Molecular signaling effects of electrical stimulation of the soleus muscle during 6 days of dry immersion

Kristina A Sharlo; Irina D Lvova; Natalya A Vilchinskaya; Sergey A Tyganov; Olga V Turtikova; Ksenia V Sergeeva; Roman O Bokov; Boris S Shenkman; Elena S Tomilovskaya; Oleg I Orlov

doi:10.1016/j.smhs.2025.06.002

. 2025 Jun 11;8(3):335–349. doi: 10.1016/j.smhs.2025.06.002

Molecular signaling effects of electrical stimulation of the soleus muscle during 6 days of dry immersion

Kristina A Sharlo ^1,^⁎, Irina D Lvova ¹, Natalya A Vilchinskaya ¹, Sergey A Tyganov ¹, Olga V Turtikova ¹, Ksenia V Sergeeva ¹, Roman O Bokov ¹, Boris S Shenkman ¹, Elena S Tomilovskaya ¹, Oleg I Orlov ¹

PMCID: PMC13159456 PMID: 42125185

Abstract

Background

Unloading of skeletal muscles triggers rapid changes in molecular signaling, leading to muscle atrophy and functional alterations. Electrical stimulation of muscles is commonly used to counteract these changes, but the precise molecular mechanisms behind its effects remain unclear.

Methods

To investigate the early changes in postural soleus muscle under unloading conditions (dry immersion, DI) and the impact of electrical stimulation during unloading, two groups of volunteers (10 men in each) underwent a 6-day DI or a 6-day DI with electrical stimulation (DI + ES). Soleus muscle samples were collected 14 days before and 6 days after DI and DI + ES.

Results

Six-day DI did not did not cause atrophy of the soleus myofibers or alter protein synthesis parameters, However, it did lead to an increase in atrogin-1 expression, a downregulation of markers for mitochondrial biogenesis and dynamics, and a decline in the mRNA expression of fast oxidative myosin isoform IIa. It also resulted in the downregulation of microRNAs mir-206 and mir-208b, which support slow fiber types. There was an upregulation of CpG methylation in the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a) promoter region and an accumulation of Ca2+/calmodulin-dependent protein kinase (p-CaMK II), indicating an increase in myoplasmic calcium levels. Electrical stimulation during the 6-day disuse period prevented the disuse-induced decreases in mitochondria-related markers and the content of mir-206 and mir-208b. It also induced a shift in myosin mRNA expression from types IId/x to IIa, counteracted the accumulation of p-CaMK II and CpG methylation in the PGC1a promoter region.

Conclusions

Electrical stimulation upregulated markers of both protein synthesis and proteolysis, as well as resulted in lower cross-sectional area of fast-type fibers compared to pre-DI + ES.

Keywords: Dry immersion, Muscle disuse, Electrical stimulation, Muscle signaling

Abbreviations

4E-BP1: eukaryotic translation initiation factor 4E-binding protein 1
AMPK: AMP-activated protein kinase
ATF: activating transcription factors
BSP: bisulfite specific primers
CaMK II: Ca²⁺/calmodulin-dependent protein kinase
COXI: cytochrome c oxidase subunit I
CpG: stretches of DNA with high guanine and cytosine content
CREB: CAMP responsive element binding protein 1
CSA: cross-sections area
DI: dry immersion
Dnmt: DNA (cytosine-5)-methyltransferase 1
DRP1: dynamin-1-like protein
ES: electrical stimulation
ETC: electron transport chain
Fis1: mitochondrial fission 1 protein
GAPDH: glyceraldehyde 3-phosphate dehydrogenase
HOCOMOCO: HOmo sapiens COmprehensive MOdel Collection
LC3B: microtubule-associated proteins 1A/1B light chain 3B
MEF: myocyte enhancer factor
Mir: microRNA
MSP: methylation-specific PCR
mtDNA: mitochondrial DNA
MuRF-1: muscle ring-finger protein-1
MYH: myosin heavy chain
NFAT: nuclear factor of activated T-cells
NRF2α: nuclear respiratory factor 2
p70s6kinase: ribosomal protein S6 kinase beta-1
PBST: phosphate-buffered saline solution with 0,1 % Tween 20
PGC1a: peroxisome proliferator-activated receptor gamma coactivator 1-alpha
ROS: reactive oxygen species
ROUT: robust regression and outlier removal
RPL13: ribosomal protein L13
RT: reverse transcription
TEAD: TEA domain family member 1
TET1: ten-eleven translocation methylcytosine dioxygenase 1
TFAM: mitochondrial transcription factor A

1. Introduction

Mammalian skeletal muscles can be divided into fast-type muscles, which predominantly control locomotion, and slow-type muscles, which predominantly maintain the position of the body or its parts. In most cases, skeletal muscles contain a mix of both slow-type and fast-type fibers. The predominant type of fibers in a muscle determines its classification (fast, slow or mixed). For example, in humans, the slow-type soleus muscle contains a high percentage (from 80% to 90%) of slow-twitch fibers. This muscle plays a crucial role in maintaining vertical body position, as well as in walking and running.

Slow- and fast-type muscle fibers have different functional properties (strength and speed of contraction), firing patterns of innervating neurons and types of metabolism. Under disuse conditions, both fiber types can undergo atrophy and loss of tone, leading to muscle weakness.¹^,² Slow-type and fast-type muscles respond differently to different types of muscle disuse: while fast-type muscles are vulnerable to the restriction of locomotor activity, slow-type muscles are more sensitive to the lack of support afferentation, leading to inactivation of slow-type fibers.2, 3, 4, 5 Since postural soleus muscle is active 11–15 hours (h) per day,⁶ the inactivation of that muscle caused by the support afferentation withdrawal leads to rapid and profound changes in its metabolism and function.³^,⁷ These changes are preceded by signaling events that occur as early as 24 h after slow-type muscle disuse and include decreases in mRNA expression of markers of mitochondrial biogenesis, shifts in myosin isoform mRNA expression patterns, down-regulation of protein synthesis, and up-regulation of proteolytic enzymes.⁷^,⁸

