Mitochondrial Maintenance in Skeletal Muscle

Abstract

Skeletal muscle is one of the tissues with the highest range of variability in metabolic rate, which, to a large extent, is critically dependent on tightly controlled and fine-tuned mitochondrial activity. Besides energy production, other mitochondrial processes, including calcium buffering, generation of heat, redox and reactive oxygen species homeostasis, intermediate metabolism, substrate biosynthesis and anaplerosis are essential for proper muscle contractility and performance. It is thus not surprising that adequate mitochondrial function is ensured by a plethora of mechanisms, aimed at balancing mitochondrial biogenesis, proteostasis, dynamics, and degradation. The fine-tuning of such maintenance mechanisms ranges from proper folding or degradation of individual proteins to the elimination of whole organelles, and, in extremis, apoptosis of cells. In this review, the present knowledge on these processes in the context of skeletal muscle biology is summarized. Moreover, existing gaps in knowledge are highlighted, alluding to potential future studies and therapeutic implications.

1. Introduction

Skeletal muscle has a remarkable ability to undergo changes in mass, fiber-type, and function, based on prevailing external and internal stimuli. For example, physical activity and high metabolic demands trigger adaptations in muscle mass, fiber type composition, mitochondrial content and mitochondrial phenotypes (Egan and Sharples 2023; Monzel et al. 2023). Functionally, mitochondria first and foremost play a critical role for energy provisioning required for prolonged muscle contractions. However, a plethora of additional processes, e.g. related to calcium homeostasis, thermoregulation, intermediate metabolism, substrate biosynthesis and anaplerosis, redox and reactive oxygens species homeostasis, are of high importance for adequate skeletal muscle functionality (Monzel et al. 2023). Accordingly, mitochondrial dysfunction has a large impact on cellular and tissue health. For example, in skeletal muscle, aberrant metabolism, muscle loss and worsened pathophysiology in various diseases and syndromes, including muscular dystrophies, sarcopenia, cachexia, cancer, and kidney disease, have been described (Russell et al. 2014; Cohen et al. 2015; De Mario et al. 2021; Wang et al. 2022). In addition, congenital mitochondrial diseases, caused by single mutations in the mitochondrial DNA (mtDNA), or in nuclear DNA (nDNA) regions that encode certain mitochondrial proteins, lead to muscle-related symptoms including muscle atrophy and myotonia (Russell et al. 2014; De Mario et al. 2021).

Mitochondrial function depends on dynamic, context-dependent organelle- and network remodeling, as well as the activation of various maintenance pathways that protect these organelles, and by proxy, muscle tissue. However, certain characteristics of skeletal muscle pose unique challenges and hence the need for specialized mitochondrial maintenance mechanisms. For example, unlike proliferating cell types where damaged mitochondria can be eliminated through dilution by cell division, post-mitotic skeletal muscle fibers rely on timely and adequate activation of degradation pathways. Moreover, due to the spatial constraints imposed by the tight packing of myofibrils, mitochondrial motility and dynamics are limited in skeletal muscle. Finally, the extraordinary high metabolic rate of this tissue, in particular in exercise, has to be fueled by adequate ATP provisioning, which can lead to exaggerated production of mitochondrial reactive oxygen species (ROS), potentially linked to damage of mtDNAs and proteins. Removal of defective mitochondria has to be tightly balanced with generation or replenishment of mitochondrial components to sustain the required metabolic rates in this context.

To cope with such constraints, mitochondria are equipped with elaborate quality control mechanisms. These processes provide support throughout all phases of the mitochondrial life cycle, starting from biogenesis, proteostasis and dynamics, to degradation, and can be escalated from intra- to inter-organellar, up to the cellular level. The aim of this review is to shed light on the current state of literature of mitochondrial maintenance mechanisms in skeletal muscle. In addition, we highlight knowledge gaps that are of particular interest for future studies that will further advance our understanding of the critical interplay between mitochondrial maintenance and skeletal muscle (patho-)physiology.

