Promote Muscle Regeneration Research Articles

MASTR directs MyoD-dependent satellite cell differentiation during skeletal muscle regeneration

In response to skeletal muscle injury, satellite cells, which function as a myogenic stem cell population, become activated, expand through proliferation, and ultimately fuse with each other and with damaged myofibers to promote muscle regeneration. Here, we show that members of the Myocardin family of transcriptional coactivators, MASTR and MRTF-A, are up-regulated in satellite cells in response to skeletal muscle injury and muscular dystrophy. Global and satellite cell-specific deletion of MASTR in mice impairs skeletal muscle regeneration. This impairment is substantially greater when MRTF-A is also deleted and is due to aberrant differentiation and excessive proliferation of satellite cells. These abnormalities mimic those associated with genetic deletion of MyoD, a master regulator of myogenesis, which is down-regulated in the absence of MASTR and MRTF-A. Consistent with an essential role of MASTR in transcriptional regulation of MyoD expression, MASTR activates a muscle-specific postnatal MyoD enhancer through associations with MEF2 and members of the Myocardin family. Our results provide new insights into the genetic circuitry of muscle regeneration and identify MASTR as a central regulator of this process.

Deubiquitinating enzyme A20 negatively regulates NF‐κB signaling in skeletal muscle inmdxmice

Duchenne muscular dystrophy (DMD) is caused by the lack of a functional dystrophin protein that results in muscle fiber membrane disruption and, ultimately, degeneration. Regeneration of muscle fibers fails progressively, and muscle tissue is replaced with connective tissue. As a result, DMD causes progressive limb muscle weakness and cardiac and respiratory failure. The absence of dystrophin from muscle fibers triggers the chronic activation of the nuclear factor of kappa B (NF-κB). Chronic activation of NF-κB in muscle leads to infiltration of macrophages, up-regulation of the ubiquitin-proteosome system, and down-regulation of the helix-loop-helix muscle regulatory factor, MyoD. These processes, triggered by NF-κB activation, promote muscle degeneration and failure of muscle regeneration. A20 (TNFAIP3) is a critical negative regulator of NF-κB. In this study, we characterize the role of A20 in regulating NF-κB activation in skeletal muscle, identifying a novel role in muscle regeneration. A20 is highly expressed in regenerating muscle fibers, and knockdown of A20 impairs muscle differentiation in vitro, which suggests that A20 expression is critically important for regeneration of dystrophic muscle tissue. Furthermore, down-regulation of the classic pathway of NF-κB activation is associated with up-regulation of the alternate pathway in regenerating muscle fibers, suggesting a mechanism by which A20 promotes muscle regeneration. These results demonstrate the important role of A20 in muscle fiber repair and suggest the potential of A20 as a therapeutic target to ameliorate the pathology and clinical symptoms of DMD.

CREB Is Activated by Muscle Injury and Promotes Muscle Regeneration

The cAMP response element binding protein (CREB) plays key roles in differentiation of embryonic skeletal muscle progenitors and survival of adult skeletal muscle. However, little is known about the physiologic signals that activate CREB in normal muscle. Here we show that CREB phosphorylation and target genes are induced after acute muscle injury and during regeneration due to genetic mutation. Activated CREB localizes to both myogenic precursor cells and newly regenerating myofibers within regenerating areas. Moreover, we found that signals from damaged skeletal muscle tissue induce CREB phosphorylation and target gene expression in primary mouse myoblasts. An activated CREB mutant (CREBY134F) potentiates myoblast proliferation as well as expression of early myogenic transcription factors in cultured primary myocytes. Consistently, activated CREB-YF promotes myoblast proliferation after acute muscle injury in vivo and enhances muscle regeneration in dystrophic mdx mice. Our findings reveal a new physiologic function for CREB in contributing to skeletal muscle regeneration.