The best-known condition for the elimination of ground support afferentation is microgravity. After 180 days of spaceflight, human soleus fibers are atrophied by 15%–20%, accompanied by a decrease in fiber maximal force; 17 days of spaceflight result in a decrease in soleus myofiber force.⁹^,¹⁰ A ground-based model called "dry immersion" (DI) was developed in the 1970s to study the effects of microgravity in humans.¹¹ In this model, the subject lies in warm water on a waterproof fabric so that the body is submerged in water up to the level of the neck. The gravitational force acting on the body under water is counterbalanced by the buoyant force, resulting in a lack of support afferentation. The effects of DI on the musculoskeletal system are similar to the effects of real microgravity.⁴

Several countermeasures have been developed to prevent the negative consequences of muscle unloading and lack of support afferentation, such as various types of exercise, plantar mechanical stimulation, centrifugation, and electrical stimulation.12, 13, 14, 15 All of these methods are quite effective, but they also have limitations and depend on the muscle type, the model of muscle disuse, and the duration of the experiment. To overcome the limitations of the methods, it is important to understand which molecular signaling cascades underlie the effects of the countermeasures. In this study, we analyzed the markers of key molecular signaling cascades that regulate proteolysis, protein synthesis, and mitochondrial biogenesis in soleus muscle during 6 days of DI with or without electrical muscle stimulation. Low-frequency stimulation was shown to activate oxidative metabolism and slow-type myosin expression,¹⁶^,¹⁷ while high-frequency electrical stimulation is often used to counteract muscle atrophy.¹⁸ High frequency electrical stimulation was shown to be an effective countermeasure for prevention of muscle atrophy under spinal cord injuries and under various health conditions requiring long-term bed rest.¹⁹ Several studies have shown that high-frequency electrical stimulation prevents atrophy or even hypertrophy of muscles under various conditions of muscle disuse, such as intensive care, knee joint immobilization, and chemotherapy.20, 21, 22, 23 Low-frequency electrical stimulation has been shown to induce hypertrophy of tissue-engineered human skeletal muscle,²⁴ but the most frequently observed effects of low-frequency electrical stimulation include prevention of downregulation of mitochondrial biogenesis and muscle fatigue caused by disuse.²⁵^,²⁶ In the present study, we used combined low-intensity, low-frequency and high-frequency sessions of electrical stimulation to target both pro-atrophic signaling and destabilization of muscle oxidative phenotype in human soleus muscle under unloading conditions.

2. Methods

2.1. Ethical approval

The study was approved by the Committee of Biomedicine Ethics of the Institute of Biomedical Problems of the Russian Academy of Science (Protocols № 594 from September 6^th, 2021 and № 620 from July 12^th, 2022). All participants provided written, signed informed consent, and all investigations were performed in accordance with the principles listed in the Declaration of Helsinki and its amendments.

2.2. Participants of the study

Ten men aged 25–38 years participated in a 6-day control DI (median height 1.78 m [interquartile range 1.71–1.79 m]; body mass 70 kg [64–74 kg]; and body mass index 24 kg⋅m⁻² [21–24 kg⋅m⁻²]). Another ten men aged 26–39 years participated in a 6-day DI with neuromuscular electrical stimulation (DI + ES) (height 1.74 m [1.70–1.76 m]; body mass 73 kg [70–79 kg]; and body mass index 24 kg⋅m⁻² [23–26 kg⋅m⁻²]). No significant differences between body mass, body mass index, age, composition and size of soleus muscle fibers were observed between the two groups of participants before the DI.

To evaluate the effects of ES, changes induced by DI (pre-DI vs. post-DI) were compared with changes induced by DI + electrical stimulation (pre-DI + ES vs. post-DI + ES). Soleus muscle samples were obtained from the medial part of the soleus muscle of the left leg using a Bergstrom needle with aspiration under local anesthesia (2 mL 2% lidocaine) 14 days before and 6 days after DI and DI + ES. (at 10:00 a.m., 3 h after a standardized light breakfast: 5.2 g protein, 2.7 g fat, 55 g carbohydrates, 1 253 kJ). The samples were immediately frozen in liquid nitrogen and stored at −80 °C for further biochemical analysis.

2.3. Dry immersion method

The participants lay on a waterproof fabric and were immersed in a deep bath up to the level of the neck, in a supine position (Fig. 1). The surface area of the fabric considerably exceeded the surface area of water. The folds of the waterproof fabric allowed the person's body to be enveloped from all sides freely. The bath, measuring 200 cm × 100 cm × 100 cm, was filled with water. The water temperature in the bath was kept constant at (33 ± 0.5) °C. During electrical stimulation sessions, eating and sleeping the participants remained in the bath and were only removed from the bath for 15–20 minutes (min) each day for sanitary and hygienic procedures. Previously a 56-day dry immersion experiment proved the applicability and safety of the DI model for reproducing long-term microgravity effects.¹¹

2.4. Neuromuscular electrical stimulation

During DI + ES, both low-intensity/low-frequency and low-intensity/high-frequency electrical stimulation of the calf muscles was performed daily, with low-frequency stimulation sessions in the morning (11 a.m., 40–45 min) and high-frequency stimulation sessions in the afternoon (5 p.m.,10 min). The protocol was determined based on the previous pre-clinical studies that showed good efficacy and no serious adverse effects for both types of electrical stimulation.²⁷^,²⁸ A pair of electrodes (PG-901, Fiab, Italy) was placed on the proximal and distal thirds of the calf for the anterior (13 cm × 5 cm) and posterior (13 cm × 5 cm) surfaces. For the posterior surface of the calf, one electrode was placed in the proximal part of the calf (13 cm × 5 cm), and another electrode (9 cm × 5 cm) was placed 5 cm distal to the gastrocnemius muscles. The low-frequency protocol: 1 second (s) of bipolar symmetrical rectangular pulses (1 ms, 25 Hz), followed by a 2-s pause. High-frequency stimulation protocol: 10 s of bipolar symmetrical pulses (2.5 kHz with a sinusoidal modulating frequency of 50 Hz), followed by a 50-s pause. The intensity of high-frequency stimulation was individually adjusted to the point of causing unpleasant sensations.

