Redox Control of Proteolysis During Inactivity-Induced Skeletal Muscle Atrophy

Abstract

Significance: Skeletal muscles play essential roles in key body functions including breathing, locomotion, and glucose homeostasis; therefore, maintaining healthy skeletal muscles is important. Prolonged periods of muscle inactivity (e.g., bed rest, mechanical ventilation, or limb immobilization) result in skeletal muscle atrophy and weakness.

Recent Advances: Disuse skeletal muscle atrophy occurs due to both accelerated proteolysis and decreased protein synthesis with proteolysis playing a leading role in some types of inactivity-induced atrophy. Although all major proteolytic systems are involved in inactivity-induced proteolysis in skeletal muscles, growing evidence indicates that both calpain and autophagy play an important role. Regulation of proteolysis in skeletal muscle is under complex control, but it is established that activation of both calpain and autophagy is directly linked to oxidative stress.

Critical Issues: In this review, we highlight the experimental evidence that supports a cause and effect link between reactive oxygen species (ROS) and activation of both calpain and autophagy in skeletal muscle fibers during prolonged inactivity. We also review the sources of oxidant production in muscle fibers during inactivity-induced atrophy, and provide a detailed discussion on how ROS activates both calpain and autophagy during disuse muscle wasting.

Future Directions: Future studies are required to delineate the specific mechanisms by which ROS activates both calpain and autophagy in skeletal muscles during prolonged periods of contractile inactivity. This knowledge is essential to develop the most effective strategies to protect against disuse muscle atrophy. Antioxid. Redox Signal. 33, 559–569.

Introduction

Skeletal muscle comprises ∼40% of total body mass and plays an essential role in numerous vital functions, including breathing, locomotion, and glucose homeostasis. Therefore, maintaining healthy skeletal muscles is important. In this regard, prolonged skeletal muscle inactivity (e.g., limb immobilization, prolonged mechanical ventilation, or long-term bed rest) leads to fiber atrophy and muscular weakness that culminate in a decreased quality of life. It is widely agreed that skeletal muscle inactivity results in fiber atrophy due to both increased proteolysis and a decreased rate of protein synthesis. Although debate continues as to whether accelerated proteolysis or depressed protein synthesis is the dominant factor in promoting inactivity-induced muscle atrophy, it is clear that accelerated proteolysis plays a key role in the loss of skeletal muscle protein in some types of inactivity-induced muscle wasting. For example, the rapid fiber atrophy that occurs in inspiratory muscles (e.g., diaphragm) during mechanical ventilation ensues within the first 12–18 h of ventilator support; therefore, this swift rate of muscle atrophy is clearly dominated by accelerated proteolysis. In reference to the mechanism(s) responsible for protease activation during disuse muscle atrophy, evidence reveals that increased production of reactive oxygen species (ROS) and disturbed redox signaling in skeletal muscle fibers activate all four major proteolytic systems (ubiquitin proteasome pathway [UPP], caspase-3, calpain, and autophagy) (10, 12, 25, 84, 85).

In regard to oxidative stress and activation of proteases, a voluminous literature exists describing the nexus between oxidant stress and activation of the UPP and caspase-3, and these findings have been summarized in several recent reviews (10, 12, 84–86). Importantly, emerging data also implicate oxidative stress in the activation of both calpain and autophagy in skeletal muscle fibers undergoing atrophy due to prolonged disuse. Because of these recent advances connecting oxidative stress with the regulation of both calpain and autophagy, this review summarizes the molecular mechanisms responsible for oxidative stress-mediated activation of both calpain and autophagy in skeletal muscle fibers during prolonged inactivity. We begin with an overview of the clinically relevant forms of inactivity-induced muscle atrophy and discuss the preclinical models used to investigate the mechanisms responsible for disuse muscle atrophy.

Preclinical Models to Investigate Inactivity-Induced Atrophy

Extended bed rest, limb immobilization, and prolonged mechanical ventilation are among the most common forms of disuse muscle atrophy encountered in the clinic. Investigating the mechanisms responsible for disuse muscle atrophy in humans is challenging due to the need for invasive procedures (i.e., muscle biopsy) and therefore, preclinical (animal) models are frequently used to study muscle atrophy. Indeed, several laboratory animal models have been developed to simulate prolonged bed rest, limb immobilization, and mechanical ventilation in humans. For example, the rodent model of hind-limb suspension is widely used as a preclinical model to investigate bed rest-induced limb muscle atrophy in humans. Similarly, a rodent model of hind-limb immobilization (i.e., casting) is commonly used to investigate the impact of limb immobilization on muscle atrophy. Finally, animal models of mechanical ventilation are commonly employed to mimic the unique muscle atrophy that occurs in inspiratory muscles when patients are exposed to prolonged mechanical ventilation.

