Redox Control of Skeletal Muscle Regeneration

Abstract

Skeletal muscle shows high plasticity in response to external demand. Moreover, adult skeletal muscle is capable of complete regeneration after injury, due to the properties of muscle stem cells (MuSCs), the satellite cells, which follow a tightly regulated myogenic program to generate both new myofibers and new MuSCs for further needs. Although reactive oxygen species (ROS) and reactive nitrogen species (RNS) have long been associated with skeletal muscle physiology, their implication in the cell and molecular processes at work during muscle regeneration is more recent. This review focuses on redox regulation during skeletal muscle regeneration. An overview of the basics of ROS/RNS and antioxidant chemistry and biology occurring in skeletal muscle is first provided. Then, the comprehensive knowledge on redox regulation of MuSCs and their surrounding cell partners (macrophages, endothelial cells) during skeletal muscle regeneration is presented in normal muscle and in specific physiological (exercise-induced muscle damage, aging) and pathological (muscular dystrophies) contexts. Recent advances in the comprehension of these processes has led to the development of therapeutic assays using antioxidant supplementation, which result in inconsistent efficiency, underlying the need for new tools that are aimed at precisely deciphering and targeting ROS networks. This review should provide an overall insight of the redox regulation of skeletal muscle regeneration while highlighting the limits of the use of nonspecific antioxidants to improve muscle function. Antioxid. Redox Signal. 27, 276-310.

Keywords: muscle stem cells; oxidative stress; skeletal muscle regeneration.

FIG. 1.

Overview of skeletal muscle regeneration. Left panel presents HE staining of uninjured and regenerating muscle (mouse tibialis anterior muscle) after an injury triggered by the injection of the venom toxin cardiotoxin. Day 1 after injury, myofibers undergo necrosis (pale pink staining) whereas the first inflammatory cells (neutrophils and monocytes/macrophages) enter the injured area (purple cells). Day 2 after injury, the number of infiltrating immune cells has increased and is mainly composed of monocytes/macrophages (large purple cells), which phagocyte the necrotic debris and necrotic myofibers. Meanwhile (nonvisible on HE staining), both MuSCs and FAPs activate and expand. Day 4 after injury, MuSCs differentiate and fuse to form new myofibers (dark pink structures containing one or two large nuclei with a “fried egg” appearance, arrow) whereas angiogenesis takes place (nonvisible on HE staining). Macrophages are still very numerous, and they surround the regenerating myofibers. Day 7 after injury, the number of interstitial cells (FAPs and macrophages) has dropped whereas the regenerating myofibers grow in size (through both fusion of differentiated MuSCs and hypertrophy). One month after injury, the muscle has come back to its normal appearance, except that the regenerating myofibers are characterized by nuclei in a central—and not peripheral—location (arrow). In this model, functionality of the muscle is fully recovered 2 months after injury. FAP, fibro-adipogenic progenitor; HE, hematoxylin-eosin; MuSCs, muscle stem cells.

FIG. 2.

Adult myogenesis. At the quiescent state, most MuSCs express high levels of the transcription factors Pax7 and Myf5. On activation, MuSCs enter the cell cycle, still express Pax7 and Myf5, and express MyoD while they are proliferating. Self-renewing MuSCs (gray arrows toward the left) dowregulate MyoD expression while upregulating Pax7 expression to return to quiescence. The majority of expanding MuSCs enter into myogenic differentiation that is sustained by the extinction of Pax7 and Myf5 expression together with the increase of Myogenin and MRF4 expression, which are synthetized once the cells have exited the cell cycle. Later on, contractile proteins are produced in the fully differentiated myogenic cells that fuse to form new myofibers. Maturation (not shown) of the myofibers is achieved by re-innervation through neuromuscular junctions.

FIG. 3.

Various cell types are present around MuSCs during skeletal muscle regeneration. During the time course of postinjury muscle regeneration, several cell types participate in the MuSC adult myogenesis program. In the model of cardiotoxin injury, MuSCs expand for a couple of days and then start to differentiate. At day 4, new regenerating myofibers are visible. FAPs are also activated on injury and rapidly expand during the first days of regeneration, after which their number rapidly decreases. After injury, the vascular bed is altered and a few days later, a reorganization of ECs is observed to form new functional vessels concomitantly to the formation of the new myofibers. Monocytes from the circulation enter into the injured area a few hours after the injury and initially give rise to inflammatory macrophages during the first 1–2 days. Then, on phagocytosis of the muscle tissue debris, they transform into anti-inflammatory/restorative macrophages that proliferate and stay numerous until the end of the regeneration process; next, their number drops back to normal values. ECs, endothelial cells.