2.5. Nucleic acid analysis

The extraction of nucleic acids from the biopsy samples was performed by AllPrep DNA/RNA/miRNA extraction kit (Qiagen, Germany) following the manufacturer's recommendations. The content of total RNA and DNA in the samples was determined by measuring the optical density of the sample at 260 nm and 280 nm. The purity of the samples was determined by the ratio of the wavelengths at 260 nm and 280 nm. For reverse transcription (RT) of total RNA and cDNA synthesis, an OT-1 reverse transcription kit (Sintol, Russia) was used according to the manufacturer's recommendations. Ribosomal protein L13 (RPL13) gene expression was used as a reference.

For miRNA RT, miRCURY LNA RT Kit (Qiagen, Germany) was used. micro-RNA real-time PCR was performed by human primers vs. 499-miRNA, 208, and 206 miRNA from Qiagen, and UnSp6 Qiagen primer assay was used as internal reference control.

To determine the content of mitochondrial DNA in samples, 120 ng DNA fractions were taken as a matrix for real-time PCR analysis, cytochrome c oxidase subunit I (COXI) primers were used as mtDNA primers, and RPL13 DNA content was used as a reference.

Reverse transcription of total RNA and real-time PCR, as well as bisulfite conversion and Methylation-specific PCR (MSP) were performed as described previously.²⁹ Bisulfite specific primers (BSP) were used for reference control. Primers used in the study are listed in Table 1. Melting curves of the PCR-products of all primers were analyzed to avoid non-specific primers usage.

Table 1.

Primers used in the analysis.

Primer name	Primer sequence	Gene bank number
Drp1-F	3′-CGACTCATTAAATCATATTTTCTCATTGTCAG-5′	NG_012219.1
Drp1-R	3′-TGCATTACTGCCTTTGGCACACT-5′	NG_012219.1
Fis1-F	3′-CCTGGTGCGGAGCAAGTACAA-5′	NC_000007.14
Fis1-R	3′-TCCTTGCTCCCTTTGGGCAG-5′	NC_000007.14
RPL13A -F	3′-T CCTGGAGGAGAAGAGGAAAGAGA-5′	NC_000019.10
RPL13A -R	3′-TTGAGGACCTCTGTGTATTTGTCAA-5′	NC_000019.10
MuRF-1 F	3′-CCTGAGAGCCATTGACTTTGG-5′	NG_033268.1
MuRF-1 R	3′-CTTCCCTTCTGTGGACTCTTCCT-5′	NG_033268.1
Atrogin-1-F	3′-GCAGCTGAACAACATTCAGATCAC-5′	NC_000008.11
Atrogin-1 R	3′-CAGCCTCTGCATGATGTTCAGT-5′	NC_000008.11
LC3B-F	3′-ACCATGCCGTCGGAGAAGAC-5′	NG_029030.2
LC3B-R	3′-TCTCGAATAAGTCGGACATCTTCTACTCT-5′	NG_029030.2
Mitofusin-1-F	3′-CTGAGGATGATTGTTAGCTCCACG-5′	NC_000003.12
Mitofusin-1- R	3′-CAGGCGAGCAAAAGTGGTAGC-5′	NC_000003.12
Mitofusin-2-F	3′-ATGGCCAGCTGTATTCTGAGGT-5′	NG_007945.1
Mitofusin-2- R	3′-AGGCAAAGCAGGATGGAACA-5′	NG_007945.1
COX I-F	3′-AAGCCTCCTTATTCGAGCCG-5′	NC_012920.1
COX I- R	3′-GGGGGCACCGATTATTAGGG-5′	NC_012920.1
COX IV-F	3′-GCAGAATGTTGGCTACCAGG-5′	NG_042280.1
COX IV- R	3′-GATACCGAGCGCGGTGAAAC-5′	NG_042280.1
TFAM-F	3′-GATTCACCGCAGGAAAAGCTG-5′	NG_053006.1
TFAM-R	3′-ATCACAGAACACCGTGGCTT-5′	NG_053006.1
NRF2α -F	3′-TTCCGGAGTGGGACTGATCC-5′	NC_000002.12
NRF2α -R	3′-TGATTCAGCATGGTGGGCAT-5′	NC_000002.12
PGC1-F	3′-CCAAAGGATGCGCTCTCGTTCA-5′	NG_028250.2
PGC1-R	3′-CGGTGTCTGTAGTGGCTTGACT-5′	NG_028250.2
PGC1-MSP-F	3′-GTAAGGGGGAGGATTAAGTTTATAC-5′	NG_028250.2
PGC1-MSP-R	3′-TAAAAAAAATACATTCACAAACGAC-5′	NG_028250.2
BSP-F	3′-TATTTTAAGGTAGTTAGGGAGGAAA-5′	NG_028250.2
BSP-R	3′-ATAACAATAAAAAATACCAACTCCC-5′	NG_028250.2
MYH 7-F	3′-TGTGTCACCGTCAACCCTTA-5′	NG_007884.1
MYH 7-R	3′-TGGCTGCAATAACAGCAAAG-5′	NG_007884.1
MYH 2-F	3′-GATGGCACAGAAGTTGCTGA-5′	NG_013014.1
MYH 2 -R	3′-CTTCTCGTAGACGGCTTTGG-5′	NG_013014.1
MYH 1-F	3′-GACAACTCCTCTCGCTTTGG-5′	NC_000017.11
MYH 1 -R	3′-GGCATAATCGTATGGGTTGG-5′	NC_000017.11
TET 1-F	3′-TCATGGGTGTCCAATTGCTA-5′	NC_000010.11
TET1-R	3′-GATGAGCACCACCATCACAG-5′	NC_000010.11
TET-2-F	3′-GGACATGATCCAGGAAGAGC-5′	NG_028191.1
TET-2-R	3′-CCCRCAACATGGTTGGTTC-5′	NG_028191.1
Dnmt1-F	3′-GAGCCACAGATGCTGACAAA-5′	NC_000019
Dnmt1-R	3′-GACACAGGTGACCGTGCTTA-5′	NC_000019
Dnmt3b-F	3′-GGGAGGTGTCCAGTCTGCTA-5′	NG_007290.1
Dnmt-3b-R	3′-GGCTTTCTGAACGAGTCCTG-5′	NG_007290.1

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2.6. Protein analysis

To assess the content of total and phosphorylated proteins, the total protein fraction of muscle samples was extracted and protein content quantified as described elsewhere.²⁹ The content of total and phosphorylated forms of proteins was assessed using polyacrylamide gel electrophoresis followed by Western blot. For polyacrylamide gel electrophoresis (PAGE), samples were diluted in 2× sample buffer (5.4 mM Tris-HCl (pH 6.8), 4% Ds -Na, 20% glycerol, 0.02% bromophenol blue) and were applied to the gel pockets (45 μg of total protein per lane). Electrophoresis was carried out in 10% separating PAGE. Samples from each group were loaded onto the same gel with control samples. Electrophoresis was carried out at 15 mA per gel in a mini system (Bio-Rad Laboratories) at room temperature.