In reference to mechanical ventilation, this important clinical intervention provides respiratory support for patients unable to maintain adequate alveolar ventilation on their own. Common clinical indications for prolonged mechanical ventilation include chronic obstructive pulmonary disease, critical illness, heart failure, stroke, and recovery from surgery. In human medicine, controlled mechanical ventilation is a life-saving intervention for many patients suffering from respiratory failure. During this mode of ventilator support, the ventilator delivers all of the breaths while the patients' inspiratory muscles (e.g., diaphragm) are completely inactive. Both human and animal studies demonstrate that prolonged mechanical ventilation results in rapid inspiratory muscle (i.e., diaphragm) atrophy. For example, as few as 12–18 h of mechanical ventilation results in a ∼15%–20% reduction in diaphragm fiber size in both humans and rodents (38, 58, 99, 102, 103). This extremely rapid rate of muscle atrophy is unique among other types of disuse muscle atrophy because a comparable level of fiber atrophy in hind-limb muscles would require ∼96 h of inactivity (108). The next segment debates the relative roles that accelerated proteolysis and decreased protein synthesis play in inactivity-induced muscle atrophy.

Is Disuse Muscle Atrophy Dominated by Accelerated Proteolysis or Decreased Protein Synthesis?

Skeletal muscle mass is regulated by the balance between the rates of protein synthesis and degradation. Animal studies consistently report that inactivity-induced atrophy in both locomotor and respiratory skeletal muscles occurs due to both increased proteolysis and decreased muscle protein synthesis (10, 12, 25, 88). For example, studies indicate that the rate of protein synthesis decreases rapidly (i.e., within 6 h) after the beginning of muscle inactivity and reaches a new “lower” steady state of muscle protein synthesis within 18–48 h after initiation of muscle disuse (32, 98, 108). Further, investigations regularly conclude that disuse muscle atrophy in experimental animals is accompanied by a large increase in proteolysis [reviewed in Powers et al. (85)]. Therefore, in animal models, it is established that the net loss of skeletal muscle protein during prolonged muscle disuse occurs due to both decreased protein synthesis and increased protein breakdown.

In difference to the belief that inactivity-induced muscle atrophy occurs due to both decreased protein synthesis and increased proteolysis, some investigators dispute this conclusion and maintain that the principal determinant of disuse muscle atrophy in human limb muscle is decreased protein synthesis (81, 92). In support of this position, studies consistently conclude that limb skeletal muscle inactivity in humans results in decreased muscle protein synthesis [reviewed in Phillips and McGlory (82)]. Nonetheless, several studies also conclude that prolonged bed rest or immobilization-induced human limb muscle atrophy is associated with increases in protease activity (26, 31, 41, 90, 110). Similarly, many animal studies demonstrate that prolonged muscle inactivity is associated with augmented protease activation (67, 68, 70, 72, 83, 99, 103–105, 108, 113). Nonetheless, the dominant mechanism responsible for disuse muscle atrophy is difficult to discern because dissimilarities in the rates of skeletal muscle protein synthesis and proteolysis are challenging to measure in humans (91). Therefore, additional studies are required to cogently determine the role that proteolysis plays in disuse muscle atrophy in human limb muscle (62).

In contrast to the controversy about the influence of proteolysis on human limb muscle atrophy, studies investigating the impact of mechanical ventilation-induced muscle inactivity on diaphragmatic atrophy consistently demonstrate that prolonged inactivity activates all major proteolytic systems (e.g., calpain, caspase-3, UPP, and autophagy) in the human diaphragm (34, 57, 58). Identical findings are reported in animal studies of mechanical ventilation-induced diaphragmatic atrophy (20, 99, 113). Together, these findings support the fact that prolonged inactivity of both human and animal respiratory muscles results in accelerated proteolysis. Importantly, oxidative stress is a key trigger of protease activation in the diaphragm during prolonged mechanical ventilation. The next segment highlights the discovery that oxidative stress is an important factor that promotes disuse muscle atrophy.