FIG. 4.

Adult myogenesis during skeletal muscle regeneration is regulated by neighboring cells. The various cell types neighboring MuSCs (Fig. 3) exert specific functions on MuSCs by delivering specific cues to MuSCs in a timely manner. Proliferation/expansion of activated MuSCs is sustained by FAPs and inflammatory (damage-associated) macrophages. The next step of myogenesis, that is, differentiation, is stimulated by anti-inflammatory (restorative) macrophages as well as by ECs. Finally, the fusion of MuSCs into multinucleated structures is boosted by restorative macrophages. Return to quiescence of a small subset of MuSCs is supported by the action of peri-ECs such as smooth muscle cells and pericytes, which also maintain the stability of vessels/capillaries.

FIG. 5.

Muscular dystrophies–the DGC complex. Inherited muscular dystrophies originate from mutations in one of the components of the DGC, which links the actin cytoskeleton of the myofiber to the extracellular matrix. The main proteins are the extracellular α-DG and laminin, the sarcolemmal β-DG, SGCs and Sar, and the cytosolic proteins dystrophin, SYN, DYB, actin, and nNOS. The DGC complex provides stability to the membrane structure of skeletal muscle fibers during contraction/relaxation cycles. When mutations target DGC components, the stability is not ensured and fragility of the membrane leads to repeated injuries of the myofibers during contraction. α-DG, α-dystroglycan; β-DG, β-dystroglycan; DGC, dystrophin-glycoprotein complex; DYB, dystrobrevin; nNOS, neural NO synthase; Sar, sarcospan; SGCs, sarcoglycans; SYN, syntrophins.

FIG. 6.

Permanent regeneration during muscular dystrophies. After an acute injury in normal muscle (top left part of the picture), the process of skeletal muscle regeneration occurs due to synchronous regenerating cues delivered by cells neighboring MuSCs. In muscular dystrophies, repeated injuries, occurring throughout the muscle, lead to the asynchronous delivery of regenerating cues, preventing the various cell types from being able to carry out the timely orchestration of the process of skeletal muscle regeneration, which is, thus, profoundly affected. The permanent attempts of regeneration together with the cycles of necrosis lead to failed myogenesis, chronic inflammation, fibrosis, and alterations in the vascular bed.

FIG. 7.

ROS metabolism and biochemistry. Chemical characteristics of ROS reveal their biological properties. Oxygen molecule (O₂) univalent reduction leads to the formation of reactive superoxide anion (O₂^•−). This primary ROS is a weak oxidant and a weak reductant agent. O₂^•− interacts with NO^•, transition metals and can naturally dismute in H₂O₂. SODs accelerate the rate of this reaction by 10,000. H₂O₂ is a nonradical molecule exhibiting a low reactivity and the ability to cross the membrane, suggesting a crucial role in signaling. By contrast, hydroxyl radical (HO^•) derived from metals and H₂O₂ interactions is highly reactive and is an unstable molecule that damages unspecified compounds that are located close to its site of production. H₂O₂, hydrogen peroxide; NO^•, nitric oxide; ROS, reactive oxygen species; SOD, superoxide dismutase.

FIG. 8.

Intracellular sources of ROS and RNS and antioxidant network in skeletal muscle cells. Nucleus, mitochondria, ER, and peroxisomes are the main sources of ROS in the skeletal muscle cell. In addition, enzymatic complexes generate ROS such as XO and NOX. Antioxidant molecules are mainly localized in the cytosol and in mitochondria. In addition, lipophilic antioxidants (vitamin E and tocopherols) are contained within the lipidic membrane. Dietary antioxidants such as vitamin C and carotenoids enter into the cytosol after food ingestion. ER, endoplasmic reticulum; NOX, NADPH oxidase; RNS, reactive nitrogen species; XO, xanthine oxidase.

FIG. 9.

Major oxidative thiol modifications in redox-sensitive cysteine residues. Thiol moiety of redox-sensitive cysteine residues (SH) is vulnerable to oxidation and can lead to various post-translational modifications that are reversible or irreversible depending on the degree of oxidation. The initial reaction product with ROS is sulfenic acid (SOH). Further oxidation of sulfenic acid leads to the formation of irreversible modifications such as sulfinic acid (SO₂H) and, subsequently, sulfonic acid (SO₃H). The reaction of thiol with ROS can lead to the formation of both intra- and intermolecular disulfide bonds. Thiol reaction with GSH and RNS leads to the formation of glutathionylation (-SGSH) and nitrosothiols (-SNO), respectively. Full and dotted arrows represent oxidation and reduction, respectively. GSH, glutathione.