The role of multifunctional delivery scaffold in the ability of cultured myoblasts to promote muscle regeneration

Many cell types of therapeutic interest, including myoblasts, exhibit reduced engraftment if cultured prior to transplantation. This study investigated whether polymeric scaffolds that direct cultured myoblasts to migrate outwards and repopulate the host damaged tissue, in concert with release of angiogenic factors designed to enhance revascularizaton of the regenerating tissue, would enhance the efficacy of this cell therapy and lead to functional muscle regeneration. This was investigated in the context of a severe injury to skeletal muscle tissue involving both myotoxin-mediated direct damage and induction of regional ischemia. Local and sustained release of VEGF and IGF-1 from macroporous scaffolds used to transplant and disperse cultured myogenic cells significantly enhanced their engraftment, limited fibrosis, and accelerated the regenerative process. This resulted in increased muscle mass and, improved contractile function. These results demonstrate the importance of finely controlling the microenvironment of transplanted cells in the treatment of severe muscle damage.

A Rodent Model to Advance the Field Treatment of Crush Muscle Injury During Earthquakes and Other Natural Disasters

Approximately 170 earthquakes of 6.0 or higher magnitude occur annually worldwide. Victims often suffer crush muscle injuries involving impaired blood flow to the affected muscle and damage to the muscle fiber membrane. Current rescue efforts are directed toward preventing acute kidney injury (AKI), which develops upon extrication and muscle reperfusion. But field-usable, muscle-specific interventions may promote muscle regeneration and prevent or minimize the pathologic changes of reperfusion. Although current rodent crush injury models involve reperfusion upon removal of the crush stimulus, an analysis of their methodological aspects is needed to ensure adequate simulation of the earthquake-related crush injury. The objectives of this systematic review are to (a) describe rodent crush muscle injury models, (b) discuss the benefits and limitations of these models, and (c) offer a recommendation for animal models that would increase our understanding of muscle recovery processes after an earthquake-induced crush muscle injury. The most commonly used rodent model uses a clamping or pressing crush stimulus directly applied to murine hindlimb muscle. This model has increased our understanding of muscle regeneration but its open approach does not adequately represent the earthquake-related crush injury. The model we recommend for developing field-usable, muscle-specific interventions is a closed approach that involves a nonclamping crush stimulus. Findings from studies employing this recommended model may have greater relevance for developing interventions that lessen the earthquake's devastating impact on individual and community health and quality of life, especially in developing countries.

Macrophage-Specific Expression of Urokinase-Type Plasminogen Activator Promotes Skeletal Muscle Regeneration

Macrophages (Mp) and the plasminogen system play important roles in tissue repair following injury. We hypothesized that Mp-specific expression of urokinase-type plasminogen activator (uPA) is sufficient for Mp to migrate into damaged muscle and for efficient muscle regeneration. We generated transgenic mice expressing uPA only in Mp, and we assessed the ability of these mice to repair muscle injury. Mp-only uPA expression was sufficient to induce wild-type levels of Mp accumulation, angiogenesis, and new muscle fiber formation. In mice with wild-type uPA expression, Mp-specific overexpression further increased Mp accumulation and enhanced muscle fiber regeneration. Furthermore, Mp expression of uPA regulated the level of active hepatocyte growth factor, which is required for muscle fiber regeneration, in damaged muscle. In vitro studies demonstrated that uPA promotes Mp migration through proteolytic and nonproteolytic mechanisms, including proteolytic activation of hepatocyte growth factor. In summary, Mp-derived uPA promotes muscle regeneration by inducing Mp migration, angiogenesis, and myogenesis.