Oxidative Stress Is Required for Inactivity-Induced Muscle Atrophy

The landmark finding that muscular contraction (i.e., exercise) results in an acute increase in muscle radical production was reported almost four decades ago (18). This observation was quickly supported by a second study; together, these experiments launched the concept that ROS production in noncontracting (i.e., resting) skeletal muscle is low, and that ROS production is increased by contractile activity (39). However, this concept was challenged when the first report linking oxidative stress to disuse muscle atrophy appeared in 1991 (49). Specifically, Kondo and colleagues discovered that prolonged skeletal muscle disuse is associated with increased lipid peroxidation, and that this inactivity-induced muscle atrophy can be partially averted by the antioxidant vitamin E (49). Although these investigators provided several additional studies to confirm their original finding (50–53), the discovery that prolonged muscle inactivity promotes increased production of ROS was essentially unnoticed by researchers for several years. Nonetheless, experiments over the past decade have firmly established that prolonged skeletal muscle inactivity promotes chronic oxidative stress in the inactive fibers, and that ROS are key players in inactivity-induced muscle atrophy (3, 6, 113). The fact that oxidative stress promotes disuse muscle atrophy has propelled research to identify the source(s) of ROS production and to investigate the signaling pathways linking oxidative stress to muscle atrophy.

Sites of ROS Production in Inactive Skeletal Muscle Fibers

The search for the site(s) of ROS production in inactive muscle fibers has spanned three decades. This quest has been difficult because of the technical challenges associated with the detection of ROS production at specific cellular locations. Nonetheless, significant progress has been made within the last decade, and evidence indicates that prolonged disuse in skeletal muscle results in increased superoxide production by three mechanisms that include NADPH oxidase, xanthine oxidase, and mitochondria (69, 72, 83, 107, 112) (Fig. 1). A brief overview of the evidence supporting a role for each of the sources of ROS production in skeletal muscle follows.

FIG. 1.

Simplified diagram illustrating the three major sites of ROS production in skeletal muscle fibers exposed to prolonged inactivity. Specifically, evidence indicates that prolonged disuse in skeletal muscle results in increased superoxide production primarily by three mechanisms, including NADPH oxidase, xanthine oxidase, and mitochondria. NOX2, NADPH oxidase 2; ROS, reactive oxygen species. Color images are available online.

Studies indicate that xanthine oxidase activity is increased in skeletal muscle fibers during prolonged inactivity. For example, xanthine oxidase activity is elevated within inactive muscle fibers during limb immobilization (53), hind-limb suspension (19, 64), and in diaphragm muscle fibers during prolonged mechanical ventilation (112). Nonetheless, whether ROS emission from xanthine oxidase is a key player in promoting inactivity-induced muscle atrophy remains a debated topic. For example, two studies have concluded that pharmacological blockage of xanthine oxidase activity during hind-limb suspension partially protects against oxidative stress and reduces muscle atrophy by ∼20% (19, 77). In contrast, an opposing study concludes that inhibition of xanthine oxidase does not protect against hind-limb suspension-induced muscle atrophy (64). Clearly, additional research is required to resolve this conflict.

The only known function of NADPH oxidase in muscle fibers is the production of ROS (27), and increased NADPH oxidase activity contributes to oxidative stress and limb muscle atrophy in several chronic diseases, including muscular dystrophy, heart failure, and chronic kidney disease (1, 4, 8, 9, 14, 46, 111). Although the role that NADPH oxidase plays in inactivity-induced limb muscle atrophy remains unclear, emerging evidence suggests that NADPH oxidase participates in the diaphragmatic atrophy associated with prolonged mechanical ventilation. Indeed, mechanical ventilation activates NADPH oxidase in diaphragm muscle fibers (69). Further, pharmacological inhibition of NADPH oxidase activity via apocynin protects the diaphragm from both ventilator-induced oxidant damage and fiber atrophy (69). Unfortunately, apocynin also exhibits several off-target effects in skeletal muscles, and this experimental artifact complicates the interpretation of experiments using apocynin to inhibit NADPH oxidase activity.