FIG. 10.

Redox-sensitive targets during adult myogenesis. Each step of adult myogenesis during skeletal muscle regeneration (Fig. 2) is regulated in MuSCs by redox activity. Some redox-related factors have been shown to directly regulate MuSC survival, activation, proliferation, self-renewal, and differentiation (upper panel). Moreover, various signaling pathways have been shown to control MuSC fate on the one hand, and they are known to be targets of redox regulation on the other hand (lower panel).

FIG. 11.

Macrophages during skeletal muscle regeneration. On skeletal muscle injury, monocytes/macrophages are recruited from the blood and invade the tissue as damage-associated macrophages (Ly6C^pos cells). They release pro-inflammatory molecules (TNFα, IL-6, IL-1β, VEGF) to stimulate MuSC proliferation. Then, on phagocytosis, these macrophages skew their phenotype to acquire a restorative phenotype and secrete compounds that stimulate myogenic differentiation (IGF-1, TGFβ). Macrophage skewing is regulated by a series of effectors, which are regulated by redox mechanisms. Moreover, restorative macrophages express high levels of SOD1 and Trx, which are associated with the reduction of the alarmin HGMB1 that participates in MuSC migration and differentiation. See text for details. IGF-1, insulin growth factor-1; IL, interleukin; TGFβ, transforming growth factor-beta; Trx, thioredoxin; VEGF, vascular endothelial growth factor.

FIG. 12.

Redox alteration of myogenesis in aged muscle. Fate of MuSCs during postinjury myogenesis is altered in aged muscle, including reduced activation, reduced proliferation, reduced self-renewal, and increased differentiation. A series of redox effectors are deregulated in aged MuSCs and are listed in the box. In italics are shown the signaling pathways that are altered in aged MuSCs and known to be redox regulated. ↗↘ Stand for increase and decrease, respectively.

FIG. 13.

ROS in MuSCs in muscular dystrophies. The upper circle represents a synchronous regeneration process triggered by a single injury. The lower circle shows that in muscular dystrophies, repeated injuries trigger the delivery of asynchronous regenerative cues, leading to a failure of the regeneration process, associated with a whole increase of ROS. MuSCs from dystrophic muscle have been shown to be more susceptible to oxidative stress than normal MuSCs. However, only a few molecular determinants underlying this susceptibility have been evidenced so far, those are listed on the right. See text for details. ↗↘ Stand for increase and decrease, respectively.

FIG. 14.

Therapeutic strategies in aged and dystrophic muscles—effects on MuSCs. Antioxidant supplementation on whole body (upper panel) increases the detection of antioxidant biomarkers, triggers some beneficial effects in muscular dystrophies, but no improvement of muscle function in both aged and diseased muscle is observed. A variety of antioxidant molecules exert beneficial effects on MuSCs in vitro (middle panel), notably they decrease some abnormalities observed in muscular dystrophies. The analysis of MuSCs in vivo after treatment in mouse models (lower panel) indicates an overall benefit for MuSC homeostasis via increased energetic metabolism and myogenesis. ↗↘≈ stand for increase, decrease, and no effect, respectively.

FIG. 15.

Future strategies for the study of ROS. The scheme presents interrelated strategies and developing tools that will help to identify precisely which ROS and ROS-dependent signaling are involved in a given biological context. Such knowledge will further allow to define specific targets that are potentially used in pharmacological trials. Detection and identification of ROS is performed through the development of specific probes (upper left). Several chemical probes have been developed to investigate redox mechanisms, mainly composed of a fluorophore that emits or is liberated on oxidation (351). More recently, genetically encoded fluorescent redox sensors or injected exomarkers allow the detection and visualization of live variations of ROS or redox-regulated molecules (e.g., GSH, NAD) both in vitro and in vivo (upper right) (5, 68, 98, 197). ROS-induced PTMs have recently been described due to the development of redox proteomics, thanks to the setup of cysteine residue labeling (bottom right). Together with crystallography, which describes protein modifications on a redox reaction, these biochemical strategies allowed the identification of new molecular targets of redox reactions (bottom right) (60, 61, 186, 213). The development of this large variety of tools for in vitro and in vivo investigation of redox biology, together with the improvement of the probes and of their specificity will enable the development of antioxidant probes to be tested in preclinical and clinical trials (bottom left). PTMs, post-translational modifications.