Electrotransfer of proteins was carried out on a nitrocellulose membrane at 100 V at 4 °C for 120 min in a mini trans-Blot system (Bio-Rad Laboratories). After electrotransfer, the NC membranes were incubated in a solution of 5% dry milk (Bio-Rad Laboratories) in phosphate-buffered saline solution with 0.1% Tween 20 (PBST) for 1 h at room temperature. To detect protein bands, primary antibodies were used: total CaMK II (CSB-PA061493, Cusabio, Wuhan, China, 1:1 000), phospho-CaMK II (CSB-PA283993, Cusabio, Wuhan, China, 1:1 000), p-p70S6kinase (Thr 389) (1:500; Santa Cruz Biotechnology, Dallas, TX, USA, #sc-11759), p70S6kinase (1:1 000, Cell Signaling Technology, Danvers, MA, USA, #9202), phospho-AMPK and total-AMPK (Q13131, S496, 1:1 000, Cusabio, Wuhan, China, 1:1 000), p-4E-BP1 (Thr37/46) (1:1 000, Cell Signaling Technology, USA, #2855) and 4E-BP-1 (1:2 000, Cell Signaling Technology, USA, #9452, GAPDH (#2118, 1:10 000, Cell Signaling, Canada) secondary antibodies goat-anti-rabbit, goat-anti-mouse conjugated with horseradish peroxidase (“Santa Cruz”, 1:30 000). Incubation of blots with primary antibodies was carried out in 5% milk in PBST overnight at 4 °C, with secondary antibodies incubation was 1 h at room temperature. The blots were washed 3 times for 10 min in PBST. Detection of protein bands was carried out using the ImmunStar kit Substrate Kit (BioRad Laboratories) and C-DiGit scanner Blot Scanner (LI-COR Biotechnology, USA). Protein bands were analyzed using Studio Digits software Ver. 4.0. The amount of phosphorylated protein was normalized to the total amount of the same protein to determine the level of phosphorylation.

2.7. Immunohistochemistry

On transverse cryosections of biopsy specimens, immunostaining of slow-type and fast-type fibers was performed as described previously.²⁹ Primary antibodies used in this study were myosin heavy chain (MyHC) I(β) slow, 1:100 (Sigma, USA) and MyHC fast, 1:60 (DSMZ, Germany). The anti-MyHC fast antibody used in this study does not distinguish between different fast MyHC isoforms. Secondary antibodies used were Alexa Fluor 546 (1:1 000) and Alexa Fluor 488 (1:1 000) (Waltham, Massachusetts, USA). The sections were examined and photographed using Leica Q500MC fluorescence microscope with an integrated digital camera (TCM 300F, Leica, Braunschweig, Germany) at 20× objective magnification. The analysis of fast-type and slow-type cross-sections areas (CSA) was performed using ImageJ 1.52a software. At least 10 cross-sections per sample were analyzed to determine the percentage of different muscle fiber types in the sample (n = 8). Fast myosin negative fibers were counted as slow-type fibers, while slow myosin-negative fibers were counted as fast-type fibers. The sum of slow-type and fast-type myofiber percentages may exceed 100% due to the presence of double-positive hybrid fibers.

2.8. PGC1a promoter region analysis

HOmo sapiens COmprehensive MOdel COllection (HOCOMOCO, v12) was used for transcription factors (TF) binding motifs search within the peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a) promoter region analyzed for CpG (stretches of DNA with high guanine and cytosine content) methylation in our experiment. Activating transcription factors (ATF2, ATF3, ATF4), CAMP responsive element binding protein 1 (CREB1), myocyte enhancer factor (MEF2A, MEF2B, MEF2C, MEF2D), nuclear factor of activated T-cells (NFATC1), and TEA domain family member 1 (TEAD1) binding motifs were searched. For the presence of the TF binding motif the p-value less than 0.000 1 (1E-4) was accounted as statistically significant.

2.9. Statistics

Statistical analysis was carried out using the Prism 8 program. The significance of differences between groups was determined using a paired t-test after checking if the data are normally distributed. Robust regression and outlier removal (ROUT) method was used to identify the outliers. We've calculated statistical power for n = 10 (or less, if there were outliers in a particular PCR, Western blot or other analysis) using G∗power 3.1.9.7 software. For all the comparisons, where the data significantly differed from one another, the power value was > 0.8. Data are expressed as a percentage of the pre-DI or pre-DI + ES group and are presented as the mean ± SEM, except fiber CSA and percentage of slow and fast fibers.

3. Results

After 6-day dry immersion in soleus muscle samples, there was no statistically significant decrease in CSA of either slow or fast type myofibers in the post-DI vs. the pre-DI. We also did not detect any differences in the percentage of slow-type or fast-type fibers (Fig. 2A–C, E,G). After 6 days of DI + ES, we did not detect any changes in the CSA of slow-type fibers or any differences in the percentage of slow-type or fast-type fibers, but the CSA of fast-type fibers was significantly lower in the post-DI + ES samples than in the pre-DI + ES samples (Fig. 2F).

