Regulation of calcium homeostasis in endoplasmic reticulum–mitochondria crosstalk: implications for skeletal muscle atrophy

Abstract

This review comprehensively explores the critical role of calcium as an essential small-molecule biomessenger in skeletal muscle function. Calcium is vital for both regulating muscle excitation–contraction coupling and for the development, maintenance, and regeneration of muscle cells. The orchestrated release of calcium from the endoplasmic reticulum (ER) is mediated by receptors such as the ryanodine receptor (RYR) and inositol 1,4,5-trisphosphate receptor (IP3R), which is crucial for skeletal muscle contraction. The sarcoendoplasmic reticulum calcium ATPase (SERCA) pump plays a key role in recapturing calcium, enabling the muscle to return to a relaxed state. A pivotal aspect of calcium homeostasis involves the dynamic interaction between mitochondria and the ER. This interaction includes local calcium signaling facilitated by RYRs and a “quasi-synaptic” mechanism formed by the IP3R-Grp75-VDAC/MCU axis, allowing rapid calcium uptake by mitochondria with minimal interference at the cytoplasmic level. Disruption of calcium transport can lead to mitochondrial calcium overload, triggering the opening of the mitochondrial permeability transition pore and subsequent release of reactive oxygen species and cytochrome C, ultimately resulting in muscle damage and atrophy. This review explores the complex relationship between the ER and mitochondria and how these organelles regulate calcium levels in skeletal muscle, aiming to provide valuable perspectives for future research on the pathogenesis of muscle diseases and the development of prevention strategies.

Introduction

The opening and closing of calcium channels modulate various key biological processes. Although 99% of calcium is stored in bones, it is essential for neuromuscular function. The role of calcium extends beyond excitation–contraction coupling (ECC) in muscles and is also imperative for the development, maintenance, and regeneration of skeletal muscle [1].

The contraction of skeletal muscles necessitates the release and absorption of calcium from the sarcoplasmic reticulum (SR), a process intricately supported by store-operated calcium entry (SOCE) [2]. This process is mediated by the stromal interaction molecule (STIM) in the SR membrane and the calcium release-activated calcium modulator 1 (ORAI) on the plasma membrane. Upon the activation of RYR1 and the resulting depletion of the SR calcium store, the STIM undergoes conformational changes. It assembles into oligomers, activates ORAI, and promotes the influx of extracellular calcium. This influx compensates for the reduction in SR calcium, thus preventing severe calcium loss in myofibers and mitigating muscle fatigue during ECC [3]. It also requires considerable amounts of ATP during the contraction and relaxation phases to meet its high metabolic needs and sustain intracellular calcium homeostasis [4]. As a key regulator of ATP production, mitochondria act not only as powerhouses of the cell but also as central hubs of cellular calcium signaling, determining cell viability [5]. Although mitochondrial calcium uptake is vital for muscle cells, it is a double-edged sword, as it plays a critical role in both cell survival and death. In skeletal muscle cells, mitochondria are closely adjacent to the endoplasmic reticulum (ER), comprising 10–15% of the fiber volume, and are densely arranged [4]. Under physiological conditions, mitochondrial calcium uptake is substantial and rapid, significantly influencing calcium signals in the cytoplasm of skeletal muscles. This uptake also serves as a calcium buffer and enhances ATP synthesis. However, owing to the heterogeneity of muscle tissue, the permissible range of mitochondrial calcium concentrations under physiological conditions continues to be a subject of debate and investigation. Moreover, mitochondrial calcium uptake regulates muscle mass by triggering two skeletal muscle hypertrophy pathways, namely, PGC-1α4 and IGF1-AKT/PKB [6]. Therefore, any imbalance in mitochondrial calcium uptake and its dynamics can precipitate mitochondrial dysfunction and muscle cell deterioration [4, 7] (Fig. 1).

Fig. 1

It presents a detailed schematic of calcium transport within skeletal muscle cells. The ER features three primary calcium transport channels: the SERCA, RYR, and IP3R. These channels facilitate the efflux of calcium from the ER, playing a crucial role in calcium delivery to both the cytoplasm and mitochondria. This process is integral to skeletal muscle contraction and the activation of the mitochondrial TCA cycle, which in turn promotes ATP production. Among these channels, the IP3R forms a ‘quasi-synaptic’ connection with mitochondrial membrane proteins, namely VDAC and the MCU, through the bridging molecule Grp75. This structure facilitates the direct transfer of calcium from the ER to the mitochondria. A dysregulation in this calcium transport can lead to mitochondrial matrix calcium ([Ca²⁺]m) overload. In response, the MPTP opens, resulting in the release of ROS and cytochrome C. Such events can precipitate skeletal muscle atrophy

Research has demonstrated that approximately 5–20% of the outer mitochondrial membrane (OMM) is in close proximity to the ER [8]. The distance between the smooth ER and mitochondria is approximately 10–50 nm, whereas that between the rough ER and mitochondria is 50–80 nm [9]. This interorganellar distance is pivotal for calcium transfer and strongly influences cell survival and apoptosis [8, 10]. Therefore, this review aims to focus on the regulation of calcium transfer from the ER to mitochondria under both physiological and pathological conditions, clarify the mechanisms underlying skeletal muscle atrophy-related myopathies, and provide therapeutic perspectives.