Without using apocynin, a recent study provides new evidence implicating NADPH oxidase in ventilator-induced diaphragmatic oxidative stress and atrophy (56). This study reveals that mechanical activation of angiotensin II type 1 receptors (AT1Rs) plays a key role in promoting inactivity-induced mitochondrial dysfunction and oxidative stress in the diaphragm. More specifically, this investigation revealed that pharmacological blockade of AT1Rs activation protects the diaphragm against ventilator-induced oxidative stress, mitochondrial dysfunction, and fiber atrophy (56). These data suggest that the ventilator-induced passive length change of diaphragm fibers provides tension to the fiber membrane to activate AT1Rs, which then activates the sarcolemma bound isoform of NADPH oxidase 2 (NOX2). Experimental support that AT1R signaling activates NADPH oxidase is robust as deletion of NOX2 in mice skeletal muscle prevents angiotensin II-induced oxidative stress and muscle atrophy (42).

Moreover, studies reveal that activation of NOX2-induced ROS production results in a cross-talk between NOX2 and mitochondria that increases mitochondrial ROS production (11, 15, 22). This redox-mediated cross-talk between NOX2 and mitochondria exacerbates mitochondrial ROS production and disrupts cellular redox homeostasis. Several mechanisms can contribute to this NOX2 and mitochondrial cross-talk and the reader is referred recent reports for details (11, 15, 22, 116). NADPH oxidase and mitochondrial cross-talk is physiologically significant because prolonged skeletal muscle inactivity results in mitochondrial damage (i.e., uncoupling) and increased mitochondrial ROS production [reviewed in Hyatt et al. (36), Ji and Yeo (40), Powers et al. (87), and Romanello and Sandri (94, 95)]. Importantly, this inactivity-induced increase in mitochondrial ROS emission is associated with activation of all major proteolytic systems in skeletal muscle (85, 87).

In regard to the relative contributions of xanthine oxidase, NADPH oxidase, and mitochondria to the total ROS production within inactive muscles, several lines of evidence implicate mitochondria as a primary source of ROS production in skeletal muscles during prolonged disuse (72, 83, 107). For example, compared with mitochondria from skeletal muscles of active rodents, mitochondria isolated from muscle exposed to prolonged periods of disuse release significantly more ROS (45, 72, 107). Further, treatment of animals with a mitochondrial-targeted antioxidant prevents inactivity-induced oxidative stress in skeletal muscle fibers (72, 83, 107). Collectively, these findings support the notion that mitochondria are a dominant source of ROS production in skeletal muscles during prolonged periods of inactivity.

Redox Control of Autophagy

As introduced earlier, four major proteolytic systems exist in skeletal muscle fibers. Furthermore, mitochondria contain proteases (e.g., Lon protease), but limited information exists about the role that these proteases play in disuse muscle atrophy (76). Numerous studies confirm that oxidative stress stimulates proteolysis within skeletal muscle fibers in at least three ways. First, redox disturbances in muscle fibers lead to increased cytosolic free calcium and the subsequent activation of both calpain and caspase-3. Second, oxidative stress often activates transcriptional activators to promote gene expression of key proteins involved in proteolytic systems. Finally, redox disturbances can also accelerate proteolysis by the oxidative modification of proteins, which increases their susceptibility to proteolytic breakdown.

During the past several decades, numerous reports have highlighted the role that oxidative stress plays in activation of both the UPP and caspase-3 in skeletal muscle. By contrast, research investigating the role of oxidative stress in the activation of both autophagy and calpain in skeletal muscle is a relatively new field with most of the research emerging during the past 5–10 years. Therefore, the remainder of this review will focus on the redox control of autophagy and calpain in skeletal muscles exposed to prolonged inactivity.

Overview of autophagy-mediated protein degradation in skeletal muscles

Removal of damaged cellular components is important for normal cell function (47). In this regard, autophagy is a lysosomal proteolytic pathway for the degradation of damaged cytosolic proteins and organelles in skeletal muscle and other cells (47, 115). The delivery of damaged proteins and organelles to lysosomes can occur by three different forms of autophagy: (i) microautophagy, (ii) chaperone-mediated autophagy, and (iii) macroautophagy. Our focus will be macroautophagy, but a brief introduction to both microautophagy and chaperone-mediated autophagy is warranted to provide context for the discussion of the proteolytic role that macroautophagy plays in skeletal muscles.

Microautophagy occurs when the cellular components to be degraded are directly engulfed by the lysosome and degraded (59). Microautophagy is a constitutively active form of autophagy involved in the turnover of long-lived cellular proteins, including membrane proteins (71).