2. Mitochondrial biogenesis

Mitochondrial biogenesis is not only important for the escalation of mitochondrial number and density in specific contexts such as exercise training, but also crucial for the replacement of damaged or dysfunctional mitochondria. This complex process is controlled by various cellular perturbations, e.g in energy, ROS or calcium, relayed by a number of different signaling cascades, engaging sensors and effector proteins such as the adenosine monophosphate-activated protein kinase (AMPK), calcium/calmodulin-dependent protein kinases (CaMK), p38 mitogen-activated protein kinases (p38 MAPK) or the deacetylase sirtuin 1 (SIRT1), which all, at least in part, converge on the peroxisome proliferator-activated receptor γ co-activator-1 proteins (PGC-1α, PGC-1β and PGC-1) (Wu et al. 2002; Zhang et al. 2014; Hood et al. 2016; Popov 2020). These three coactivator proteins all control mitochondrial biogenesis and oxidative metabolism, albeit in different contexts (Lin et al. 2005; Villena 2015). Activation of PGC-1s gives rise to transcription and translation of mtDNA, and the production, import and assembly of mitochondrial proteins encoded by nDNA. For example, PGC-1α is activated by various external and internal perturbations, including inadequate mitochondrial function, which evoke higher energetic demands (Figure 1). Subsequently, PGC-1α, together with different transcription factor binding partners, coordinates the transcription and translation of mitochondrial genes encoded in nDNA and mtDNA to remodel mitochondria, increase biogenesis and boost function. A description of the numerous pathways involved in this regulation can be found elsewhere (Kupr and Handschin 2015; Jannig et al. 2022). Moreover, previously published reviews cover the general aspects of mitochondrial biogenesis in more detail (Hock and Kralli 2009; Hood et al. 2016; Popov 2020). Of note, due to the high demand on protein synthesis, mitochondrial biogenesis poses a major proteostatic challenge, for which multiple lines of quality control are set in place.

Figure 1. Regulation of mitochondrial biogenesis by PGC-1α.

PGC-1α integrates the consequence of external perturbations that increase energetic demands with internal sensing of the cellular energy state, and retrograde feedback signaling on mitochondrial health and function. Once activated, both in terms of gene expression as well as protein posttranslational modifications, this coregulator subsequently coordinates the transcriptional program of mitochondrial biogenesis to boost oxidative metabolism and ATP synthesis. To do so, PGC-1α associates with different transcription factors, initiating the transcription of nuclear genes that encode mitochondrial proteins (NuGEMPs) and regulators of mitochondrial biogenesis, including its own gene expression. These NuGEMPs include subunits of the electron transport chain (ETC) (depicted in green) and regulators such as Mitochondrial Transcription Factor A (TFAM) (depicted in purple), which enable the expression of mitochondrial-encoded ETC subunits. Additionally, components like mitochondrial ribosomal proteins are imported and incorporated into the mitochondrial translation machinery. This ensures the production of all necessary ETC subunits from both genomic sources, forming a functional ETC in a well-controlled and coordinated manner, with feedback mechanisms to modulate this process.

3. Proteostasis

Most proteins found within mitochondria are nuclear-encoded and translated in the cytosol. During their import into the organelle, these proteins must remain in an unfolded state to pass through the translocases located on both the inner and outer mitochondrial membranes. Subsequently, proteases cleave off the leader sequence, enabling the proper folding as a critical step to enable proteins to execute their function. Key complexes of the electron transport chain (ETC) consist of protein subunits that are in part encoded by the nuclear genome, and in part by the mitochondrial genome (Moehle et al. 2019; Soto et al. 2022a). Thus, to ensure stoichiometric assembly and proper function, it is vital to balance and coordinate transcription and protein synthesis from both genomes. If dysregulated, accumulation of unassembled and misfolded proteins in the mitochondrial translocases and matrix can lead to altered mitochondrial morphology, dissipation of the proton gradient, and functional deterioration (Soto et al. 2022b). For example, this balance is assailed by reduced mitochondrial translation rates in skeletal muscle aging (de Smalen et al. 2023). Moreover, due to the high metabolic activity of mitochondria, often linked to the creation of ROS, mitochondrial proteins are prone to sustain aberrant posttranslational modifications and protein oxidation, potentially compromising enzyme function and mitochondrial morphology (Gibson 2005). Various mitochondrial protein quality control mechanisms, interlinked with the corresponding cytosolic counterparts, help to overcome such proteostatic challenges.