Fig. 2 — Slow-type fibers cross-sectional area (CSA) in pre-DI and post-DI samples (A), slow-type fibers CSA in pre-DI + ES and post-DI + ES samples (B), slow-type fibers % in pre-DI and post-DI samples (C), slow-type fibers % in pre-DI + ES and post-DI + ES samples (D), fast-fast fibers CSA in pre-DI and post-DI samples (E), fast-type fibers CSA pre-DI + ES and post-DI + ES samples (F), fast-type fibers % pre-DI and post-DI samples (G), fast-type fibers % in pre-DI + ES and post-DI + ES samples (H), representative microphotographs of anti-myosins staining (above panel). White scale bar is 200 μm. The data are represented in absolute values (for CSAs) and as percentage of the total number of myofibers analyzed in the sample (for fiber-type %). ∗ - p < 0.05 (paired t-test). The number of samples (excl. outliers) is indicated as dots on the graphs.

The content of phosphorylated p70s6kinase (ribosomal protein S6 kinase beta-1) did not differ in the post-DI samples vs. the pre-DI samples (Fig. 3 A). After 6-day DI + ES the content of p-p70s6kinase was significantly higher in the post-DI + ES samples vs. the pre-DI + ES samples (Fig. 3B).

The content of phosphorylated 4E-BP (eukaryotic translation initiation factor 4E-binding protein 1) did not differ between the pre-DI vs. the post-DI or between the pre-DI + ES vs. the post-DI + ES samples (Fig. 3C and D).

There was no change in muscle ring-finger protein-1 (MuRF-1) mRNA levels after 6 days of DI or 6 days of DI + ES. (Fig. 4A and B). Atrogin-1 mRNA content was significantly higher in the post-DI samples vs. the pre-DI samples as well as in the post-DI + ES vs the pre-DI + ES (Fig. 4D and E). Microtubule-associated proteins 1A/1B light chain 3B (LC3B) mRNA content did not differ between the pre-DI vs. post-DI samples; at the same time, it was significantly higher in the post-DI + ES samples vs. the pre-DI + ES samples (Fig. 4E and F).

Neither 6-day DI + ES nor 6-day DI affected myh7 mRNA levels in soleus muscle (Fig. 5A and B). Myh2 mRNA levels were significantly lower in the post-DI samples vs. the pre-DI samples. In the post-DI + ES samples, myh2 mRNA expression did not differ from the pre-DI + ES sample group (Fig. 5C and D). 6-day DI did not affect myh1 mRNA expression, while in the post-DI + ES the expression of myh1 mRNA was significantly lower than in the pre DI + ES samples (Fig. 5E and F).

TFAM (mitochondrial transcription factor A) and PGC1a mRNA expression were significantly lower in both post-DI + ES and post-DI vs. pre-DI (Fig. 6A and B; E-F). NRf2α (nuclear respiratory factor 2) expression was significantly lower in the pre-DI samples vs. the post-DI samples. After 6-day DI with electrical stimulation NRf2α expression in the post-DI + ES samples did not differ from the pre-DI + ES samples (Fig. 6C and D).

The content of COXI mRNA and COX IV mRNA was significantly lower in the post-DI samples compared to the pre-DI samples (Fig. 6G and H), while there were no significant differences between the pre-DI + ES vs. post-DI + ES samples (Fig. 6I and J). The content of mitochondrial DNA did not significantly differ between pre-DI vs. post-DI or between pre-DI + ES vs. post-DI + ES (although there was a trend towards a decrease in post-DI + ES vs. pre-DI + ES, p = 0.078).

Mitofusin-1 mRNA content was significantly lower in the post-DI samples compared to the pre-DI samples, while it did not differ between pre-DI + ES and post-DI + ES (Fig. 7A and B). Mitofusin-2 mRNA content did not differ in both sample groups after the immersion vs. pre-immersion samples (Fig. 7C and D). Fis1 (mitochondrial fission 1 protein) mRNA content was significantly lower in the post-DI samples vs. the pre-DI samples, but it did not differ between the pre-DI + ES and post-DI + ES sample groups (Fig. 7E and F). Dynamin-1-like protein Drp1 (dynamin-1-like protein) mRNA content was significantly lower in both post-DI + ES and post-DI vs. pre-DI (Fig. 7G and H)

Fig. 7 — Mitofusin-1 mRNA in pre-DI and post-DI samples (A), mitofusin-1 mRNA in pre-DI + ES and post-DI + ES samples (B), mitofusin-2 mRNA in pre-DI and post-DI samples (C), mitofusin-2 mRNA in pre-DI + ES and post-DI + ES samples (D), Fis1 (mitochondrial fission 1 protein) mRNA in pre-DI and post-DI samples (E), Fis1 mRNA in pre-DI + ES and post-DI + ES samples (F), drp1 (dynamin-1-like protein) mRNA in pre-DI and post-DI samples (G), drp1 mRNA in pre-DI + ES and post-DI + ES samples (H). The data are represented as mean ± *SEM* from pre-DI or from pre-DI + ES samples, respectively. ∗ - p < 0.05 (paired t-test). The number of samples (excl. outliers) is indicated as dots on the graphs.

The level of methylated DNA in the PGC1a promoter region was significantly higher in the post-DI samples than in the pre-DI samples. (Fig. 8 A). After DI with electrical stimulation, the level of methylated DNA in the PGC1a promoter region was not significantly different from the pre-DI + ES samples (Fig. 8 B). The search for transcription factor binding motifs within the DNA of the PGC1a promoter region, analyzed by BSP-PCR, revealed the NFATc1 (NFAT2) and TEAD1 binding motifs in the region. (Fig. 8 C).

TET1 (ten-eleven translocation methylcytosine dioxygenase 1) mRNA content was significantly lower in the post-DI samples vs. the pre-DI samples, while in the post-DI + ES samples TET1 mRNA content did not significantly differ from the pre-DI + ES samples. TET2, Dnmt1 (DNA (cytosine-5)-methyltransferase 1) and Dnmt3b mRNA content did not differ between pre-DI and post-DI (Fig. 9).