Regulation of mitochondrial calcium by the endoplasmic reticulum

The function of skeletal muscle relies on the availability of calcium. The SR in skeletal muscle is excellent for the uptake, storage, and regulated release of calcium, serving as the central hub for calcium dynamics [11]. In striated muscle, the primary source of calcium is the intracellular SR calcium reserve rather than the extracellular medium. The ER and mitochondria form a tightly interconnected network, where the mitochondria-associated endoplasmic reticulum membrane (MAM) facilitates calcium transfer from the ER to mitochondria. The spatial dynamics between the ER and mitochondria may be a key determinant of mitochondrial calcium uptake [12]. In addition, excessive release of calcium from the ER creates a state of calcium overload in mitochondria, which in turn activates mitochondrial permeability transition pore (MPTP). This activation leads to fragmentation, swelling, and eventual death of the organelles [13, 14]. Therefore, the regulated release and efficient cycling of calcium in the ER are associated with the maintenance of mitochondrial calcium homeostasis.

Ryanodine receptor 1 (RYR1)

Ryanodine receptors (RYRs) consist of three isoforms encoded by distinct genes, namely, RYR1, RYR2, and RYR3. RYR1 is predominantly expressed in skeletal muscle, RYR2 in cardiac muscle, and RYR3 in smooth muscle, the brain, and the developing diaphragm [15]. RYR1, a six-transmembrane homotetrameric protein with four 550 kDa subunits located in the SR, is the principal calcium channel in the ER of skeletal muscle [16]. During ECC, RYR1 interacts locally and directly with dihydropyridine receptors in the plasma membrane to facilitate physiological calcium release from skeletal muscle [17]. A highly dynamic physical distance of 10–50 nm is achieved by specific tethering proteins, including IP3R–Grp75–VDAC, PACS2, DRP1, MFN2 and the Mmm1–Mdm10–Mdm12–Mdm34 complex. These proteins facilitate close SR–mitochondria interactions and allow local calcium communication between mitochondria and RYRs [18,19,20,21,22]. RYR-mediated calcium release creates a high-calcium microdomain between the SR and mitochondria, promoting rapid mitochondrial calcium uptake from this localized source. Consequently, this mechanism does not significantly alter overall cytoplasmic calcium concentrations [23]. In skeletal muscle cells, Brini et al. [24] utilized caffeine to induce SR calcium release and observed that mitochondria accumulated 15% of the calcium released from the SR.

Reports have indicated that there is a direct correlation between RYR protein expression and muscle strength. Pelletier et al. [25] performed calcium flux assays in isolated myofibers and revealed that a reduction in RYR1 protein levels led to reduced SR calcium release, abnormal mitochondrial distribution in skeletal muscle, and progressive atrophy of myofibers. Compared with wild-type mice, RYR-deficient mice presented 20% and 50% reductions in body weight and spontaneous running distance, respectively, within the first 20 weeks of life [26]. Moreover, RYR-deficient mice presented significant mitochondrial mismatch and myofibrillar disarray in their skeletal muscle. Studies have shown that RYR1 dysfunction can result in a substantial and sustained increase in cytoplasmic calcium, leading to cell death by affecting mitochondrial matrix calcium at rest [27]. These findings are consistent with the phenotype of RYR1-associated myopathy (RYR1-RM), which is characterized by persistent leakage of cytosolic calcium from myocytes at rest, elevated mitochondrial oxidative stress, hypotonia, mild facial weakness, and joint laxity [28].

Together, these findings imply that RYR1 plays a crucial role in releasing calcium and influencing mitochondrial calcium uptake. This process occurs via either the propagation of cytoplasmic calcium signals to the mitochondrial matrix or the establishment of local calcium domains directly with the mitochondria, thereby regulating essential cellular functions.

Inositol 1,4,5-trisphosphate receptor (IP3R)

The inositol 1,4,5-trisphosphate receptor (IP3R), a tetrameric calcium channel composed of four subunits with a molecular weight of approximately 310 kDa, is ubiquitously expressed in the ER membrane. The three isoforms of IP3Rs (IP3R1, IP3R2, and IP3R3), which are encoded by three distinct ITPR genes, have an overall similarity of 75–80% [29, 30]. IP3Rs are crucial for normal skeletal muscle activity and regulates gene expression, energy metabolism, and mitochondrial function [31].