3.1. Mitochondrial protein import

A number of mitochondrial import mechanisms in skeletal muscle were recently reviewed (Richards et al. 2023). These are complemented by multiple other protein import mechanisms, mostly identified in lower organisms, which also are protected from clogging and disruption in various ways (Wiedemann and Pfanner 2017). First, cytosolic chaperones such as mitochondrial-type heat shock protein 70 (mtHSP70) keep proteins unfolded to prevent the blockage of translocases of the inner and outer membrane. In skeletal muscle, GrpE protein homolog 2 (GRPEL2) is an essential co-chaperone in mitochondrial protein import, loss of which causes proteotoxic stress leading to muscle atrophy (Neupane et al. 2022). Second, clogged translocases can be cleared by AAA ATPases. Similarly, blockages in mitochondrial translocases due to premature protein folding, translation arrest or dissipation of the membrane potential can be resolved by ubiquitination of the corresponding proteins by the cytosolic valosin containing protein (VCP), also known as p97, which has a similar function in protein degradation in the endoplasmic reticulum. Stalled and ubiquitinated proteins are subsequently degraded by the proteasome (Piccirillo and Goldberg 2012). This mechanism is likely relevant for skeletal muscle as well, since loss of VCP in flies correlates with impaired mitochondrial and muscle function (Johnson et al. 2015). Moreover, several human myopathies are associated with VCP mutations (Custer et al. 2010).

If unresolved, disrupted mitochondrial protein import can trigger stress-adaptive responses. Cytosolic accumulation of activating transcription factor (ATF) 5, normally internalized into mitochondria and degraded by Lon protease 1 (LONP1), gives rise to translocation of ATF5 to the nucleus and engages the mitochondrial unfolded protein response (mtUPR) (Slavin et al. 2022). As a next level of escalation, dysfunctional mitochondria caused by protein import stress can be removed by mitophagy. Compromised import and degradation of PTEN-induced putative kinase protein 1 (PINK1) leads to its stabilization and accumulation on the outer mitochondrial membrane, which activates a cascade that initiates mitophagy (Narendra et al. 2010). Similarly, nod-like receptor X1 (NLRX1) retention in the cytosol induces mitophagosome formation, which was found to be required for mitophagy following acute exercise in skeletal muscle (Killackey et al. 2022). In animals and humans, the expression of translocase of the outer membrane 22 (TOM22) increases with age, which may provide a compensatory mechanism to reduce degradation of mitochondria (Joseph et al. 2010; Joseph et al. 2012). In addition, TOM22 phosphorylation by the casein kinase 2 (CSNK2) is important for the induction of mitophagy in skeletal muscle (Kravic et al. 2018). Interestingly, protein import rates differ between subsarcolemmal and intramyofibrillar mitochondria, but the functional consequence of these divergent rates is largely unknown (Takahashi and Hood 1996; Takahashi et al. 1998; Singh and Hood 2011).

3.2. Mitochondrial Unfolded Protein Response (mtUPR)

The mtUPR is a stress-responsive pathway that becomes activated via retrograde signaling upon accumulation of un- or misfolded proteins in the mitochondria. Transcriptional regulation by factors ATF4/5 and C/EBP-homologous protein (CHOP) leads to increased expression of various mitochondrial chaperones and proteases that enhance protein folding and clearance of unsalvageable proteins in the mitochondria (Melber and Haynes 2018). In the context of aging, mild mitochondrial stress might be beneficial for mitochondrial function due to hermetic activation of the mtUPR. However, similar to mtUPR insufficiency, overactivation of this pathway can be detrimental (Lima et al. 2022).

Since the discovery of the mtUPR in lower organisms, the significance of this pathway in mammalian systems has become increasingly appreciated. Indeed, growing evidence links mtUPR dysregulation to various human diseases, including neurodegenerative and cardiovascular pathologies (Zhou et al. 2022). Until now, the mtUPR is understudied in skeletal muscle, despite novel indications of high relevance for the sustenance of mitochondrial function and biogenesis, and exercise adaptation in this tissue (reviewed in (Richards et al. 2023)).

