the body weight on the ground in contrast with the force of gravity. Thus, determining improvements in a cluster of parameters severely decompensated in MS patients.

Key Words: Balance, multiple sclerosis, neuromuscular exercise, muscular strength

References

Halabchi F, Alizadeh Z, Sahraian MA, Abolhasani M. Exercise prescription for patients with multiple sclerosis; potential benefits and practical recommendations. BMC Neurol. 2017. Sep 16;17(1):185. doi: 10.1186/s12883-017-0960-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

Mental health disorders of relatives of oncohematological patients

Igor Reverchuk ^1,^✉, Dmitriy Miklin ¹, Konstantin Yazykov ¹

Abstract

The aim of the study is to study the features of the mental health of the closest relatives of oncohematological patients. The sample consisted of 274 subjects aged 18 to 65 years. The experimental groups were patients and their closest relatives at different periods of the course of the disease: at the onset, at three and six months. The number of people in the sample under consideration progressively decreased due to the mortality rate of patients with the underlying disease (92 at the onset of the disease to 70 people on each side at the end of the study). The following research methods were used: clinical and psychological (a conversation aimed at collecting psychological anamnesis and identifying biosocial characteristics of patients, assessment of the current mental status, assessment of the severity of the condition-the Karnovsky index); testing (methods SF-36, MFA-20, 4DSQ, coping behavior in stressful situations by Norman S.); instrumental studies (assessment of the Kerdo vegetative index); mathematical and statistical. The co-dependent relationships and the influence of cancer patients and their close relatives on the mental health of the latter during the six months of the development of hemoblastosis were studied. First identified predictors of mental health of the next of kin hematooncology at different stages of the disease of acute leukemia. New data on the structure and dynamics of the influence of various factors on the mental health of relatives of oncohematological patients through latent variables were obtained. The variants of disadaptation of the closest relatives of oncohematological patients during the first six months of the disease course are indicated. For the first time, a comprehensive approach to the problem of providing medical and psychological care is justified, including the mental health of the closest relatives of oncohematological patients in relation to the psychoemotional status of patients with hemoblastoses. The mechanisms of functioning of relatives of oncohematological patients, which have an impact on their mental health, are revealed, which is of differential importance in correctional and psychotherapeutic practice.

Key Words: Relatives of cancer patients, distress, coning strategies, mental health, anxiety, depression, codependent relationships, quality of life, oncology, psychotherapy

References

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Eur J Transl Myol. 2022 Mar 10;32(1):10440.

The state of helplessness in preschool children with mental retardation and its correction

Anna Zavertyaeva ¹, Igor Reverchuk ^1,^✉

Abstract

The presentation deals with the problem of helplessness in preschool children with mental retardation (ANW). Became widely known studies of so-called "learned helplessness" Seligmena, in the national psychology studies "of personal helplessness" Ciring, etc., but work in assessing the role and place of helplessness (just helplessness, learned, personal) in preschool children in general and delayed mental development in particular is virtually non-existent. At the base of the research based on the ideas of Vygotsky about the features of development of children, as well as whole-value approach and the biopsychosocionoetic model of human nature, its health and development, proposed by one of the authors of the article. The authors put forward a hypothesis to test done quite voluminous study of the phenomenon of helplessness, which brought together 140 children and their families (mothers). Of these, 90 children with CRA and 50 without such a diagnosis. 45 children with CRA entered the pilot group, which on the second stage of the study, participated in the psychocorrective program proposed by the authors of the study. The monitoring group comprised 45 children with CRA and 50 children without the CRA. Based on the quantitative and qualitative analyses of the results obtained was differentially 3 groups of children with varying degrees of severity, the condition of helplessness: no signs of the condition of helplessness, with moderately expressed signs of helplessness and the lack thereof. The results of the second cut after the implementation of the program of psychocorrective indicate the possibility, under certain conditions, absorb any movement state of helplessness.

Key Words: Helplessness, learned helplessness, personal helplessness, children of pre-school age, development, techniques, attitude, family, parents, psychological support

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PERMALINK

The 2022 On-site Padua Days on Muscle and Mobility Medicine hosts the University of Florida Institute of Myology and the Wellstone Center, March 30 - April 3, 2022 at the University of Padua and Thermae of Euganean Hills, Padua, Italy: The collection of abstracts

H Lee Sweeney

Stefano Masiero

Ugo Carraro

Abstract

Ethical Publication Statement

Figure 1.

Acknowledgments

List of acronyms

Funding Statement

Contributor Information

References

LECTURE: The crucial role of force transmission in muscle function: Focus on cytoskeleton and myotendinous junction Part A: Cytoskeletal specialization and mechanoprotection in fast and slow skeletal muscles. Insights from single-fiber proteomics and comparison with cardiac muscle

Stefano Schiaffino

Leonardo Nogara

Jane-Lise Samuel

Martina Baraldo

Bert Blaauw

Carlo Reggiani

Marta Murgia

Abstract

References

Part B: The myotendinous junction: the weakest link of the muscle-skeleton force transmission chain?

Abigail L Mackey

Abstract

Figure 1.

References

SESSION I: Mechanisms of muscle atrophy: from UPS implication in rodent models to human biomarkers

Daniel Taillandier

Dulce Peris Moreno

Cécile Polge

Abstract

References

A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes

Pascal Maire

Novel players in muscle mass regulation

Marco Sandri

Abstract

Loss of MuRF1 prevents skeletal muscle wasting and weakness, and slows the rate of tumor growth, in mice bearing pancreatic tumors

Daria Nereid

Orlando Latino

Christopher Lopez

David W Hammers

H Lee Sweeney

Glenn A Walter

Sarah M Judge

Andrew R Judge

Abstract

Figure 1.

References

Insights into the mechanisms of muscle wasting at high age

Daniel J Ham

Anastasiya Börsch

Giulia Milan

Nitish Mittal

Mihaela Zavolan

Markus A Ruegg

Abstract

Molecular mechanisms of cancer-induced muscle wasting

Andrew R Judge

Andrew C D’Lugo s

Jose G Trevino

Sarah M Judge

Abstract

Figure 1.

References

Neuromuscular basis of disuse muscle atrophy and weakness

Marco Narici

Elena Monti

Martino Franchi

Fabio Sarto

Sandra Zampieri

Carlo Reggiani

Abstract

Figure 1.

Acknowledgements

References

Activation of Akt-mTORC1 signaling reverts cancer-dependent muscle wasting

Alessia Geremia