In contrast to RYRs, IP3Rs are located on the mitochondria-facing surface of the SR in muscle tissue [32, 33]. IP3Rs have been identified as a critical anchoring site for ER‒mitochondrial contact points [34]. It is theorized that upon IP3R activation, the concentration of cytosolic calcium increases to 0.5–1 µM. However, these levels are insufficient to trigger a low-affinity mitochondrial calcium uptake mechanism [35]. Advanced studies utilizing luminescent proteins derived from jellyfish and targeting the mitochondrial matrix have been performed. Their results showed that in various cell types, despite the low affinity of mitochondria for calcium, IP3-induced calcium release occurs nearby, resulting in the formation of local microdomains. In response to IP3 stimulation, the calcium concentration near the mitochondria significantly increases, leading to a significant increase in matrix calcium [36, 37]. A comparative study by Csordás et al. [35] indicated that the size of these microdomains is < 1 µM², approximately 10% of the mitochondrial surface area; however, the calcium concentration within these microdomains is ≥ 15 µM, which is 20 times greater than the general cytoplasmic calcium concentration. Furthermore, their findings demonstrated that calcium released by IP3Rs can increase mitochondrial matrix calcium levels by up to 50% [38]. This observation suggests the privileged and direct transfer of calcium released by IP3Rs between the ER and the mitochondria. Furthermore, Csordás et al. [35] noted that each mitochondrial calcium uptake site is opposite to multiple IP3Rs, which helps integrate calcium from various IP3Rs into each mitochondrial uptake site. Therefore, synchronized activation of multiple IP3Rs can optimize mitochondrial calcium uptake.

Subsequent studies have shown that the calcium liberated by IP3Rs can be transported to the mitochondria via a mechanism similar to quasi-synaptic transfer. IP3Rs form an interaction link with voltage-dependent anion channels (VDACs) located on the OMM. This interaction is facilitated by the molecular chaperone glucose-regulated protein 75 (Grp75), which forms an ER-mitochondrial tethering complex within the MAM. This complex is crucial for bidirectional calcium transfer between these two organelles [18, 39]. Furthermore, a study has shown that lipopolysaccharides can cause mitochondrial calcium overload by strengthening the IP3R-Grp75-VDAC/MCU axis, leading to cellular necrosis [40]. In adult skeletal muscle fibers, IP3Rs play a pivotal role in mediating the increase in mitochondrial calcium following depolarization. This process is essential for promoting excitation–metabolism coupling between muscle fibers and mitochondria [41]. Moreover, IP3Rs facilitate mitochondrial calcium uptake and then participates in the tricarboxylic acid (TCA) cycle. This uptake stimulates the activity of key enzymes, thereby regulating the mitochondrial production of ATP and NADH and contributing to mitochondrial bioenergetics [9]. Among the three IP3R isoforms, IP3R2 is the most efficient at transporting calcium from the ER to mitochondria, followed by IP3R3 and, ultimately, IP3R1 [42]. This efficiency is attributed to the proximity of IP3R2 to mitochondria, which enhances its ability to facilitate ER-to-mitochondria calcium release. However, a study by Mendes et al. [43] showed that silencing the IP3R3 isoform is more effective at inhibiting mitochondrial calcium signaling than the other two isoforms. These findings suggest that the isoforms may have synergistic or compensatory mechanisms in maintaining calcium homeostasis. Future research should further explore the interactions between these isoforms and their specific impacts on muscle function to reveal a more comprehensive regulatory network.

In addition, the involvement of IP3-induced calcium release from IP3Rs in the regulation of apoptosis and autophagy has been well documented [44]. IP3Rs play a dual role in these processes. First, it enhances autophagy in response to cellular stress by supplying calcium to the cytoplasm, which is necessary for autophagic flux. Second, the transfer of calcium from IP3Rs to the mitochondria inhibits autophagy at the initial stage. This inhibition is mediated by a reduction in the level of AMP-activated protein kinase (AMPK) due to sustained mitochondrial metabolic activity and energy demands [45]. A decrease in IP3R expression leads to inefficient mitochondrial ATP generation, resulting in an increase in the AMP/ATP ratio, thereby activating AMPK and subsequently inducing autophagy [46] (Fig. 2). In contrast, an increase in IP3R expression increases cytoplasmic calcium levels, causing mitochondrial calcium overload, which triggers apoptosis, necrosis, and increased autophagic activity. Thus, the expression of IP3Rs is important for modulating autophagy and apoptosis pathways, either through an indirect effect on cytoplasmic calcium levels or a direct effect on mitochondrial calcium levels in a quasi-synaptic manner. Maintaining IP3Rs within its normal physiological range is fundamental for optimal calcium uptake by the mitochondria and, hence, for the preservation of skeletal muscle function.