Fig. 9 — TET1 (ten-eleven translocation methylcytosine dioxygenase 1) mRNA in pre-DI and post-DI samples (A), TET1 mRNA in pre-DI + ES and post-DI + ES samples (B), TET2 mRNA in pre-DI and post-DI samples (C), TET2 mRNA in pre-DI + ES and post-DI + ES samples (D), Dnmt1 (DNA (cytosine-5)-methyltransferase 1) mRNA in pre-DI and post-DI samples (E), Dnmt1 mRNA in pre-DI + ES and post-DI + ES samples (F), Dnmt3b mRNA in pre-DI and post-DI samples (G), Dnmt3b mRNA in pre-DI + ES and post-DI + ES samples (H). The data are represented as mean ± *SEM* from pre-DI or from pre-DI + ES samples, respectively. ∗ - p < 0.05 (paired t-test). The number of samples (excl. outliers) is indicated as dots on the graphs.

Mir-206 and mir-208 mRNA content were significantly lower in the post-DI samples vs. the pre-DI samples, while there were no significant differences in mir-206 and mir-208 mRNA content between pre-DI + ES and post-DI + ES (Fig. 10A–D). The level of Mir-499 mRNA was not significantly different before and after the dry immersion.

Fig. 10 — mir-206 content in pre-DI and post-DI samples (A), mir-206 content in pre-DI + ES and post-DI + ES samples (B), mir-208b content in pre-DI and post-DI samples (C), mir-208b content in pre-DI + ES and post-DI + ES samples (D), mir-499 content in pre-DI and post-DI samples (E), mir-499 content in pre-DI + ES and post-DI + ES samples (F). The data are represented as mean ± *SEM* from pre-DI or from pre-DI + ES samples, respectively. ∗ - p < 0.05 (paired t-test). The number of samples (excl. outliers) is indicated as dots on the graphs.

The level of phosphorylated AMPK (AMP-activated protein kinase) did not differ between the pre-DI vs. post-DI or the pre-DI + ES vs. post-DI + ES samples, although there was a trend toward a decrease in the post-DI vs. pre-DI samples. (p = 0.079) (Fig. 11A and B).

Fig. 11 — Phosphorylated AMPK (AMP-activated protein kinase) content in pre-DI and post-DI samples (A), phosphorylated AMPK content in pre-DI + ES and post-DI + ES samples (B), phosphorylated CaMK II (Ca2+/calmodulin-dependent protein kinase) content in pre-DI and post-DI samples (C), phosphorylated CaMK II content in pre-DI + ES and post-DI + ES samples (D)The data are represented as mean ± *SEM* from pre-DI or from pre-DI + ES samples, respectively. ∗ - p < 0.05 (paired t-test). The number of samples (excl. outliers) is indicated as dots on the graphs. The data are represented as mean ± *SEM* from pre-DI or from pre-DI + ES samples, respectively. ∗ - p < 0.05 (paired t-test). The number of samples (excl. outliers) is indicated as dots on the graphs.

The level of phosphorylated Ca2+/calmodulin-dependent protein kinase (CaMK II) was significantly higher in the post-DI samples compared to the pre-DI samples, while it did not differ in the pre-DI + ES samples vs. the post-DI + ES samples. (Fig. 11C and D).

4. Discussion

4.1. Mitochondria-related and fiber-type parameters

Dry immersion led to a significant decrease in the expression of markers associated with mitochondrial biogenesis, fusion, and fission. Previous studies also showed a notable downregulation of mitochondrial translation and mitophagy markers after 6-day DI and a decline in electron transport chain (ETC) oxygen consumption after 3-day DI.²⁹^,³⁰ In contrast to the 6-day DI without countermeasures, the 6-day DI with electrical stimulation did not result in the downregulation of several mitochondrial biogenesis markers (COXI, COXIV, NRF2α) and mitochondrial dynamics markers (Drp1, Fis1, mitofusin1). Surprisingly, despite the positive effects of electrical stimulation on mitochondrial dynamics and biogenesis markers, there was a trend towards a decrease in the number of mitochondrial DNA (mtDNA) copies. Previous research has shown that electrical stimulation can increase the production of mitochondrial reactive oxygen species (ROS) and stimulate mitophagy in C2C12 myotubes.³¹^,³² The upregulation of LC3B in the post-DI + ES samples may indicate mitophagy activation, suggesting that the decreasing trend in mtDNA copy number could result from increased mitochondrial turnover due to oxidative stress in the pre-DI + ES samples.

An increase in the CpG methylation in the PGC1a promoter region was detected after 6 days of disuse (DI), while it remained unchanged after DI + ES. Disuse-induced upregulation of CpG methylation in the PGC1a promoter region has been previously observed in both humans and animals.³³^,³⁴ Although electrical stimulation had an effect on PGC1a CpG methylation, it did not prevent the decline in PGC1a mRNA expression. However, CpG methylation is not the only regulator of the decline in PGC1a mRNA expression, as previous studies have revealed the role of AMPK and nitric oxide-dependent mechanisms in the disuse-induced decline in PGC1a expression.³⁵^,³⁶ The analyzed CpG contained binding sites for NFATc1 and TEAD1, which are both activators of PGC1a expression.37, 38, 39 Therefore, an increase in the methylation of CpG in this promoter region could contribute to the extent of PGC1a downregulation during muscle disuse or certain stages of muscle disuse. An analysis of the mRNA expression of CpG methylation regulators, specifically TET1 and TET2 (ten-eleven translocases that remove methyl groups from CpGs) and Dnmt1 and Dnmt3b (DNA methyltransferases that catalyze the methylation of CpGs), revealed that TET1 mRNA expression decreased after a 6-day period of disuse (DI) but was equal to pre-DI + ES levels in the post-DI + ES samples. Previously, it was shown that silencing TET1 suppresses PGC1a expression.⁴⁰ The decline in TET1 mRNA expression could be the cause of increased CpG methylation in the PGC1a promoter region after the 6-day DI. Additionally, the prevention of this decline in the post-DI + ES group could result in no increase in CpG methylation in the PGC1a promoter region in this sample group compared to pre-DI + ES.

Dry immersion without electrical stimulation resulted in the downregulation of micro-RNAs 208 and 206, but did not affect mir-499. Mir-208b and mir-206 micro-RNAs were shown to promote slow-type myosin expression and mitochondrial biogenesis and were shown to decline in disused soleus muscle in animals.³⁴^,41, 42, 43 The prevention of mir-206 and mir-208b downregulation in the post-DI + ES samples is in good agreement with the effect of low-frequency electrical stimulation of rat soleus leading to the upregulation of mir-208/mir-499.²⁵ This may contribute to the effects of electrical stimulation on some parameters of mitochondrial biogenesis.