Chaperone-mediated autophagy involves the process of selectively targeting proteins to lysosomes for degradation (13). The selectivity of chaperone-mediated autophagy contributes to cellular quality control by eliminating only damaged or unfolded proteins. A distinctive feature of chaperone-mediated autophagy is that proteins degraded by this pathway are selected by recognition of a motif in their amino acid sequence (13).

The third and final form of autophagy is labeled as macroautophagy; this is the most researched form of autophagy and will be the focus of our discussion on redox control of autophagy. Macroautophagy is an essential, highly conserved proteolytic system that degrades cytosolic components, including soluble proteins, aggregated proteins, organelles, macromolecular complexes, and foreign bodies (114). Whereas microautophagy and chaperone-mediated autophagy are not associated with morphological changes in the cytosol of the cell, macroautophagy involves the formation of a dedicated vesicle within the cytoplasm (29). During macroautophagy (hereafter referred to as autophagy), damaged organelles (e.g., mitochondria) and cytosolic proteins are separated into double membrane vesicles called autophagosomes (29). These autophagosomes can sequester cytoplasmic proteins, including entire organelles or fragments of organelles; after formation, these autophagosomes fuse with lysosomes to form autolysosomes (29). After fusion with the lysosome, the contents of the autophagosome are degraded by lysosomal proteases (i.e., cathepsins) (48).

Our understanding of the mechanisms that regulate autophagy has advanced rapidly during the past decade. Formation of the autophagosome and its fusion with lysosomes are associated with numerous autophagy-related gene products that are commonly recognized by the acronym of “Atg” followed by a number that identifies the specific gene or gene product (e.g., Atg1, Atg2, etc.). Currently, >80 autophagy-related proteins have been identified in humans, and many of these proteins exist in other organisms (29). Autophagy involves a four-step process: (i) induction, (ii) expansion of the phagophore, (iii) completion of autophagosome, and (iv) autophagosome fusion with the lysosome followed by degradation of autophagosome cargo (i.e., proteins and organelles) (Fig. 2). A detailed discussion of the steps involved in autophagy exceeds the scope of this review and therefore, only a brief overview will be provided. For specifics on the numerous events leading to autophagy, the reader is referred to recent reviews (29, 33, 73, 114).

FIG. 2.

Illustration of the steps leading to autophagy. Autophagy progresses during a four-step process: (1) induction, (2) expansion, (3) completion of autophagosome, and (4) fusion with lysosome and degradation of autophagosome cargo (i.e., proteins and organelles). AMPK, AMP-activated protein kinase; mTORC1, mammalian target of rapamycin complex 1; PI3KC3, phosphatidylinositol 3-kinase catalytic subunit type 3; TSC2, tuberous sclerosis complex 2. Color images are available online.

The induction of autophagy begins with activation of the ULK1 complex. This complex involves several players, including ULK1, Atg13, Atg101, and FIP200 (74). The induction step is controlled by two master regulators: mammalian target of rapamycin complex 1 (mTORC1) and AMP-activated protein kinase (AMPK) (74, 93). mTORC1 is a serine/threonine kinase that acts as a central inhibitor of autophagy by phosphorylation of ULK1; phosphorylation of ULK1 at this specific site prevents activation of induction, and therefore blocks autophagy (74). It follows that inhibition of mTORC1 activation promotes autophagy. In this regard, AMPK is a primary negative regulator of mTORC1 (93). Although it is possible that AMPK can inhibit mTORC1 activation in several ways, a clearly identified path involves AMPK-mediated phosphorylation of tuberous sclerosis complex 2 (TSC2); phosphorylation of TSC2 inhibits Ras homolog enriched in brain (RHEB), an important mTORC1 activator [for more details, see Dibble and Cantley (21)]. Moreover, AMPK can directly promote the induction of autophagy by phosphorylation of ULK1 at a site on the molecule that facilitates the formation of the ULK1 complex (24).

Step two of autophagy involves the assembly of a portion of the autophagosome membrane called the phagophore; this stage is often referred to as expansion and requires the recruitment of several Atg proteins, including the essential autophagy protein Beclin-1 that interacts with phosphatidylinositol 3-kinase catalytic subunit type 3 (PI3KC3), Atg14L, and other autophagy-related proteins to create the Beclin complex leading to formation of the phagophore (Fig. 2) (74).