Fig. 2

It illustrates the role of the IP3R in mediating autophagy through calcium release. Under normal physiological conditions, calcium liberated by IP3R are predominantly taken up by mitochondria to facilitate ATP synthesis. This mitochondrial activity leads to a reduction in AMP levels, subsequently inhibiting the autophagic process. Conversely, a reduction in IP3R expression diminishes the release of calcium, insufficient for optimal mitochondrial ATP generation. This deficit triggers an increase in AMP-activated protein kinase (AMPK) activity, which serves as a cellular energy sensor and, in turn, activates autophagy

Sarcoplasmic reticulum calcium-ATPase (SERCA)

In muscle cells, the SR not only is a calcium reservoir but also plays a crucial role in regulating muscle contraction. When calcium is released through both RYRs and IP3Rs, muscle contraction begins. Subsequently, as cytoplasmic calcium levels return to those in the resting state (approximately 100 nM), SERCA actively pumps cytoplasmic calcium back into the SR in a counter-concentrative manner. This process is facilitated by the depletion of ATP and ultimately promotes relaxation of the muscle. SERCA is an integral membrane transport protein within the SR that contains three key domains, with a molecular weight of 110 kDa. Importantly, SERCA accounts for 90% of the SR membrane proteins and is the most abundant protein type in the SR [47]. SERCA is encoded by ATP2A1–3 genes and comprises three isoforms: SERCA1, SERCA2, and SERCA3. Among these isoforms, the SERCA1 isoform is expressed predominantly in fast-twitch fibers, the SERCA2 isoform is expressed in slow-twitch skeletal muscles, and the SERCA3 isoform is expressed in nonmuscle tissues [48]. The different expression patterns of these isoforms in muscles influence the dynamic changes in calcium in the SR. This, in turn, affects the rate and amount of calcium uptake, thereby influencing its storage and release from the SR in the muscle [49].

Recent investigations on SERCA in skeletal muscles have focused on SERCA regulators. Three distinct inhibitory proteins that modulate SERCA activity have been identified, including phospholamban (PLN), sarcolipin (SLN), and the newly discovered myoregulin (MLN) [50]. An investigation has shown that the elimination of any of these inhibitory proteins from healthy skeletal muscle leads to an increase in calcium uptake by the SR and an acceleration of muscle diastole [51]. Importantly, the binding characteristics of SLN to SERCA differ considerably from those of PLN. The interactions of PLN and calcium with SERCA are mutually exclusive, with PLN acting as an inhibitor only in the presence of free calcium [52]. When the cytoplasmic calcium concentration is high, calcium competes with PLN for binding to SERCA, thus inhibiting the action of PLN [53]. In contrast, SLN can inhibit SERCA both in the presence of free calcium and SERCA-bound calcium. This interaction does not alter SERCA activity but nullifies the transport of cytoplasmic calcium, augments ATP depletion, and leads to muscle thermogenesis [48]. Therefore, SLN is currently the focus of research as a nonshivering thermogenic regulator in muscles. Moreover, the inhibitory effect of SLN on SERCA modifies cytoplasmic calcium dynamics and enhances SR–mitochondrial communication. This increases the entry of calcium into the mitochondria and may cause mitochondrial calcium overload and muscle atrophy under pathological conditions (Fig. 3). Notably, in situations related to muscle wasting, atrophy, and diseases, SLN expression is upregulated, which intensifies muscle thermogenesis [54]. However, the study by Sopariwala et al. [55] presents an opposing view. They discovered that SLN overexpression in whole animals enhances endurance and resistance to muscle fatigue and improves skeletal muscle performance during prolonged periods of muscle activity. This improvement in muscle function may be attributed to the role of SLN in optimizing muscle ATP utilization, thereby enhancing muscle thermogenesis. This finding adds a novel dimension to our understanding of the function of SLN and highlights its potential to augment muscle endurance and performance.

Fig. 3

It depicts the interaction of SLN and PLN with the SERCA, and their respective roles in mediating calcium dynamics. Elevated cytoplasmic calcium concentrations impede the binding of PLN to SERCA. Contrarily, SLN exhibits a competitive binding affinity for SERCA in the presence of calcium, without significantly altering SERCA’s functional activity. The binding of SLN to SERCA results in inefficient calcium translocation, coupled with increased ATP consumption, which subsequently enhances thermogenesis within skeletal muscles. Furthermore, the binding of SLN to SERCA impedes the pump’s ability to transport calcium back into the ER. This inhibition leads to an accumulation of calcium in the cytoplasm, precipitating mitochondrial calcium overload. Such dysregulation can contribute to cellular damage and potentially instigate skeletal muscle atrophy

Furthermore, several activators of SERCA have been identified, including the dwarf open reading frame [56], the synthetic compound CDN1163 [57], and various natural dietary polyphenolic compounds [58]. It has been reported that SERCA pump dysfunction is associated with several chronic conditions, such as aging [59], denervation [60], and muscular dystrophy [61]. A prominent reduction in SERCA1 activity, coupled with an increase in oxidative stress, has been observed in both naturally aging and prematurely aging gastrocnemius muscles [62]. Interestingly, treatment with CDN1163 for 7 weeks reversed muscle atrophy and weakness in CuZnSOD-deficient mice by restoring SERCA activity [63]. Moreover, impaired SERCA activity can cause mitochondrial calcium overload and the opening of MPTP, culminating in cellular necrosis. This process is the principal pathological mechanism of dystrophic myopathies [64]. Similar phenomena have been observed in type 2 diabetes-induced muscle atrophy [65] and Duchenne muscular dystrophy (DMD) [66] models. These conditions are characterized by reduced SERCA expression, accompanied by continuous calcium influx into muscle cells and suboptimal intracellular calcium regulation, thereby leading to muscle fiber atrophy and necrosis. SERCA1 overexpression can significantly accelerate calcium reuptake in the SR and exert multiple protective effects, including protecting mitochondria from swelling, mitigating calpain activation, and providing some degree of membrane protection [64]. Additionally, this overexpression alleviates the detrimental effects of chronic calcium leakage due to channel activation and membrane rupture. Additionally, it enhances ECC of skeletal muscle, improves skeletal muscle metabolism and energy production, and ultimately leads to an overall improvement in muscle function.