Hesperedin promotes MyoD‐induced myogenic differentiation in vitro and in vivo

The bioflavonoid, hesperedin, promotes osteoblast differentiation in human mesenchymal stem cells, indicating an anabolic effect of hesperedin on bone metabolism. Murine bone marrow mesenchymal stem cells undergo myogenic differentiation as well as osteogenic differentiation. We therefore explored whether hesperedin modulates muscle cell differentiation. Myoblast C2C12 cells were differentiated into muscle cells in the presence or absence of hesperedin. The effects of hesperedin on myogenic differentiation were determined by analysing specific muscle markers in vitro using reporter gene assays, immunoblotting, RT-PCR and DNA pull-down assays. In vivo, the effects of hesperedin were assessed using the freeze injury-induced muscle regeneration model in mice and daily injections of hesperedin for 6 days. Hesperedin promoted myogenic differentiation, in a dose-dependent manner, by increasing myogenin gene expression. MyoD-induced myogenin gene transcription was enhanced by hesperedin, as this bioflavonoid augmented the nuclear localization and myogenin promoter-binding of MyoD. In addition, hesperedin increased myogenin and muscle creatine kinase gene expression during myogenic differentiation from C3H10T1/2 mesenchymal stem cells in a MyoD-dependent manner and accelerated in vivo muscle regeneration induced by muscle injury. Our results demonstrate that hesperedin promoted myogenic differentiation in vitro and in vivo through activation of MyoD-mediated myogenin expression, suggesting a beneficial role in promoting muscle regeneration, following injury.

Inhibition of TGF‐β promotes the early recovery of force following contraction‐induced skeletal muscle injury

Transforming Growth Factor-β (TGF-β) is a cytokine liked to fibrosis and other inflammatory processes in many tissues. Previous studies have shown detrimental effects of TGF-β on skeletal muscle in vivo, including fibrosis, atrophy, and decreased force. Thus, inhibition TGF-β may be beneficial to muscle function. We hypothesized that inhibition of TGF-β would promote muscle regeneration following injury. Extensor digitorum longus muscles of adult, male C57Bl/6 mice were subjected to an in situ lengthening-contraction protocol to induce injury. Intraperitoneal injections of 1D11, a murine monoclonal bio-neutralizing IgG antibody against TGF-β, or equivalent amounts of a sham murine IgG were administered immediately following injury and again every three days after injury. Peak isometric force was measured before, immediately after, and 7 days post injury. Compared with contralateral control muscles, mass of injured muscles from mice that received 1D11 were 12% lower while injured muscles from sham treated mice were 19% lower. Additionally, compared with pre-injury force values, mice receiving 1D11 showed a 30% greater recovery in force production than sham treated mice. The results indicate that the therapeutic inhibition of TGF-β following contraction-induced muscle injuries may help promote the early recovery of force production. Supported by AR-058920 and AR-055624.

Long-Term Blocking of Calcium Channels in mdx Mice Results in Differential Effects on Heart and Skeletal Muscle

The disease mechanisms underlying dystrophin-deficient muscular dystrophy are complex, involving not only muscle membrane fragility, but also dysregulated calcium homeostasis. Specifically, it has been proposed that calcium channels directly initiate a cascade of pathological events by allowing calcium ions to enter the cell. The objective of this study was to investigate the effect of chronically blocking calcium channels with the aminoglycoside antibiotic streptomycin from onset of disease in the mdx mouse model of Duchenne muscular dystrophy (DMD).Treatment in utero onwards delayed onset of dystrophic symptoms in the limb muscle of young mdx mice, but did not prevent degeneration and regeneration events occurring later in the disease course. Long-term treatment had a positive effect on limb muscle pathology, reduced fibrosis, increased sarcolemmal stability, and promoted muscle regeneration in older mice. However, streptomycin treatment did not show positive effects in diaphragm or heart muscle, and heart pathology was worsened. Thus, blocking calcium channels even before disease onset does not prevent dystrophy, making this an unlikely treatment for DMD. These findings highlight the importance of analyzing several time points throughout the life of the treated mice, as well as analyzing many tissues, to get a complete picture of treatment efficacy.