Step three (completion of autophagosome) involves several required Atg genes, including Atg3, Atg4, Atg5, Atg7, LC3, Atg12, and Atg16 resulting in closure of the autophagosome into a double membrane vesicle (74). In this phase of autophagy, the conjugation of Atg5 to Atg12 is an essential step in elongation and completion of the autophagosome. In particular, the conjugation of Atg5 to Atg12 is required for LC3 incorporation into the autophagosomal structure, and therefore prevention of the conjugation of Atg5 to Atg12 inhibits autophagy (74).

The fourth and final step of autophagy involves fusion of autophagosome with the lysosome; this is followed by the degradation of the autophagosome cargo by lysosomal proteases. This fusion step requires several proteins, including ESCRT, SNAREs, Rab7, and the class C Vps proteins (74). After fusion with the lysosome, the autophagosome contents (i.e., cytosolic proteins and organelles) are released into the lysosome for degradation by proteases that include cathepsins B, D, and L (74).

Role of autophagy in disuse muscle atrophy

Compared with the other major proteolytic systems involved in disuse skeletal muscle atrophy, autophagy has received less research attention. Nonetheless, it is established that prolonged inactivity in skeletal muscle results in increased expression of autophagy genes, activation of lysosomal proteases (i.e., cathepsins B, D, and L), and accelerated autophagy flux (5, 7, 28, 35, 43, 44, 61, 96, 104, 105, 107).

Although many studies have confirmed that the basal rate of autophagy is increased during disuse muscle atrophy, incomplete information exists about the impact of autophagy on disuse muscle atrophy. Nonetheless, a recent study reveals that accelerated autophagy is required for the diaphragmatic atrophy that occurs during prolonged mechanical ventilation (105). Specifically, to prevent autophagosome formation and blunt autophagy, this study overexpressed a mutated (dominant negative) form of Atg5 in diaphragm muscle fibers to prevent the conjugation of Atg5 to Atg12. As discussed earlier, the conjugation of Atg5 to Atg12 is required for LC3 incorporation into the early autophagosomal structure (105). Together, the results of this study, combined with the abundant evidence that prolonged inactivity increases autophagy in skeletal muscle, suggest that autophagy plays a prominent role in the proteolysis associated with prolonged muscle inactivity.

Role of ROS in promoting autophagy during disuse muscle atrophy

Both in vitro and in vivo studies consistently demonstrate a key role of ROS in stimulating autophagy in skeletal muscles (74, 79, 97, 104, 105, 107). Indeed, numerous cell culture experiments (i.e., C₂C1₂ myotubes) reveal that both superoxide and hydrogen peroxide (H₂O₂) stimulate autophagy (37, 65, 89). In theory, ROS signaling can promote autophagy in several ways. For example, oxidative stress can activate AMPK, which suppresses mTORC1 activation (74). Again, active mTORC1 inhibits the activation of ULK1, which is essential for induction of autophagy (55). Moreover, ROS can promote autophagy by increasing the expression of autophagy genes. For instance, exposure of cells to H₂O₂ increases the expression of crucial autophagy genes, including LC3 and Beclin-1; the importance of this increase in expression of Atg genes is supported by the observation that oxidative stress is also associated with an increased formation of autophagosomes [reviewed in Navarro-Yepes et al. (74)]. One of the signaling pathways implicated in the ROS-mediated expression of autophagy genes involves activation of the mitogen-activated kinase, p38 alpha/beta. Activation of p38 promotes myotube atrophy and increases the expression of several autophagy-related genes (e.g., Atg7) (65). Finally, oxidative stress can also inactivate Atg4, which plays a key role in the regulation of the Atg/LC3 lipid conjugation system, an essential step in the process of autophagy (63, 97).

In addition to in vitro studies, in vivo studies confirm that oxidative stress promotes autophagy in skeletal muscles during prolonged periods of inactivity. Indeed, prevention of immobilization-induced oxidative stress prevents the activation of forkhead boxO (FoxO) signaling in skeletal muscles; this is significant because active FoxO promotes the expression of several key autophagy-related proteins and accelerates autophagy (23, 104). Further, two independent experiments using a mitochondrial-targeted antioxidant to prevent inactivity-induced oxidative stress demonstrated that prevention of oxidative stress blocked autophagy in skeletal muscles exposed to prolonged inactivity. Specifically, treatment of animals with a mitochondrial-targeted antioxidant prevented the increase of primary biomarkers of autophagy in rat hind-limb muscles during muscle immobilization (107). Similarly, prevention of ventilator-induced oxidative stress in the diaphragm significantly reduced the increase in autophagy biomarkers in muscle fibers (105). Specifically, prevention of oxidative stress in the diaphragm during prolonged mechanical ventilation diminished the messenger RNA (mRNA) levels of LC3, Atg7, Atg12, Beclin-1, cathepsin B, cathepsin D, and cathepsin L in diaphragm muscle fibers (105). Moreover, protection against oxidative stress prevented the inactivity-induced increase in autophagic vacuoles in diaphragm fibers (105). Collectively, these in vivo experiments along with the in vitro animal experiments provide robust evidence that oxidative stress plays a required role in activating autophagy in skeletal muscles during periods of prolonged inactivity (Fig. 3).