Regulation of calcium by mitochondrial calcium channels

In skeletal muscle, the regulation of the mitochondrial calcium concentration plays a pivotal role in controlling muscle nutrition and strength [67]. Mitochondria function as essential sensors and regulators of calcium signaling and facilitate aerobic metabolism by facilitating calcium entry into the matrix to activate three TCA cycle dehydrogenases. However, calcium overload in the mitochondria can lead to the production of excessive ROS and trigger the opening of MPTP, which releases apoptotic factors and results in cell damage [68]. Furthermore, Andersson et al. [69] demonstrated a bidirectional relationship between RyR1 and mitochondria. They found that increased ROS production in mitochondria led to the oxidation of RyR1. This oxidation-induced calcium leak prompts mitochondria to produce even more ROS, thereby perpetuating a detrimental cycle. This vicious cycle may exacerbate the loss of muscle mass and contribute to disorders such as mechanical ventilation-induced diaphragmatic dysfunction [70], sarcopenia [69], spinal cord injury-induced muscle dysfunction [71], and muscle dysfunction in mdx mice [72].

Extensive literature suggests that mitochondrial dysfunction is involved in various skeletal muscle myopathies, including insulin resistance [73], muscle wasting [74], and muscular dystrophy [75]. Skeletal muscle dysfunction observed in septic mice and elderly patients with sarcopenia is correlated with a pathological increase in mitochondrial matrix calcium [76, 77]. Evidently, maintaining normal mitochondrial calcium uptake is crucial for the optimal physiological function of muscle. Therefore, the role of mitochondrial calcium delivery in skeletal muscle function should be scrutinized by investigating both mitochondrial calcium uptake and efflux channels. This exploration can provide valuable insights and contribute to the development of preventive measures for various muscle diseases.

Voltage-dependent anion channels (VDACs)

VDACs, commonly known as mitochondrial porins, are 30–35 kDa in size and are the most abundant mitochondrial outer membrane protein [78]. The mammalian VDAC gene family includes three isoforms (VDAC1, VDAC2, and VDAC3), each of which shares approximately 70% similarity with the other two family members [79]. Nevertheless, studies have shown that each of these three isoforms has unique physiological functions [80]. VDAC1-deficient mice remain viable, with only minor effects on mitochondria. The deletion of VDAC2 has been proven to be lethal, while mice lacking VDAC3 have been shown to be healthy [81]. It has been suggested that there may be channel types other than VDACs in the OMM, but these activities are considered alternative functional states of VDACs [82]. As VDAC is voltage-dependent, it can adopt various conformational states with different selectivity and permeability. Specifically, VDAC selectively uptakes calcium via channel gating with structural modifications [83]. VDAC also serves as a binding site for hexokinase and glycerol kinase and actively participates in diverse ATP-dependent oxidative phosphorylation events. This channel facilitates the transport of small-molecule metabolites, including ADP and ATP, with the pore size of VDACs being approximately 8-10-fold larger than that of calcium ions [84].

VDAC is the first regulator of mitochondrial calcium uptake and functions as a positive regulator of mitochondrial calcium accumulation [85]. Rapizzi et al. [85] demonstrated the aggregation of VDACs at the ER–mitochondrial contact site using immunofluorescence and electron microscopic studies. As a component of MAM, VDACs are closely linked to the IP3R calcium release channel on the SR [86]. When VDAC operates as a large conductance channel, it enables the calcium released by RYRs and IP3Rs to rapidly diffuse across the OMM and reach the mitochondrial intermembrane gap. This exposure activates the MCU with low calcium affinity in the inner mitochondrial membrane (IMM), leading to rapid calcium accumulation in the matrix owing to the high-calcium microdomains opened by the ER [85, 87]. There is a delay between the increase in calcium concentrations in the cytoplasm and that in the mitochondria, and the overexpression of VDAC significantly reduces this delay [88]. VDAC is a crucial determinant of calcium permeability at the MAM, and its permeability acts as a kinetic bottleneck in the process of mitochondrial calcium homeostasis. In skeletal muscle myotubes, the transient expression of VDAC increases the agonist-dependent calcium concentration in the mitochondrial matrix by mediating the rapid diffusion of calcium from ER release sites into the mitochondrial matrix [85].