Recently, the mtUPR was implicated as a link between mitochondrial and cytoplasmic proteostasis in the context of insulin sensitivity in muscle (Munoz et al. 2023). Mitochondrial protein synthesis inhibition blunted expression of mtUPR markers in muscle cells and subsequent insulin treatment led to reduced phosphorylation of Akt and AS160 (Munoz et al. 2023). Such an association could be highly relevant in muscle wasting contexts. For example, skeletal muscle aging is affected by the mtUPR regulator ATF4 (Miller et al. 2023). Inversely, exercise-induced mitochondrial biogenesis poses a protein folding challenge that requires fine-tuning by the mtUPR to produce functional mitochondria. Accordingly, acute exercise is a potent inducer of ATF5 expression (Slavin et al. 2022), and mtUPR engagement has been observed after chronic contractile activity in vitro (Mesbah Moosavi and Hood 2017). PGC-1α, a regulatory nexus for endurance exercise adaptation in skeletal muscle, therein provides a layer of coordination between cytosolic and mitochondrial UPR axes functionally interacting with ATF6a and ATF5, respectively (Wu et al. 2011; Slavin et al. 2022). Specifically, PGC-1α coactivates ATF6α, which promotes the induction of UPR genes involved in managing endoplasmic reticulum stress (Wu et al. 2011). Knockout of PGC-1α blunted expression of downstream ATF5 mtUPR targets (Slavin et al. 2022). In aged mice, where ATF5 expression is diminished and mtUPR targets are not normally induced despite proteostatic stress, exercise can still stimulate the expression of some of these mtUPR targets (de Smalen et al. 2023).

3.3. Mitochondrial proteases

Like their cytoplasmic counterparts, mitochondrial proteases degrade proteins that are unassembled, misfolded or damaged. There are more than 40 mitochondrial proteases described in lower organisms. These proteases are localized in different compartments of mitochondria, and have additional functions besides protein degradation, including metabolic control, protein import, apoptosis and stress signaling (Quiros et al. 2015; Ahola et al. 2019; Deshwal et al. 2020). The understanding of mitochondrial proteases in skeletal muscle is rudimentary, and is mainly focused on presenilins-associated rhomboid-like protein (PARL), caseinolytic protease (CLPP) and LONP. PARL for example, regulates optic atrophy 1 (OPA1) in cristae remodeling (Cipolat et al. 2006). Moreover, reduced PARL expression was observed in aged human subjects, as well as type 2 diabetic rats and human patients, and was associated to impaired insulin signaling and oxidative metabolism (Civitarese et al. 2010).

Although CLPP has been described as an inducer of the mtUPR, it may be a dispensable factor given that activation of the mtUPR was not reduced in heart tissue lacking CLPP expression (Seiferling et al. 2016). In muscle cells however, knockdown of CLPP leads to a decline in mitochondrial function and respiration, as well as cellular morphology and differentiation (Deepa et al. 2016). Surprisingly, loss of CLPP in mice evoked increased insulin sensitivity and resistance to diet-induced obesity, possibly through compensatory mechanisms (Bhaskaran et al. 2018). Similar effects were observed in Overlapping with the M-AAA protease 1 homolog (OMA1) in animals fed a control, but not when exposed to a high fat diet (Ahola et al. 2019).

LONP safeguards mitochondrial proteostasis by degrading protein aggregates in the mitochondrial matrix. Disuse and aging leads to a reduced expression of LONP in skeletal muscle, giving rise to mitochondrial dysfunction (Bota et al. 2002; Guo et al. 2022; Xu et al. 2022). Although mitochondrial proteolysis seems critical for maintenance of mitochondrial function, the causal link between an inbalance of muscle mitochondrial proteostasis and physiological outcomes remains unclear.