FIG. 3.

Steps leading to autophagy and the location of influence by ROS. Note that ROS can promote autophagy by both the activation of AMPK and the increased expression of autophagy genes. Color images are available online.

Redox Control of Calpain Activation in Skeletal Muscle During Prolonged Inactivity

Calpains are Ca²⁺-activated proteases that cleave target proteins (30, 78). While 15 calpain genes exist in humans, the two primary calpains that contribute to skeletal muscle atrophy are calpain 1 and calpain 2 (30, 78). Active calpain 1 and 2 are reported to cleave >100 different proteins, including the important cytoskeletal proteins titin and nebulin and many kinases and phosphatases (30). Interestingly, calpain can also cleave oxidized muscle contractile proteins such as actin and myosin (101). In fact, oxidation of contractile proteins greatly increases their susceptibility for degradation by calpain; hence, protein oxidation greatly expands the number of potential substrates for calpain (101).

Role of calpain in promoting disuse muscle atrophy

The specific role that calpain plays in muscle atrophy has been debated for over two decades. Historically, the dogma has been that the UPP plays the dominant role in most types of skeletal muscle atrophy because calpains were believed to degrade only a limited number of skeletal muscle proteins (30). However, this belief has changed after the discovery that oxidation of muscle contractile proteins increases their susceptibility for cleavage by calpain (101). Importantly, studies also reveal that inhibition of calpain activation protects skeletal muscles against disuse muscle atrophy. For example, prevention of calpain activation by both pharmacological inhibitors and transgenic overexpression of calpastatin (endogenous inhibitor of calpain) protects limb muscles in rodents against disuse muscle atrophy (106, 109). Further, inhibition of calpain activation in the diaphragm protects against mechanical ventilation-induced diaphragmatic atrophy (60, 75). Interestingly, inhibition of calpain activation in the diaphragm during prolonged mechanical ventilation prevents the activation of caspase-3, indicating that a calpain/caspase-3 cross-talk exists (75). Together, these studies reveal that activation of calpain plays a significant role in disuse muscle atrophy.

Role of ROS in activating calpain during disuse muscle atrophy

Numerous in vitro studies confirm that oxidative stress increases the expression of calpain 1 and 2 in several cell types. For example, oxidative stress increases the abundance of both calpain 1 and 2 in liver cells (80). Similarly, exposure of C₂C₁₂ myotubes to H₂O₂ significantly elevates calpain 1 mRNA (66). Finally, exposure of human myoblasts to ROS increases the expression of both calpain 1 and calpain 2 (16). Together, these studies confirm that ROS are capable of increasing calpain expression in cells.

Robust evidence also supports the concept that ROS can trigger calpain activation in rodent skeletal muscles in vivo. Specifically, two independent studies reveal that mitochondrial-targeted antioxidants avert immobilization-induced oxidative stress and prevent calpain activation in rodent limb muscles (72, 107). Similarly, prevention of ventilator-induced increases of mitochondrial ROS emission in diaphragm muscle fibers also prevents calpain activation (83). Collectively, these investigations confirm that oxidative stress is a required trigger to activate calpain in skeletal muscle during prolonged inactivity.

The primary mechanism responsible for ROS-mediated activation of calpain in skeletal muscle fibers is likely an increase in cytosolic free calcium (16, 17). In particular, calpain activity is regulated by both cytosolic calcium levels and the concentration of calpastatin, an endogenous inhibitor of calpain (30). In this regard, it is clear that increased cytosolic free calcium is required to activate calpain (30), and it is established that oxidative stress can increase intracellular free calcium (54). At least two potential mechanisms can account for a ROS-induced increase in cytosolic calcium. First, a potential connection between ROS and increased intracellular calcium is that ROS-mediated formation of reactive aldehydes (i.e., 4-hydroxy-2,3-trans-nonenal) can inhibit membrane calcium ATPase activity (100). This is important because inhibition of cell calcium ATPase activity hinders calcium removal from the cytosol resulting in increased intracellular calcium levels. Further, ROS-mediated oxidation of the ryanodine receptor 1 (RYR1) in skeletal muscle fibers results in a calcium leak from the sarcoplasmic reticulum leading to increased cytosolic levels of free calcium (2). Together, or independently, each of these mechanisms can promote an increase in cytosolic free calcium (Fig. 4).