A 6-day period of dry immersion did not result in changes to the ratio of slow-to-fast muscle fiber types. This can be explained by the slower development of disuse-induced processes in human skeletal muscles compared to rodent models. At the same time, the 6-day dry immersion led to a decrease in the expression of the fast oxidative myosin isoform IIa (myh2) in the post-immersion group compared to the pre-immersion one. In rodents, hindlimb unloading for 3 and 7 days also leads to a decrease in the expression of the fast oxidative myosin isoform IIa in the soleus muscle.⁴⁴ This finding aligns well with the current data.

In the post-DI + ES samples, myh2 did not change compared to pre-DI + ES. Moreover, dry immersion with electrical stimulation resulted in the downregulation of myh1 (fast glycolytic myosin isoform IId/x) mRNA expression vs. the pre-DI + ES samples. A study examining myosin isoform expression during chronic low-frequency electrical stimulation of rat and rabbit muscles found that fast oxidative myosin IIa mRNA was upregulated, while fast glycolytic myosin IId/x mRNA was downregulated after 5–10 days of daily stimulation. Changes in slow-type myosin mRNA were only observed at later stages of the experiment.⁴⁵ These results are in good agreement with our data. It has been shown that nerve activity influences the activity of NFAT transcription factor isoforms, which regulate myosin isoform expression in rat muscle.⁴⁶ The NFAT transcription factor family includes four members: NFATc1, NFATc2, NFATc3, and NFATc4. Calabria et al.showed that during slow-frequency electrical stimulation, all the four members of the NFAT family are located in myonuclei, which is accompanied by the activation of slow-type myosin isoform gene expression.⁴⁶ During high frequency electrical stimulation, only NFATc2, NFATc3 and NFATc4 are found in the nuclei, contributing to the expression of fast-type myosins. NFATc1 has been shown to suppress the expression of myosin IId/x but not myosin IIa.⁴⁷ It is possible that the combination of high-frequency and slow-frequency electrical stimulation used in the post-DI + ES group resulted in the nuclear localization of NFAT isoforms, promoting the expression of predominantly myosin IIa but not myosin IId/x.

4.2. Atrophy and proteostasis

In the present study, 6 days of dry immersion (DI) did not result in atrophy of either slow- or fast-type fibers. These findings differ from a previous 7-day dry immersion study.⁴⁸ The discrepancy could be attributed to a larger variability in initial cross-sectional areas among participants in our study compared to the study by Shenkman et al.⁴⁸ At the same time, electrical stimulation during DI led to a significant decrease in the cross-sectional area (CSA) of fast-type fibers compared to pre-DI levels. This suggests that ES enhanced fast-type muscle atrophy during dry immersion. These results align with literature data showing the absence of an anti-atrophic effect or even a decrease in fast-type fiber size during low-frequency stimulation.⁴⁹^,⁵⁰ Our data contrast with numerous studies reporting an anti-atrophic effect of high-frequency electrical stimulation during muscle disuse.20, 21, 22, 23 However, in all these studies, the anti-atrophic effect was observed in fast-type or mixed-type muscles, whereas our study focused on the slow-type soleus muscle. It should be emphasized that the properties of slow-type and fast-type fibers depend on the type of muscle they belong to, so the effect of electrical stimulation may vary for fast-type fibers from plantaris (fast muscle) or soleus (slow muscle).⁵¹ Previous research has shown that long-term high-frequency electrical stimulation did not lead to hypertrophy of normally innervated soleus muscles.⁵² Therefore, the observed decrease in the CSA of fast-type fibers may result from the lack of an anti-atrophic effect of high-frequency electrical stimulation on soleus muscle fibers.

It is important to note that fast-type fibers constitute only 10%–30% of the soleus muscle, meaning their reduction may not necessarily result in a decline in overall muscle function. Furthermore, previous research has demonstrated that in certain cases, a smaller fiber CSA can be associated with increased muscle endurance.⁵³

After 6-day dry immersion (DI), there was no downregulation of protein synthesis parameters, p-p70s6kinase or p-4E-BP, which might be attributed to the relatively short duration of the experiment. Electrical stimulation during dry immersion resulted in p-p70s6kinase upregulation vs. the pre-DI + ES samples. This finding is in good agreement with the previously reported data on protein synthesis activation in skeletal muscle via high frequency electrical stimulation.²² Consequently, the upregulation of p-p70s6kinase after electrical stimulation could be attributed to the high-frequency component of the stimulation protocol. Interestingly, the upregulation of p-p70s6kinase in the post-DI + ES group was associated with a decrease rather than an increase in fast-type fiber CSA. mTOR signaling is a key activator of exercise-induced muscle hypertrophy. However, in certain models of muscle atrophy, such as immobilization, mTOR activation can be accompanied by muscle atrophy.⁵⁴ This suggests that mTOR activation in some muscle disuse models may not be sufficient to prevent atrophy or promote hypertrophy. Six-day dry immersion resulted in an increase in the mRNA content of Atrogin-1 E3 ubiquitin ligase, but did not lead to an upregulation of MuRF-1 or LC3B (autophagy marker). Since Atrogin-1 upregulation was not accompanied by fiber atrophy after 6-day DI, this finding supports the previous hypothesis that in humans, muscle proteolysis does not play a key role in negative changes induced by disuse.⁵⁵ At the same time, electrical stimulation during dry immersion resulted in LC3B upregulation, which was not observed after 6-day DI without ES. LC3B is a marker of autophagy signaling,⁵⁶ and its increase has been shown to contribute to muscle atrophy in some experimental models.⁵⁷^,⁵⁸ Therefore, the upregulation of LC3B may explain the reduced fast-type fiber CSA in post-DI + ES compared to pre-DI + ES.