3.4. Mitochondrial long-lived proteins

Protein levels are determined by synthesis, degradation and inherent or context-dependent stability. Recent findings on mitochondrial protein half-lives provide an interesting perspective on the assumed dynamic nature of mitochondria in skeletal muscle. In post-mitotic tissues such as heart, muscle and brain, mitochondrial long-lived proteins (mtLLPs) can persist up to 4-6 months (Price et al. 2010; Fornasiero et al. 2018; Bomba-Warczak et al. 2021; Krishna et al. 2021). These mtLLPs are cristae-associated, and include ETC complex components as well as mitochondrial contact site and cristae organizing system (MICOS) proteins (Fornasiero et al. 2018; Bomba-Warczak et al. 2021; Krishna et al. 2021). Strikingly, these proteins have a much higher turnover rates in tissues such as liver and spleen (Price et al. 2010; Bomba-Warczak et al. 2021). The longevity of mtLLPs in post-mitotic tissues varies, possibly due to different metabolic demands (Fornasiero et al. 2018). Interestingly, mtLLP-containing multiprotein complexes exhibit limited subunit and complex exchange rates, and the stability of mtLLPs is increased in ETC super- compared to individual complexes(Krishna et al. 2021). It thus is possible that mtLLPs play a role in maintaining stability and function of the ETC. However, deletion of the Cytochrome C Oxidase Subunit 7C (COX7C) gene, encoding a mtLLP that anchors complex I and VI, does not result in the loss of the protein or supercomplex assembly (Krishna et al. 2021). The presence of mtLLPs may be particularly crucial in maintaining mitochondrial structure and function during cellular stress, e.g. in aging, when expression of ETC proteins is reduced. However, this concept has not been critically tested in skeletal muscle. Moreover, the mechanistic underpinnings of the stability of mtLLPs, which also confers protection from proteostatic challenges such as protein oxidation and posttranslational modifications, remains unclear. This remarkable longevity of some components of the mitochondrial proteome in skeletal muscle challenges the conventional notion that mitochondrial function is dependent on high dynamism and rapid turnover.

4. Mitochondrial dynamics

Dynamic remodeling of mitochondrial morphology provides a mode of mitochondrial maintenance, elimination and expansion, for example restricting damage by pinching off defective mitochondrial compartments, replacement of mitochondrial content through fusion with functional organelles, or increasing mitochondrial number since these organelles cannot be generated de novo. The processes of fusion and fission, and the involvement of the factors mitofusin (MFN) 1, 2 and OPA1, as well as dynamin-related protein 1 (DRP1) and fission 1 (FIS1), respectively, therein have recently been expertly reviewed (Chan 2012; Mishra et al. 2015; Pernas and Scorrano 2016), and the implications for skeletal muscle function described (Romanello and Sandri 2023). Here, we therefore focus on alternative aspects of mitochondrial dynamics, which could also contribute to mitochondrial remodeling in skeletal muscle.

4.1. Mitochondrial cristae organization

Cristae formation of the inner mitochondrial membrane markedly enlarges surface area, and helps in organizing mitochondrial respiratory complexes into structures known as supercomplexes. Cristae organization is therefore strongly associated with mitochondrial function and cellular energy metabolism. Besides changes in the amount of membrane, cristae can also be actively remodeled through widening and tightening of cristae junctions and lumen, as a way to deal with physiological cues such as energetic stress. Cristae remodeling is prominent in skeletal muscle, e.g. in endurance and resistance athletes where cristae density can increase up to 20% (Nielsen et al. 2017; Botella et al. 2023). Importantly, high density of cristae structures is required for the formation of OXPHOS supercomplexes, which also is stimulated by exercise (Cogliati et al. 2013; Greggio et al. 2017; Baker et al. 2019). In addition to the regulation of energy production, intact structural cristae integrity protects against mtDNA release and subsequent inflammation (He et al. 2022). So far, various factors have been implicated in this stabilization process, even though their role in skeletal muscle requires further study (Ikeda et al. 2013; Perez-Perez et al. 2016). Mechanical instability engages the mitochondria-localized actin motor-Myosin 19 (Myo19) to provide a tethering force for cristae morphogenesis (Shi et al. 2022). OPA1 (Frezza et al. 2006) and the MICOS complex (Stephan et al. 2020), both situated at cristae junctions, are likewise important regulators of cristae remodeling (Baker et al. 2019). Accordingly, decreased expression of OPA1 in aging and other pathological contexts is associated with mitochondrial dysfunction (Levytskyy et al. 2018), reviewed in (Noone et al. 2022). Inversely, increased OPA1 levels in swimmers after high-intensity high-volume training, but not in sprint interval training, is associated with cristae remodeling (Huertas et al. 2019). The proteases YME1-like protein 1 (YME1L), ATPase Family AAA Domain Containing 3A (ATAD3), PARL and OMA1 are also involved in this process, even though the mechanistic underpinnings remain poorly understood (Cipolat et al. 2006; Stiburek et al. 2012; Levytskyy et al. 2018; Peralta et al. 2018). Yme1L and OMA1 regulate OPA1 processing in the coordination of fusion and fission (Anand et al. 2014), but how such a regulatory axis could pertain to cristae morphology has not been described.