FIG. 4.

Simplistic overview of the role that ROS plays in promoting an increase in intracellular calcium and calpain activation in skeletal muscle. Increased ROS production in the muscle fiber increases calcium release from the sarcoplasmic reticulum and impaired activity of calcium ATPase activity, resulting in increased cytosolic calcium levels and increased activation of calpain. RYR1, ryanodine receptor 1. Color images are available online.

Conclusions

Maintaining healthy skeletal muscles is important; indeed, skeletal muscle performs many vital functions, including breathing, locomotion, and assistance with glucose homeostasis. In regard to muscle health, muscle fiber atrophy and weakness occur in response to prolonged periods of muscle inactivity, which consequently results in decreased quality of life, and increased morbidity and mortality in patients suffering from chronic diseases. Disuse skeletal muscle atrophy occurs due to both accelerated proteolysis and decreased protein synthesis with proteolysis playing the dominant role in some conditions (e.g., mechanical ventilation-induced diaphragmatic atrophy). Although all major proteolytic systems are involved in proteolysis, compelling evidence indicates that both calpain and autophagy play active roles in promoting disuse muscle atrophy. In this regard, it is established that increased production of ROS plays a causative role in activation of both calpain and autophagy in muscle fibers during prolonged inactivity.

In regard to ROS and autophagy, oxidative stress can promote autophagy in several ways. For example, ROS can activate AMPK, which suppresses the activation of the ULK1 inhibitor, mTORC1. Moreover, increased ROS production in the muscle promotes autophagy by increasing the expression of numerous autophagy genes (e.g., Atg4, Atg7, LC3, Beclin-1). Finally, oxidative stress in inactive muscle fibers can also inactivate Atg4, which plays an important role in the regulation of the Atg/LC3 lipid conjugation system, an essential step in the process of autophagy.

Similar to autophagy, oxidative stress in inactive skeletal muscles can activate calpain 1 and 2. This ROS-mediated activation of calpain is likely linked to increased cytosolic levels of free calcium. In this regard, ROS can increase cytosolic calcium in at least two ways. First, ROS-mediated formation of reactive aldehydes (i.e., 4-hydroxy-2,3-trans-nonenal) can inhibit membrane calcium ATPase activity, which consequently results in increased intercellular calcium levels. Further, ROS-mediated oxidation of the ryanodine receptor in skeletal muscle fibers can promote a calcium leak from the sarcoplasmic reticulum leading to increased cytosolic levels of free calcium.

In regard to oxidative stress and activation of autophagy and calpain in skeletal muscles, many unanswered questions remain. For example, the mechanisms responsible for activation of NADPH oxidase in skeletal muscle during disuse remain generally unknown. Moreover, specific details about the role that NADPH oxidase plays in promoting increased mitochondrial ROS production in skeletal muscle are missing. Furthermore, additional research is required to fully understand if both subsarcolemmal and intermyofibrillar mitochondria play equal roles in ROS emission and activation of proteases in skeletal muscles during prolonged periods of inactivity. Clearly, there is much more to be learned about this exciting field.

Abbreviations Used

AMPK

AMP-activated protein kinase

AT1Rs

angiotensin II type 1 receptors

FoxO

forkhead boxO

H₂O₂

hydrogen peroxide

mRNA

messenger RNA

mTORC1

mammalian target of rapamycin complex 1

NOX2

NADPH oxidase 2

PI3KC3

phosphatidylinositol 3-kinase catalytic subunit type 3

RHEB

Ras homolog enriched in brain

ROS

reactive oxygen species

RYR1

Ryanodine receptor 1

TSC2

tuberous sclerosis complex 2

UPP

ubiquitin proteasome pathway

Funding Information

Our work in this research area has been supported by National Institutes of Health Grants R21AR064956 and R21AR073956 awarded to Scott K. Powers.