Nevertheless, VDAC plays a role in pathological calcium signaling in mitochondria, as it is the primary impediment to mitochondrial calcium uptake [89]. Investigations have revealed that tissue-specific deficiency of the VDAC1 isoform in human skeletal muscle leads to rare mitochondrial encephalomyopathy [90]. Individuals affected by this condition display various malformations, including reduced muscle tone, impaired substrate oxidation, and decreased ATP production, which is fatal in childhood. In the context of DMD, mice with this condition exhibited significant downregulation of VDAC3 mRNA levels, which signifies the potential involvement of VDAC3 expression in early pathogenic events in mdx muscular dystrophy [91]. In both DMD patients and mdx mouse models, VDAC1 deficiency is associated with impaired energy metabolism in skeletal muscle [92]. Furthermore, VDAC plays a crucial role in regulating skeletal muscle function by modulating mitochondrial autophagy. By recruiting the ubiquitin ligase targeting mitochondria parkin to the mitochondria, VDAC facilitates the elimination of damaged mitochondrial proteins via mitochondrial autophagy [93]. Gouspillou et al. [94] observed a decrease in the parkin/VDAC ratio in the skeletal muscles of elderly individuals, which led to the accumulation of damaged mitochondria and adversely affected skeletal muscle mass and function. Moreover, VDACs have recently been associated with cytopathology. VDAC undergoes extensive conformational changes, which facilitate the release of cytochrome C, thereby mediating apoptosis under oxidative stress conditions [95]. VDAC is also involved in apoptotic calcium signaling by interacting with proapoptotic and antiapoptotic proteins of the Bcl-2 family [96]. Thus, the regulation of mitochondrial calcium uptake by VDACs is important for both the physiological function and dysfunction of skeletal muscle.

Mitochondrial calcium uniporter complex (MCUcx)

MCUcx, located in the IMM, has a molecular mass of approximately 480 kDa and is instrumental in facilitating mitochondrial calcium uptake [97]. It comprises pore-forming subunits (MCU and MCUb) and an essential MCU regulator (EMRE). These components collectively interact with the mitochondrial calcium uptake-regulating subunits (MICUs: MICU1, MICU2, and MICU3) to orchestrate the overall process of mitochondrial calcium uptake [98]. The ionic permeability of the IMM is low, requiring a potential difference of approximately − 160 to -180 mV across the two sides of the membrane. This substantial electrochemical gradient supports the function of MCUcx in mediating calcium influx into the mitochondrial matrix [99]. Furthermore, the latest progress made by Gottschalk et al. [100] through super-resolution microscopy studies indicates that the cristae membranes (CMs) within the IMM maintain a lower membrane potential than the inner boundary membranes (IBMs). When calcium enters the mitochondrial matrix, this differential in potential becomes particularly significant, altering the membrane potential and facilitating the voltage-dependent generation of ATP in skeletal muscle [101]. However, subsequent studies revealed that the MCUcx channel has a remarkable ability to bind calcium with nanomolar affinity, which facilitates a large amount of calcium influx into the mitochondrial matrix [102]. Therefore, mutations in MCUcx can severely impair the routine mechanisms of calcium uptake in mouse muscle mitochondria, triggering a series of events that culminate in muscle dysfunction disorders [103].

MCU, a protein with a molecular mass of approximately 40 kDa, is widely expressed and localized in the IMM. As a highly selective channel, MCU promotes mitochondrial calcium uptake [104]. The indispensability of MCU extends to the maintenance of mitochondrial energy metabolism and tissue function, especially in skeletal muscle [105]. MCU-mediated mitochondrial calcium uptake is necessary for the regulation of muscle size, rigidity, trophic utilization, and sarcolemma repair [6, 106]. Studies have revealed that mice lacking MCU exhibit significantly impaired locomotor function. The resting mitochondrial calcium level is reduced by 75%, which leads to muscle atrophy [6, 107]. Notably, Kwong et al. [108] demonstrated that the targeted knockout of MCU in skeletal muscle during the embryonic, postnatal, and adult stages inhibited acute mitochondrial calcium influx and calcium ion-stimulated mitochondrial respiration, resulting in decreased locomotor activity; however, MCU deficiency had no obvious effect on muscle growth or maturation. Furthermore, MCU deficiency altered the metabolic substrate preferences of mice, favoring fatty acid metabolism and enhancing muscle performance under fatigue conditions [109]. However, the effect of MCU deficiency on muscle fiber types remains controversial [104, 109]. MCU overexpression induces muscle hypertrophy by activating two major hypertrophic pathways in skeletal muscle, namely, PGC-1α4 and IGF1-AKT/PKB [6]. MCU overexpression further promotes the trophic utilization of skeletal muscle and offered protection against denervation-induced muscle atrophy. Importantly, a positive correlation is observed between MCU content and muscle size in the elderly, and intense physical activity increases the protein level of MCU in skeletal muscle [110]. Interestingly, Higashitani et al. [77] reported an age-related elevation in the expression of MCU in nematodes, which is associated with increased mitochondrial calcium uptake and autophagy, ultimately resulting in the occurrence of sarcopenia. Hence, the multifaceted role of MCU-mediated mitochondrial calcium uptake in skeletal muscle homeostasis remains debatable, and no consensus has been reached.

