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Cardiac mononuclear phagocytic cells (Cardiac MPCs) participate in maintaining homeostasis and orchestrating cardiac responses upon injury. However, the function of specific MPC subtypes and the related cell fate commitment mechanisms remain elusive in regenerative and nonregenerative hearts due to their cellular heterogeneities. Using spatiotemporal single-cell epigenomic analysis of cardiac MPCs in regenerative (P1) and nonregenerative (P10) mouse hearts after injury, we found that P1 hearts accumulate reparative [Arg1.sup.+] macrophages, while proinflammatory [S100a9.sup.+][Ly6c.sup.+] monocytes are uniquely abundant during nonregenerative remodeling. Moreover, blocking chemokine CXCR2 to inhibit the specification of the [S100a9.sup.+][Ly6c.sup.+]-biased inflammatory fate in P10 hearts resulted in elevated wound repair responses and marked improvements in cardiac function after injury. Single-cell RNA-Seq further confirmed an increased [Arg1.sup.+] macrophage subpopulation after CXCR2 blockade, which was accomplished by increased expression of wound repair- related genes and reduced expression of proinflammatory genes. Collectively, our findings provide instructive insights into the molecular mechanisms underlying the function and fate specification of heterogeneous MPCs during cardiac repair and identify potential therapeutic targets for myocardial infarction., Introduction Cardiovascular disease is the primary cause of global mortality (1). Upon myocardial infarction (MI), the heart undergoes a diverse range of reparative processes, including the initiation of proinflammatory responses, [...]

Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells.sup.1-4. Macrophages and satellite cells communicate in different ways.sup.1-5, but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles. Mouse models of muscle injuries and ageing characterized by low levels of intra-tissue glutamine are ameliorated by macrophage-specific deletion or systemic pharmacological inhibition of glutamate dehydrogenase 1, which results in constitutively high activity of glutamine synthetase., Author(s): Min Shang [sup.1] [sup.2] , Federica Cappellesso [sup.1] [sup.2] , Ricardo Amorim [sup.1] [sup.2] [sup.3] [sup.4] [sup.5] , Jens Serneels [sup.1] [sup.2] , Federico Virga [sup.1] [sup.2] [sup.6] [sup.7] [...]

Interactions between stem cells and their microenvironment, or niche, are essential for stem cell maintenance and function. Our knowledge of the niche for the skeletal muscle stem cell, i.e., the satellite cell (SC), is incomplete. Here we show that β1-integrin is an essential niche molecule that maintains SC homeostasis, and sustains the expansion and self-renewal of this stem cell pool during regeneration. We further show that β1-integrin cooperates with fibroblast growth factor 2 (Fgf2), a potent growth factor for SCs, to synergistically activate their common downstream effectors, the mitogen-activated protein (MAP) kinase Erk and protein kinase B (Akt). Notably, SCs in aged mice show altered β1-integrin activity and insensitivity to Fgf2. Augmenting β1-integrin activity with a monoclonal antibody restores Fgf2 sensitivity and improves regeneration after experimentally induced muscle injury. The same treatment also enhances regeneration and function of dystrophic muscles in mdx mice, a model for Duchenne muscular dystrophy. Therefore, β1-integrin senses the SC niche to maintain responsiveness to Fgf2, and this integrin represents a potential therapeutic target for pathological conditions of the muscle in which the stem cell niche is compromised., Sarcopenia, the slow progressive loss of skeletal muscle mass concomitant with advancing age, affects elderly people. Age-related muscle wasting is characterized by loss of muscle quantity and quality due to [...]