4.2. Inter-mitochondrial communication

In contrast to many other cell types, skeletal muscle cells are densely packed with myofibrils, which inherently limits mitochondrial motility and dynamic flexibility. To overcome these structural limitations, skeletal muscle mitochondria utilize alternative communication and quality control mechanisms to compensate for a reduced frequency of fusion events, for example when compared to muscle progenitor cells devoid of myofibrils (Eisner et al. 2014).

Intermitochondrial junctions (IMJs) are H⁺-permeable contact sites of outer mitochondrial membranes that enhance membrane potentials across organelles, thereby optimizing energy production without the need for bona fide fusion (Glancy et al. 2017). Importantly, such an organization facilitates rapid physical and electrical decoupling in the case of local dysfunction, containing aberrant membrane potentials from propagating (Glancy et al. 2017). IMJs are characterized by alignment of cristae (Picard et al. 2015), and, like cristae morphology, are regulated by exercise in skeletal muscle mitochondria (Picard et al. 2013a). In addition to IMJs, mitochondrial ‘kissing’ helps to compensate for compromised spatial dynamic flexibility. Mitochondrial ‘kissing’, initially described in (cardio)myocytes, encompasses transient fusion events in which mitochondrial content is exchanged (Liu et al. 2009; Huang et al. 2013). Similar intimate contacts have been observed in skeletal muscle (Picard et al. 2013b; Lavorato et al. 2017). However, the regulation of this process, and the payload that is exchanged are currently unknown.

The necessity for direct contact can be circumvented by mitochondrial nanotunnels. These structures connect distant mitochondria through long protrusions, enabling exchange of mitochondrial matrix content without fusion of two organelles (Vincent et al. 2017). Nanotunnels allow passage of larger proteins, however are inpermissive for the transfer of mtDNA nucleoids due to size limitations (Vincent et al. 2016; Vincent et al. 2019). Currently, little is known about the regulation of nanotunnel formation, including the signaling mechanisms that initiate their formation. In cardiomyocytes, calcium dysregulation is a potent trigger for nanotunnel formation (Lavorato et al. 2017). Muscle biopsies of mtDNA disease patients show increased nanotunnel formation, which implicate nanotunnels in mitochondrial stress mitigation through functional complementation (Vincent et al. 2019). Whether exercise-induced perturbations affect nanotunnels in skeletal muscle is unclear. The fact that fusion rates are lower in glycolytic muscle fibers raises the question whether nanotunnel formation, which may provide a compensatory mechanism upon metabolic strain, is fiber-type dependent (Mishra et al. 2015). Curiously, muscle nanotubes are affected by age, with increased nanotunnel formation at 1 year, followed by a reduction in 2-year old mice (Vue et al. 2022). The regulation of and coordination between of the formation of IMJs, mitochondrial “kissing” and nanotube dynamics in skeletal muscle in health and disease are currently unknown.

Mitochondrial dynamics also facilitate between-organelle exchange in individual cells. Such inter-cellular horizontal transfer of mitochondria was observed in mesenchymal stem cells, vascular smooth muscle cells, cardiomyocytes and -fibroblasts, as well as in the transition of primary myoblasts to myofibers. Stem cells, mediators of tissue resilience and regeneration, are often the donors in this process (Tavi et al. 2010; He et al. 2011; Vallabhaneni et al. 2012; Shen et al. 2018). For example, the integration of isolated mitochondria into myoblasts in microfluidic devices led to improved differentiation into myotubes(Sun et al. 2022). Such a process could be important for tissue differentiation and repair, but could also have therapeutic implications in skeletal muscle pathologies (Dong et al. 2023).