In contrast to MCU deficiency, several studies have shown that MICU1 deficiency leads to skeletal muscle atrophy and dysfunction. Clinically, individuals with MICU1 gene mutations manifest early-onset proximal muscle weakness and progressive extrapyramidal dyskinesia [103]. This deficiency lowers the mitochondrial calcium uptake threshold, resulting in calcium overload under basal conditions. Therefore, both mice and patients with MICU1 deficiency show muscle weakness, atrophy, and impaired sarcolemma repair [106]. The differential expression of MCU and MICU1 has distinct impacts on skeletal muscle function. To address this issue, Paillard et al. [111] proposed a hypothesis that mitochondrial calcium uptake in skeletal muscle depends on the relative abundance of MICU1 and MCU. The researchers suggested that different tissues that perform diverse functions present varying physiological MICU1/MCU ratios. Subsequent experimental studies support this hypothesis. Previous research revealed that a decrease in the MICU1/MCU ratio in the tibialis anterior muscle of septic mice resulted in an increase in mitochondrial matrix calcium at the basal level, leading to muscle atrophy [76]. This phenomenon is similar to that of embryonal rhabdomyosarcoma [112]. Consequently, a balanced interaction between MICU1 and MCU must be maintained for optimal skeletal muscle functionality (Fig. 4).

Fig. 4

It illustrates the impact of varying MICU1/MCU ratios on mitochondrial calcium uptake dynamics. Ordinarily, in skeletal muscle, the MICU1/MCU ratio is maintained at an optimal level, facilitating the precise regulation of mitochondrial calcium uptake. During pathological conditions, this balance is disrupted due to a decrease in the MICU1/MCU ratio. This alteration enables a rapid influx of cytoplasmic calcium ions into the mitochondria. Such unregulated entry of calcium, even in a resting state, precipitates mitochondrial calcium overload, which plays a critical role in the pathogenesis of muscle atrophy

MICU1 has two paralogous homologs, namely, MICU2 and MICU3, which play important roles in regulating mitochondrial calcium uptake. In the presence of MICU1, MICU2 elevates the mitochondrial calcium uptake threshold, thereby preventing mitochondrial calcium overload under basal cytoplasmic calcium conditions [113]. In contrast, MICU3 functions as a positive regulator of MCUcx channels, and the MICU1-MICU3 heterodimer collectively lowers the threshold of mitochondrial calcium uptake [114]. Historically, MICU3 was identified primarily in the nervous system and has limited expression in skeletal muscle; therefore, research has been conducted mainly in brain neurons [115]. Nevertheless, a recent study by Roman et al. [114] shows that the deletion of MICU3 negatively impacts calcium handling and locomotion in skeletal muscle, thus affecting the composition of muscle fiber types. Additionally, MICU1.1, an alternative splice isoform of MICU1, is expressed mainly in skeletal muscle [116]. Compared with MICU1, MICU1.1 has a higher affinity for calcium and binds calcium with an efficiency one order of magnitude greater than MICU1. Consequently, after stimulation, MICU1.1 induces a more significant increase in mitochondrial calcium uptake [117]. Although MICU family members exhibit differential functions, the interactions between these proteins and their regulation of calcium signaling demonstrate a complex synergistic effect, which may be crucial for maintaining intracellular calcium homeostasis and adapting to physiological demands.

Mitochondrial permeability transition pore (MPTP)

The mitochondria-mediated signaling network is involved not only in providing energy for skeletal muscle but also in determining the viability and apoptosis of muscle fibers [74]. At the heart of mitochondrial function is MPTP, a critical initiator of apoptosis and necrosis. MPTP is a conductance channel responsible for maintaining the mitochondrial membrane potential, spanning both the outer and the inner mitochondrial membranes. While previous studies have identified that MPTP is a protein complex comprising VDAC, adenine nucleotide translocator protein (ANT), and cyclophilin D (CypD), Baines et al. [118] challenged this finding by demonstrating that VDAC is dispensable for MPTP induction, thus reigniting the debate regarding the precise nature of MPTP. These findings also confirmed that only CypD plays a definitive role in the molecular mechanism of MPTP, while the involvement of ANT and VDAC is relatively secondary [119]. Moreover, it has been proposed that MPTP may not be a complex composed of specific proteins but rather arises from unspecific interactions among mitochondrial membrane proteins [120]. Consequently, the molecular identity of MPTP remains under debate.