The X chromosome-encoded histone demethylase UTX (also known as KDM6A) mediates removal of repressive trimethylation of histone H3 lysine 27 (H3K27me3) to establish transcriptionally permissive chromatin. Loss of UTX in female mice is embryonic lethal. Unexpectedly, male UTX-null mice escape embryonic lethality due to expression of UTY, a paralog that lacks H3K27 demethylase activity, suggesting an enzyme-independent role for UTX in development and thereby challenging the need for active H3K27 demethylation in vivo. However, the requirement for active H3K27 demethylation in stem cell-mediated tissue regeneration remains untested. Here, we employed an inducible mouse KO that specifically ablates UtX in satellite cells (SCs) and demonstrated that active H3K27 demethylation is necessary for muscle regeneration. Loss of UTX in SCs blocked myofiber regeneration in both male and female mice. Furthermore, we demonstrated that UTX mediates muscle regeneration through its H3K27 demethylase activity, as loss of demethylase activity either by chemical inhibition or knock-in of demethylase-dead UTX resulted in defective muscle repair. Mechanistically, dissection of the muscle regenerative process revealed that the demethylase activity of UTX is required for expression of the transcription factor myogenin, which in turn drives differentiation of muscle progenitors. Thus, we have identified a critical role for the enzymatic activity of UTX in activating muscle-specific gene expression during myofiber regeneration and have revealed a physiological role for active H3K27 demethylation in vivo., Introduction The development of complex organisms from a single genome requires that cell-specific gene expression programs be tightly controlled both spatially and temporally. This transcriptional control is partially mediated through [...]

Conditions such as muscular dystrophies (MDs) that affect both cardiac and skeletal muscles would benefit from therapeutic strategies that enable regeneration of both of these striated muscle types. Protocols have been developed to promote induced pluripotent stem cells (iPSCs) to differentiate toward cardiac or skeletal muscle; however, there are currently no strategies to simultaneously target both muscle types. Tissues exhibit specific epigenetic alterations; therefore, source-related lineage biases have the potential to improve iPSC-driven multilineage differentiation. Here, we determined that differential myogenic propensity influences the commitment of isogenic iPSCs and a specifically isolated pool of mesodermal iPSC-derived progenitors (MiPs) toward the striated muscle lineages. Differential myogenic propensity did not influence pluripotency, but did selectively enhance chimerism of MiP-derived tissue in both fetal and adult skeletal muscle. When injected into dystrophic mice, MiPs engrafted and repaired both skeletal and cardiac muscle, reducing functional defects. Similarly, engraftment into dystrophic mice of canine MiPs from dystrophic dogs that had undergone TALEN-mediated correction of the MD-associated mutation also resulted in functional striatal muscle regeneration. Moreover, human MiPs exhibited the same capacity for the dual differentiation observed in murine and canine MiPs. The findings of this study suggest that MiPs should be further explored for combined therapy of cardiac and skeletal muscles., Introduction Induced pluripotent stem cells (iPSCs) represent a promising contribution to regenerative medicine (1). Despite the regulatory hurdles and safety issues involved, reprogramming patients' cells into iPSCs for autologous cell [...]

The progressive loss of muscle regenerative capacity with age or disease results in part from a decline in the number and function of satellite cells, the direct cellular contributors to muscle repair (1-11). However, little is known about the molecular effectors underlying satellite cell impairment and depletion. Elevated levels of inflammatory cytokines, including interleukin-6 (IL-6), are associated with both age-related and muscle-wasting conditions (12-15). The levels of STAT3, a downstream effector of IL-6, are also elevated with muscle wasting (16, 17), and STAT3 has been implicated in the regulation of self-renewal and stem cell fate in several tissues (18-21). Here we show that IL-6-activated Stat3 signaling regulates satellite cell behavior, promoting myogenic lineage progression through myogenic differentiation 1 (Myodl) regulation. Conditional ablation of Stat3 in Pax7-expressing satellite cells resulted in their increased expansion during regeneration, but compromised myogenic differentiation prevented the contribution of these cells to regenerating myofibers. In contrast, transient Stat3 inhibition promoted satellite cell expansion and enhanced tissue repair in both aged and dystrophic muscle. The effects of STAT3 inhibition on cell fate and proliferation were conserved in human myoblasts. The results of this study indicate that pharmacological manipulation of STAT3 activity can be used to counteract the functional exhaustion of satellite cells in pathological conditions, thereby maintaining the endogenous regenerative response and ameliorating muscle-wasting diseases., Chronic inflammation is a hallmark of several muscle-wasting diseases and impairs the normal regenerative response. IL-6 is among the inflammatory cytokines present during the initial stages of muscle repair and [...]