4.3. Mitochondria-associated membranes (MAMs)

Mitochondria-associated membranes are critical sites for the maintenance of mitochondrial morphology, as well as for metabolic function and cellular homeostasis. Contact sites between mitochondria and the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR) are best described in muscle (reviewed in (Narendra et al. 2010)). These sites facilitate the exchange of ions and lipids, coordinate the activation of signaling pathways, and are implicated in glucose homeostasis. Aging, obesity and type 2 diabetes are associated with a loosening of contacts between mitochondria and the ER and an ensuing alteration of morphology in skeletal muscle (Tubbs et al. 2018; Lu et al. 2022). Contact sites between mitochondrial membranes and other organelles such as endosomes, lysosomes and peroxisomes exert a variety of functions, including spatial positioning, dynamics and transfer of lipids, calcium, iron and metabolites (reviewed in (Lackner 2019; Harper et al. 2020)). The frequency, plasticity and function of such contact sites have yet to be elucidated in skeletal muscle.

Degradation

Dysfunctional and defective mitochondria possess an escalating arsenal of repair mechanisms. First, as discussed above, proteostatic stress can be relieved by different pathways, for example the degradation of proteins located in the mitochondria matrix and intermembrane space by proteases, and VCP/p97-assisted clearance of outer membrane proteins by the proteasome. However, if insufficient, these processes are complemented or replaced by more drastic measures.

4.4. Mitophagy

Mitophagy constitutes the main quality control mechanism to eliminate damaged mitochondria. To do so, a PINK/Parkin-dependent signaling pathway triggers the engulfment of whole mitochondria in an autophagosome, which subsequently fuses with a lysosome, thereby leading to the degradation of the defective organelle. If successful, mitochondrially-initiated apoptosis, the last resort of defense against mitochondrial dysfunction, can be averted in certain contexts. Interestingly, in contrast to wholesome mitophagy, recent evidence shows that piecemeal or bit-by-bit removal of mitochondria through the formation of mitochondrial-derived vesicles (MDVs) provides an additional means for mitochondrial quality control. MDVs are small vesicles, formed upon mitochondrial stress, that can expel cargo such as oxidized proteins from mitochondria. The vesicles bud off from the outer mitochondrial membrane, and fuse with lysosomes in the endosomal degradative pathway. Alternatively, MDVs can also enter the extracellular space through the secretory pathway (reviewed in (Picca et al. 2023)). Piecemeal mitophagy might prevent the wasteful degradation of whole mitochondria. When all of these processes fail or turn out to be inadequate, mitochondria are pushed beyond a point of repair by Parkin-mediated selective degradation of outer mitochondrial membrane proteins by the proteasome. This causes rupture of the mitochondrial membrane, thereby triggering mitophagy as a mean of clearance of whole organelles (Chan et al. 2011; Yoshii et al. 2011). Moreover, Parkin-mediated mitophagy was associated with proteasome activation on a larger scale, leading to denervation atrophy in slow twitch muscles (Furuya et al. 2014).

4.5. Apoptotic pathways

As a last response to mitochondrial damage that cannot be mitigated, apoptotic pathways are induced. In skeletal muscle, this predominantly leads to muscle protein loss and fiber atrophy, opposed to the programmed cell death as is normally observed in other cell types (reviewed in (Dupont-Versteegden 2006; Marzetti et al. 2010; Powers et al. 2012)). The molecular mechanisms that underlie muscle wasting caused by mitochondrial damage are understudied and not well understood across different muscle atrophy conditions. In many contexts, caspases, in particular caspase 3, are the main inducers of apoptosis. In response to cytochrome c release from dysfunctional mitochondria, caspase 3 is activated and elevates proteosomal activity in muscle cells (Wang et al. 2010). In addition, caspase 3 directly contributes to muscle protein loss due to its proteolytic activity on actin-myosin contractile complexes (Du et al. 2004). However, contrasting findings of unchanged caspase 3 activity in apoptosis have been described, suggesting a context- or disease-dependent engagement of this and other caspases or factors to exert pro-apoptotic functions (Leeuwenburgh et al. 2005; Siu et al. 2005; Plant et al. 2009). Indeed, caspase-independent pathways, for example mediated via Endonuclease G (EndoG) and Apoptosis Inducible Factor (AIF), cause DNA fragmentation and thereby stimulate apoptosis in muscle (Leeuwenburgh et al. 2005; Siu and Alway 2005). Moreover, it has been proposed that apoptosis in skeletal muscle is induced in a cell-type specific manner (Dupont-Versteegden 2006). Thus, for the development of strategies to counteract muscle atrophy, it is imperative to understand the contribution of mitochondrial dysfunction and the activation of apoptotic pathways in detail, and how these differ in their activation across (patho)physiological contexts.