Sca-1 influences the innate immune response during skeletal muscle regeneration

Efficient muscle regeneration requires the clearance of dead and dying tissue via phagocytosis before remodeling. We have previously shown that mice lacking stem cell antigen-1 (Sca-1) display a defect in skeletal muscle regeneration characterized by increased fibrosis and decreased turnover of the extracellular matrix. In the present study we demonstrate that Sca-1(-/-) mice have a defect in their capacity to recruit soluble IgM, and subsequently C3 complement, to damaged muscle. We hypothesize that this defect in recruitment delays or decreases phagocytosis by macrophages, contributing to the previously observed fibrotic phenotype of these mice. As the primary source of soluble IgM is peritoneal B-1a cells, which are a subset of self-renewing B cells, we analyzed this cell population and observed a significant reduction in B-1a cells in Sca-1(-/-) animals. Interestingly, these mice are protected from ischemia-reperfusion injury, an acute inflammatory reaction also mediated by IgM and C3 complement that has been linked to a deficit in B-1a cells in previous studies. Collectively, these data reveal a novel role for Sca-1 in innate immunity during muscle regeneration and indicate that further elucidation of immuno-myogenic processes will help to better understand and promote muscle regeneration.

Relaxin Regulates MMP Expression and Promotes Satellite Cell Mobilization During Muscle Healing in Both Young and Aged Mice

The polypeptide hormone relaxin has been proven to be effective in promoting both the remodeling and regeneration of various tissues, including cardiac muscle. In addition, our previous study demonstrated that relaxin is beneficial to skeletal muscle healing by both promoting muscle regeneration and preventing fibrosis formation. However, the molecular and cellular mechanisms of relaxin in regulating both myogenic cell differentiation and muscle healing process are still unclear. In this study, C2C12 mouse myoblasts and primary human myoblasts were treated with relaxin to investigate its potential effect in vitro; relaxin was also injected intramuscularly into the injured site of the mouse on the second day after injury to observe its function in vivo, especially in the aged muscle. Results showed that relaxin promoted myogenic differentiation, migration, and activation of matrix metalloproteinases (MMPs) of cultured myoblasts in vitro. In the injured muscle, relaxin administration promoted the activation of Pax7-positive skeletal muscle satellite cells and increased its local population compared with nontreated control muscles. Meanwhile, both angiogenesis and revascularization were increased, while the extended inflammatory reaction was repressed in the relaxin-treated injured muscle. Moreover, relaxin similarly promoted muscle healing in mice with aged muscle. These results revealed the multiple effects of relaxin in systematically improving muscle healing as well as its potential for clinical applications in patients with skeletal muscle injuries and diseases.

Macrophages recruited via CCR2 produce insulin‐like growth factor‐1 to repair acute skeletal muscle injury

CC chemokine receptor 2 (CCR2) is essential to acute skeletal muscle injury repair. We studied the subpopulation of inflammatory cells recruited via CCR2 signaling and their cellular functions with respect to muscle regeneration. Mobilization of monocytes/macrophages (MOs/MPs), but not lymphocytes or neutrophils, was impaired from bone marrow to blood and from blood to injured muscle in Ccr2(-/-) mice. While the Ly-6C(+) but not the Ly-6C(-) subset of MOs/MPs was significantly reduced in blood, both subsets were drastically reduced in injured muscle of Ccr2(-/-) mice. Expression of insulin-like growth factor-1 (IGF-I) was markedly up-regulated in injured muscle of wild-type but not Ccr2(-/-) mice. IGF-I was strongly expressed by macrophages within injured muscle, more prominently by the Ly-6C(-) subset. A single injection of IGF-I, but not PBS, into injured muscle to replace IGF-I remarkably improved muscle regeneration in Ccr2(-/-) mice. CCR2 was not detected in myogenic cells or capillary endothelial cells in injured muscle to suggest its direct involvement in muscle regeneration or angiogenesis. We conclude that CCR2 is essential to acute skeletal muscle injury repair primarily by recruiting Ly-6C(+) MOs/MPs. Within injured muscle, these cells conduct phagocytosis, contribute to accumulation of intramuscular Ly-6C(-) macrophages, and produce a high level of IGF-I to promote muscle regeneration.