The opening of MPTP is intricately associated with various cellular processes, notably apoptosis induction [121]. Physiologically, the transient opening of MPTP allows the reversible transfer of 0.3 kDa molecules, which facilitates the release of metabolites from the mitochondrial matrix. This controlled opening helps maintain appropriate mitochondrial homeostasis, which is a vital aspect of cellular physiology [122]. Nonetheless, when MPTP undergoes sustained opening, irreversible passive diffusion of 1.5 kDa ions and solutes across the IMM occurs, which leads to collapse, swelling, and eventual rupture of the mitochondrial membrane potential. This ultimately leads to necroptosis, a form of cell death associated with several degenerative disorders of skeletal muscle [122,123,124]. Although the regulation of MPTP gating displays tissue-specific variations, the accumulation of mitochondrial matrix calcium is universally acknowledged as the key trigger for MPTP opening. Other factors, such as ROS and pH, also play regulatory roles in MPTP opening [121]. In denervated mice with skeletal muscle atrophy, increased calcium leakage from myotubes, along with abnormal SR morphology and function, results in elevated cytoplasmic calcium concentrations. Hence, calcium overload occurs in mitochondria, leading to concurrent MPTP opening [74, 125]. Karam et al. [74] reported that the absence of physiologic cytoplasmic calcium transients following denervation leads to a lack of dynamic calcium uptake in mitochondria. This, in turn, triggers the opening of MPTP and culminates in mitochondrial dysfunction. In addition, various exercise modalities indirectly influence mitochondrial function in skeletal muscle via their effects on MPTP. It has been documented that prolonged eccentric exercise affects mitochondrial function in mouse skeletal muscle by increasing sensitivity to calcium-induced MPTP opening, thereby contributing to skeletal muscle injury and dysfunction [126]. In contrast, moderate endurance exercise, such as running training, has been demonstrated to improve mitochondrial bioenergetics in the gastrocnemius muscle of Wistar male rats. This improvement may be attributed to increased tolerance to calcium-induced MPTP opening [127].

Extensive research has explored the role of MPTP as an upstream mechanism of muscle atrophy [128]. The opening of MPTP triggers muscle fiber atrophy, since it releases ROS and caspase-3 and simultaneously upregulates protein degradation pathways. It has been reported that inhibiting the opening of MPTP can improve muscle function by enhancing mitochondrial calcium retention capacity. ALS, a fatal neurodegenerative disease of motor neurons (MNs), is associated with MPTP activation, which triggers MN degeneration [129]. In a prospective randomized placebo-controlled drug trial, Martin et al. [130] examined the therapeutic potential of GNX-4728 in a transgenic mouse model of ALS. This study revealed that GNX-4728 effectively inhibited MPTP opening, thereby preserving the number of MNs and the integrity of MN mitochondria and neuromuscular junctions in ALS mice and ultimately improving the survival rate of these mice. The opening of MPTP has also been implicated in various pathological conditions. For example, cyclosporin A prevents MPTP-mediated muscular toxicity induced by the anesthetic drug bupivacaine via the inhibition of MPTP opening [131]. Oxidative stress-induced mitochondrial dysfunction is a relevant factor in the pathogenesis of several diseases, including muscle diseases. Melatonin has been identified as a protective agent that prevents oxidative stress-mediated MPTP opening and myotube death in skeletal muscle cells by inhibiting tert-butyl hydroperoxide-induced mitochondrial depolarization [132]. Furthermore, insulin-resistant skeletal muscle often presents with mitochondrial swelling, malformation, and membrane damage. The inhibition of MPTP opening has been shown to inhibit muscle mitochondrial dysfunction induced by high glucose levels [133]. These results emphasize the multiple therapeutic implications of modulating MPTP in the context of muscle-related and neurodegenerative diseases.

Conclusion

Skeletal muscle accounts for more than 40% of total body weight and is vital for maintaining overall health [134]. The contraction of skeletal muscle is intricately associated with cytosolic calcium signaling and ATP generation. The crux of this process is the highly structured interface between the terminal cisternae of the SR and the transverse tubules (T-tubules). This junction houses calcium release units, which are essential for facilitating the rapid increase in cytoplasmic calcium concentrations during ECC of skeletal muscle. Thus, the depolarization of the T-tubule membrane triggers calcium release from the SR via the RYR1 channel, subsequently inducing muscle contraction. This process is reversed by SERCA, which uses ATP to promote muscle relaxation and the reuptake of calcium into the ER [135].

Mitochondria are crucial intracellular organelles that play a central role in calcium uptake via VDAC and MCUcx, simultaneously facilitating ATP synthesis via aerobic respiration [136]. The transfer and signaling of calcium are vital for maintaining physiological functions, particularly in skeletal muscle. Impairments in calcium signaling pathways can significantly reduce the mass and function of skeletal muscle, thereby reducing quality of life and exacerbating the risk of morbidity and mortality [137].

This review explores the complex relationship between the ER and mitochondria in regulating calcium homeostasis and its consequent effects on muscle function, laying emphasis on the role of calcium influx and efflux channels within these organelles and the examination of their interactions. While the current understanding provides valuable insights into calcium signaling mechanisms, future research critically addressing the pathological implications of calcium homeostasis disruption in skeletal muscle is warranted. Specifically, it is necessary to investigate how aging, metabolic disorders, and other chronic conditions influence these interactions. Additionally, exploring therapeutic strategies that target calcium signaling pathways may offer new avenues for the prevention and treatment of muscle-related diseases. These insights could be instrumental in deepening the understanding of muscle physiology and developing effective interventions to mitigate muscle atrophy and improve overall health outcomes.