4.3. AMPK and calcium signaling

In recent years, it has been demonstrated that the primary signaling pathways regulating muscle proteostasis, fiber type, and oxidative metabolism include ATP/ADP/AMPK and calcium/CaMK pathways.59, 60, 61, 62 AMPK inactivation occurs at the early stage of soleus muscle unloading in humans after 3-day dry immersion and in rats after 1-day and 3-day hindlimb suspension.⁶³^,⁶⁴ In this study, no significant changes in p-AMPK content were observed after 6-day DI, possibly due to the experiment's duration. However, considering the decreasing trend in post-DI versus pre-DI and previous findings showing p-AMPK reduction in soleus muscle after 3-day DI,⁶³ it is plausible that p-AMPK content might have decreased during earlier stages of DI. Previous research in animal models has shown calcium accumulation in the myoplasm of unloaded soleus muscle fibers and an increase in calcium-dependent p-CaMK II content.65, 66, 67 These findings are in good agreement with the observed increase in p-CaMK II content after DI in our experiment. Interestingly, samples after 6-day DI with electrical stimulation did not exhibit p-CaMK II content upregulation, unlike samples after 6-day DI without stimulation. Since phosphorylation of CaMK II (Thr 287) is regulated by intracellular calcium concentration,⁶⁸ it can be suggested that electrical stimulation during DI prevented p-CaMK II accumulation by inhibiting the DI-induced increase in myoplasmic calcium levels. Such effect of the ES could be explained by a possible downregulation of ATP accumulation in myofibers of the unloaded soleus muscle due to muscle contractions. This effect of electrical stimulation could be attributed to the possible downregulation of ATP accumulation in unloaded soleus muscle myofibers due to muscle contractions. Previous studies have shown that calcium overload in unloaded slow-type fibers occurs due to Na⁺/K⁺ ATPase dysfunction, leading to L-type calcium channel activation.⁶⁹ Na⁺/K⁺ ATPase dysfunction, in turn, is triggered by ATP accumulation, resulting in AMPK inactivation.⁷⁰ Therefore, electrical stimulation during DI could lead to AMPK activation through muscle contractions, thereby counteracting Na⁺/K⁺ ATPase dysfunction and L-type calcium channel opening. This possible AMPK activation could also contribute to LC3B expression, as AMPK can activate both LC3B mRNA expression and LC3B-mediated increase in autophagy.⁷¹

Prevention of myoplasmic calcium overload during rodent hindlimb suspension can lead to upregulation of mitochondrial biogenesis parameters.⁷² Moreover, calcium overload and AMPK inactivation could be involved in the upregulation of CpG methylation by blocking TETs and Dnmts activation.⁷³^,⁷⁴ Therefore, we cannot exclude the possibility that the effects of electrical stimulation during dry immersion on CpG methylation in the PGC1a promoter region and on the mRNA expression of several mitochondrial biogenesis parameters could be mediated by preventing myoplasmic calcium overload. In conclusion, we can summarize that 6-day dry immersion resulted in the downregulation of mitochondrial biogenesis and dynamics markers, a decline in fast oxidative myosin isoform IIa mRNA expression, and an accumulation of p-CaMK II content. The sessions of electrical stimulation during the 6-day DI prevented the DI-induced decreases in COXI, COXIV, Nrf2α, myosin IIa, mitofisin-1 and Fis-1 mRNA expression, and counteracted p-CaMK II accumulation.

CRediT authorship contribution statement

Kristina A. Sharlo: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation. Irina D. Lvova: Writing – review & editing, Methodology, Investigation, Formal analysis. Natalya A. Vilchinskaya: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Sergey A. Tyganov: Writing – review & editing, Methodology, Investigation. Olga V. Turtikova: Writing – review & editing, Methodology, Investigation, Data curation. Ksenia V. Sergeeva: Writing – review & editing, Methodology, Investigation. Roman O. Bokov: Visualization, Software, Investigation, Formal analysis, Data curation. Boris S. Shenkman: Writing – review & editing, Supervision, Project administration, Conceptualization. Elena S. Tomilovskaya: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. Oleg I. Orlov: Writing – review & editing, Supervision, Project administration, Conceptualization.

Ethical approval approvement

The study was approved by the Committee of Biomedicine Ethics of the Institute of Biomedical Problems of the Russian Academy of Science (Protocols № 594 from September 6th, 2021 and № 620 from July 12, 2022). All participants provided written, signed informed consent, and all investigations were performed in accordance with the principles listed in the Declaration of Helsinki and its amendments.

Funding details

The study was supported by the Ministry of Science and Higher Education of the Russian Federation under agreement № 075-15-2022-298 from April 18, 2022 about the grant in the form of subsidy from the federal budget to provide government support for the creation and development of a world-class research center, the “Pavlov Center for Integrative Physiology to Medicine, High-tech Healthcare and Stress Tolerance Technologies”.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors are grateful to Timur M. Mirzoev for English language editing and proofreading.

Footnotes

Peer review under the responsibility of Editorial Board of Sports Medicine and Health Science

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.smhs.2025.06.002.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1

mmc1.pdf^{(179.9KB, pdf)}

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PERMALINK

Molecular signaling effects of electrical stimulation of the soleus muscle during 6 days of dry immersion

Kristina A Sharlo

Irina D Lvova

Natalya A Vilchinskaya

Sergey A Tyganov

Olga V Turtikova

Ksenia V Sergeeva

Roman O Bokov

Boris S Shenkman

Elena S Tomilovskaya

Oleg I Orlov

Abstract

Background

Methods

Results

Conclusions

Abbreviations

1. Introduction

2. Methods

2.1. Ethical approval

2.2. Participants of the study

2.3. Dry immersion method

Fig. 1.

2.4. Neuromuscular electrical stimulation

2.5. Nucleic acid analysis

Table 1.

2.6. Protein analysis

2.7. Immunohistochemistry

2.8. PGC1a promoter region analysis

2.9. Statistics

3. Results

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 9.

Fig. 10.

Fig. 11.

4. Discussion

4.1. Mitochondria-related and fiber-type parameters

4.2. Atrophy and proteostasis

4.3. AMPK and calcium signaling

CRediT authorship contribution statement

Ethical approval approvement

Funding details

Declaration of competing interest

Acknowledgements

Footnotes

Appendix A. Supplementary data

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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