Stem Cell Therapies to Treat Muscular Dystrophy

Muscular dystrophies are heritable, heterogeneous neuromuscular disorders and include Duchenne and Becker muscular dystrophies (DMD and BMD, respectively). DMD patients exhibit progressive muscle weakness and atrophy followed by exhaustion of muscular regenerative capacity, fibrosis, and eventually disruption of the muscle tissue architecture. In-frame mutations in the dystrophin gene lead to expression of a partially functional protein, resulting in the milder BMD. No effective therapies are available at present. Cell-based therapies have been attempted in an effort to promote muscle regeneration, with the hope that the host cells would repopulate the muscle and improve muscle function and pathology. Injection of adult myoblasts has led to the development of new muscle fibers, but several limitations have been identified, such as poor cell survival and limited migratory ability. As an alternative to myoblasts, stem cells were considered preferable for therapeutic applications because of their capacity for self-renewal and differentiation potential. In recent years, encouraging results have been obtained with adult stem cells to treat human diseases such as leukemia, Parkinson's disease, stroke, and muscular dystrophies. Embryonic stem cells (ESCs) can be derived from mammalian embryos in the blastocyst stage, and because they can differentiate into a wide range of specialized cells, they hold potential for use in treating almost all human diseases. Several ongoing studies focus on this possibility, evaluating differentiation of specific cell lines from human ESCs (hESCs) as well as the potential tumorigenicity of hESCs. The most important limitation with using hESCs is that it requires destruction of human blastocysts or embryos. Conversely, adult stem cells have been identified in various tissues, where they serve to maintain, generate, and replace terminally differentiated cells within their specific tissue as the need arises for cell turnover or from tissue injury. Moreover, these cells can participate in regeneration of more than just their specific tissue type. Here we describe multiple types of muscle- and fetal-derived myogenic stem cells, their characterization, and their possible use in treating muscular dystrophies such as DMD and BMD. We also emphasize that the most promising possibility for the management and therapy of DMD and BMD is a combination of different approaches, such as gene and stem cell therapy.

Regulatory interactions between muscle and the immune system during muscle regeneration

Recent discoveries reveal complex interactions between skeletal muscle and the immune system that regulate muscle regeneration. In this review, we evaluate evidence that indicates that the response of myeloid cells to muscle injury promotes muscle regeneration and growth. Acute perturbations of muscle activate a sequence of interactions between muscle and inflammatory cells. The initial inflammatory response is a characteristic Th1 inflammatory response, first dominated by neutrophils and subsequently by CD68(+) M1 macrophages. M1 macrophages can propagate the Th1 response by releasing proinflammatory cytokines and cause further tissue damage through the release of nitric oxide. Myeloid cells in the early Th1 response stimulate the proliferative phase of myogenesis through mechanisms mediated by TNF-alpha and IL-6; experimental prolongation of their presence is associated with delayed transition to the early differentiation stage of myogenesis. Subsequent invasion by CD163(+)/CD206(+) M2 macrophages attenuates M1 populations through the release of anti-inflammatory cytokines, including IL-10. M2 macrophages play a major role in promoting growth and regeneration; their absence greatly slows muscle growth following injury or modified use and inhibits muscle differentiation and regeneration. Chronic muscle injury leads to profiles of macrophage invasion and function that differ from acute injuries. For example, mdx muscular dystrophy yields invasion of muscle by M1 macrophages, but their early invasion is accompanied by a subpopulation of M2a macrophages. M2a macrophages are IL-4 receptor(+)/CD206(+) cells that reduce cytotoxicity of M1 macrophages. Subsequent invasion of dystrophic muscle by M2c macrophages is associated with progression of the regenerative phase in pathophysiology. Together, these findings show that transitions in macrophage phenotype are an essential component of muscle regeneration in vivo following acute or chronic muscle damage.