5. Concluding Remarks

Mitochondrial maintenance is intrinsically tied to skeletal muscle function. Accordingly, mitochondrial dysfunction, and the inability for adequate repair and compensation, is observed in various muscle pathologies. It therefore is unsurprising that muscle mitochondria use the full repertoire of proteostatic stress relief and organelle repair to be able to meet the highly variable metabolic rates of this tissue at rest and when exercised (summarized in Figure 2). The special cellular morphology of skeletal muscle fibers, with high spatial constraints due to the myofibrillar and sarcomeric structures, and the mostly post-mitotic nature of these cells constitute challenges that are unique to this tissue. Piecemeal mitophagy, mitochondrial-derived vesicles, nanotubes, mitochondrial “kissing” or IMJs are exciting processes that have recently described, and which complement and expand the biogenesis, fusion/fission and degradation pathways. Unfortunately, to date, many of these mechanisms have mostly been studied in lower organisms, and detailed insights into mammalian biology, in particular that of skeletal muscle, remain elusive. It, however, is clear that such insights have the potential to challenge pre-existing notions of mitochondrial biology, for example, the dichotomy of the postulated high rates of dynamics and turnover in light of the existence of mtLLPs.

Figure 2. Overview of mitochondrial maintenance mechanisms in skeletal muscle.

(A) In basal conditions, chaperones assist the import of translated nuclear genes encoding mitochondrial proteins (NuGEMPs) into mitochondria during mitochondrial biogenesis upon transcriptional activation by peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α), estrogen-related receptor α (ERRα), Nuclear Respiratory Factor 1 and 2 (NRF-1/2) and other regulators. The formation of the electron transport chain (ETC) depends on the expression and assembly of subunits encoded by both the nuclear and mitochondrial DNA. Chaperones and proteases located in the mitochondrial matrix help to prevent unassembled or misfolded proteins from accumulating. Certain cristae-associated proteins, so-called mitochondrial long-lived proteins (mtLLPs), have remarkably long half-lives, providing the ETC with higher stability. Remodeling of cristae by increasing their density or tightening of cristae junctions fine-tunes metabolic activity. Nanotunnels, inter-mitochondrial junctions (IMJs) and contact sites with other organelles such as the endoplasmic reticulum (ER) are ways that allow for dynamic remodeling of the mitochondrial network, and sharing of mitochondrial content, proton gradient, metabolites and ions such as calcium.

(B) Mild stress, such as clogging of mitochondrial import channels, leads to cytosolic accumulation of activating transcription factor 5 (ATF5), which translocates to the nucleus and transcriptionally activates the mitochondrial unfolded protein response (mtUPR) through coactivation of C/EBP-homologous protein (CHOP) and other factors. This increases the expression of proteases and chaperones to clear damaged proteins. Alternatively, these proteins can be expelled via mitochondria derived vesicles (MDVs). Damaged sub-compartments of mitochondria, or MDVs, are degraded by piecemeal mitophagy.

(C) Upon further damage, whole organelles are engulfed and degraded by mitophagy. Finally, apoptotic pathways are induced via factors including caspase 3 (Casp3), Endonuclease G (EndoG) and Apoptosis Inducible Factor (AIF). This give rise to increased proteosomal activity and DNA fragmentation, ultimately leading to muscle atrophy.

Aging has been associated with changes in various distinct maintenance mechanisms discussed in this review, including mitochondrial translation, mtUPR, protease expression, MAM contact sites and cristae-associated proteins. However, the etiology of sarcopenia, particularly concerning mitochondrial dysfunction, and the underlying mechanisms that lead to the failure of these mitochondrial maintenance systems, remain largely unknown. In a more general sense, mitochondrial dysfunction has been reported in a large number of pathologies, from type 2 diabetes to neurodegenerative disorders, metabolic syndrome or cancer, basically almost all of the most prevalent diseases (San-Millán 2023). Future work will show the implication of mitochondrial maintenance as disease-causing event, as well as the potential to leverage the corresponding processes as therapeutic targets. Thus, undoubtedly, the coming years will yield breakthroughs in the understanding of mitochondrial maintenance and function, with high relevance not only for our understanding of muscle plasticity evoked by exercise, but also the patho-etiological events in muscle pathologies and aging.