Functional muscle regeneration with combined delivery of angiogenesis and myogenesis factors

Regenerative efforts typically focus on the delivery of single factors, but it is likely that multiple factors regulating distinct aspects of the regenerative process (e.g., vascularization and stem cell activation) can be used in parallel to affect regeneration of functional tissues. This possibility was addressed in the context of ischemic muscle injury, which typically leads to necrosis and loss of tissue and function. The role of sustained delivery, via injectable gel, of a combination of VEGF to promote angiogenesis and insulin-like growth factor-1 (IGF1) to directly promote muscle regeneration and the return of muscle function in ischemic rodent hindlimbs was investigated. Sustained VEGF delivery alone led to neoangiogenesis in ischemic limbs, with complete return of tissue perfusion to normal levels by 3 weeks, as well as protection from hypoxia and tissue necrosis, leading to an improvement in muscle contractility. Sustained IGF1 delivery alone was found to enhance muscle fiber regeneration and protected cells from apoptosis. However, the combined delivery of VEGF and IGF1 led to parallel angiogenesis, reinnervation, and myogenesis; as satellite cell activation and proliferation was stimulated, cells were protected from apoptosis, the inflammatory response was muted, and highly functional muscle tissue was formed. In contrast, bolus delivery of factors did not have any benefit in terms of neoangiogenesis and perfusion and had minimal effect on muscle regeneration. These results support the utility of simultaneously targeting distinct aspects of the regenerative process.

Urokinase-type plasminogen activator increases hepatocyte growth factor activity required for skeletal muscle regeneration

AbstractThe plasminogen system plays a crucial role in the repair of a variety of tissues, including skeletal muscle. We hypothesized that urokinase-type plasminogen activator (uPA) promotes muscle regeneration by activating hepatocyte growth factor (HGF), which, in turn, stimulates proliferation of myoblasts required for regeneration. In our studies, levels of active HGF and phosphorylation of the HGF receptor c-met were increased after muscle injury in wild-type mice. Compared with wild-type animals, mice deficient in uPA (uPA−/−) had markedly reduced HGF levels and c-met activation after muscle damage. This reduced HGF activity in uPA−/− animals was associated with decreased cell proliferation, myoblast accumulation, and new muscle fiber formation. On the other hand, HGF activity was enhanced at early time points in PAI-1−/− mice compared with wild-type mice and the PAI-1−/− animals exhibited accelerated muscle fiber regeneration. Furthermore, administration of exogenous uPA rescued HGF levels and muscle regeneration in uPA−/− mice, and an HGF-blocking antibody reduced HGF activity and muscle regeneration in wild-type mice. We also found that uPA promotes myoblast proliferation in vitro through its proteolytic activity, and this process was inhibited by an HGF-blocking antibody. Together, our findings demonstrate that uPA promotes muscle regeneration through HGF activation and subsequent myoblast proliferation.

Chemokine CXCL16 Regulates Neutrophil and Macrophage Infiltration into Injured Muscle, Promoting Muscle Regeneration

Only a few specific chemokines that mediate interactions between inflammatory and satellite cells in muscle regeneration have been identified. The chemokine CXCL16 differs from other chemokines because it has both a transmembrane region and active, soluble chemokine forms. Indeed, we found increased expression of CXCL16 and its receptor, CXCR6, in regenerating myofibers. Muscle regeneration in CXCL16-deficient (CXCL16KO) mice was severely impaired compared with regeneration in wild-type mice. In addition, there was decreased MyoD and myogenin expression in regenerating muscle in CXCL16KO mice, indicating impaired satellite cell proliferation and differentiation. After 1 month, new myofibers in CXCL16KO mice remained significantly smaller than those in muscle of wild-type mice. To understand how CXCL16 regulates muscle regeneration, we examined cells infiltrating injured muscle. There were more infiltrating neutrophils and fewer macrophages in injured muscle of CXCL16KO mice compared with events in wild-type mice. Moreover, absence of CXCL16 led to different expression of cytokines/chemokines in injured muscles: mRNAs of macrophage-inflammatory protein (MIP)-1alpha, MIP-1beta, and MIP-2 were increased, whereas regulated on activation normal T cell expressed and secreted, T-cell activation-3, and monocyte chemoattractant protein-1 mRNAs were lower compared with results in muscles of wild-type mice. Impaired muscle regeneration in CXCL16KO mice also resulted in fibrosis, which was linked to transforming growth factor-beta1 expression. Thus, CXCL16 expression is a critical mediator of muscle regeneration, and it suppresses the development of fibrosis.

MOR23 Promotes Muscle Regeneration and Regulates Cell Adhesion and Migration

Odorant receptors (ORs) in the olfactory epithelium bind to volatile small molecules leading to the perception of smell. ORs are expressed in many tissues but their functions are largely unknown. We show multiple ORs display distinct mRNA expression patterns during myogenesis in vitro and muscle regeneration in vivo. Mouse OR23 (MOR23) expression is induced during muscle regeneration when muscle cells are extensively fusing and plays a key role in regulating migration and adhesion of muscle cells in vitro, two processes common during tissue repair. A soluble ligand for MOR23 is secreted by muscle cells in vitro and muscle tissue in vivo. MOR23 is necessary for proper skeletal muscle regeneration as loss of MOR23 leads to increased myofiber branching, commonly associated with muscular dystrophy. Together these data identify a functional role for an OR outside of the nose and suggest a larger role for ORs during tissue repair.

Development of a Nitric Oxide-Releasing Analogue of the Muscle Relaxant Guaifenesin for Skeletal Muscle Satellite Cell Myogenesis

Nitric oxide (NO) mediates activation of satellite precursor cells to enter the cell cycle. This provides new precursor cells for skeletal muscle growth and muscle repair from injury or disease. Targeting a new drug that specifically delivers NO to muscle has the potential to promote normal function and treat neuromuscular disease, and would also help to avoid side effects of NO from other treatment modalities. In this research, we examined the effectiveness of the NO donor, iosorbide dinitrate (ISDN), and a muscle relaxant, methocarbamol, in promoting satellite cell activation assayed by muscle cell DNA synthesis in normal adult mice. The work led to the development of guaifenesin dinitrate (GDN) as a new NO donor for delivering nitric oxide to muscle. The results revealed that there was a strong increase in muscle satellite cell activation and proliferation, demonstrated by a significant 38% rise in DNA synthesis after a single transdermal treatment with the new compound for 24 h. Western blot and immunohistochemistry analyses showed that the markers of satellite cell myogenesis, expression of myf5, myogenin, and follistatin, were increased after 24 h oral administration of the compound in adult mice. This research extends our understanding of the outcomes of NO-based treatments aimed at promoting muscle regeneration in normal tissue. The potential use of such treatment for conditions such as muscle atrophy in disuse and aging, and for the promotion of muscle tissue repair as required after injury or in neuromuscular diseases such as muscular dystrophy, is highlighted.

Muscle CD31(−) CD45(−) Side Population Cells Promote Muscle Regeneration by Stimulating Proliferation and Migration of Myoblasts

CD31(-) CD45(-) side population (SP) cells are a minor SP subfraction that have mesenchymal stem cell-like properties in uninjured skeletal muscle but that can expand on muscle injury. To clarify the role of these SP cells in muscle regeneration, we injected green fluorescent protein (GFP)-positive myoblasts with or without CD31(-) CD45(-) SP cells into the tibialis anterior muscles of immunodeficient NOD/scid mice or dystrophin-deficient mdx mice. More GFP-positive fibers were formed after co-transplantation than after transplantation of GFP-positive myoblasts alone in both mdx and NOD/scid muscles. Moreover, grafted myoblasts were more widely distributed after co-transplantation than after transplantation of myoblasts alone. Immunohistochemistry with anti-phosphorylated histone H3 antibody revealed that CD31(-) CD45(-) SP cells stimulated cell division of co-grafted myoblasts. Genome-wide gene expression analyses showed that these SP cells specifically express a variety of extracellular matrix proteins, membrane proteins, and cytokines. We also found that they express high levels of matrix metalloproteinase-2 mRNA and gelatinase activity. Furthermore, matrix metalloproteinase-2 derived from CD31(-) CD45(-) SP cells promoted migration of myoblasts in vivo. Our results suggest that CD31(-) CD45(-) SP cells support muscle regeneration by promoting proliferation and migration of myoblasts. Future studies to further define the molecular and cellular mechanisms of muscle regeneration will aid in the development of cell therapies for muscular dystrophy.