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine whether endogenous [c-kit.sup.+] cells contribute differentiated cardiomyocytes to the heart during development, with ageing or after injury in adulthood. A complementary DNA encoding either Cre recombinase or a tamoxifen-inducible MerCreMer chimaeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous [c-kit.sup.+] cells did produce new cardiomyocytes within the heart, although at a percentage of approximately 0.03 or less, and if a preponderance towards cellular fusion is considered, the percentage falls to below approximately 0.008. By contrast, [c-kit.sup.+] cells amply generated cardiac endothelial cells. Thus, endogenous [c-kit.sup.+] cells can generate cardiomyocytes within the heart, although probably at a functionally insignificant level., The adult mammalian heart was originally proposed to be essentially incapable of renewal after injury or with ageing; although some recent studies have shown that the heart is capable of [...]

Damage to the adult mammalian heart is irreversible, and lost cells are not replaced through regeneration. In neonatal mice, prior to P7, heart tissue can be regenerated after injury; however, the factors that facilitate cardiac regeneration in the neonatal heart are not known. In this issue of the JCI, Aurora and colleagues evaluated the immune response following myocardial infarction in P1 mice compared with that in P14 mice, which have lost their regenerative capacity, and identified a population of macrophages as mediators of cardiac repair. Further understanding of the immune modulators that promote the regenerative properties of this macrophage subset could potentially be exploited to recapitulate regenerative function in the adult heart., Repair without regeneration in the damaged adult heart Myocardial infarction (MI), commonly known as heart attack, results in the loss of around 1 billion heart muscle cells and destruction of [...]

Precise epigenetic modifications in stem cells control developmental programs and cell fate decisions. In particular, the addition or removal of trimethylation of histone 3 lysine 27 (H3K27me3) at lineage-specific genes has been linked to the repression of gene expression, and a precise balance of methyltransferases and demethylases within cells determines H3K27me3 levels. The demethylase UTX is essential for development and tissue homeostasis; however, a role for UTX in stem cell-mediated tissue regeneration is unknown. In this issue of theJCI, Dilworth and colleagues reveal that UTX and its demethylase activity are required in the muscle stem cell lineage for muscle regeneration in response to injury. Specifically, UTX mediates the removal of H3K27me3 in the promoter of the transcription factor myogenin, which regulates myogenic differentiation. The results of this study provide important insight into the contribution of epigenetic regulation in stem cell-mediated regeneration of adult tissues., H3K27me3 in development and tissue homeostasis Modifications of histone proteins are among the most well studied epigenetic features of stem cells. One of these histone modifications, trimethylation of histone 3 [...]

Skeletal muscle injury activates adult myogenic stem cells, known as satellite cells, to initiate proliferation and differentiation to regenerate new muscle fibers. The skeletal muscle-specific microRNA miR-206 is upregulated in satellite cells following muscle injury, but its role in muscle regeneration has not been defined. Here, we show that miR-206 promotes skeletal muscle regeneration in response to injury. Genetic deletion of miR-206 in mice substantially delayed regeneration induced by cardiotoxin injury. Furthermore, loss of miR-206 accelerated and exacerbated the dystrophic phenotype in a mouse model of Duchenne muscular dystrophy. We found that miR-206 acts to promote satellite cell differentiation and fusion into muscle fibers through suppressing a collection of negative regulators of myogenesis. Our findings reveal an essential role for miR-206 in satellite cell differentiation during skeletal muscle regeneration and indicate that miR-206 slows progression of Duchenne muscular dystrophy., Introduction Adult skeletal muscle can regenerate in response to exercise, injury, and disease. Skeletal muscle regeneration relies on a small population of stem cells, known as satellite cells (SCs), which [...]

Regeneration of motor systems involves reestablishment of central control networks, reinnervation of muscle targets by motoneurons, and reconnection of neuromodulator)' circuits. Still, how these processes are integrated as motor function is restored during regeneration remains ill defined. Here, we examined the mechanisms underlying motoneuronal regeneration of neuromuscular synapses related to feeding movements in the pulmonate snail Helisoma trivolvis. Neurons B19 and B110, although activated during different phases of the feeding pattern, innervate similar sets of muscles. However, the percentage of muscle fibers innervated, the efficacy of excitatory junction potentials, and the strength of muscle contractions were different for each cell's specific connections. After peripheral nerve crush, a sequence of transient electrical and chemical connections formed centrally within the buccal ganglia. Neuromuscular synapse regeneration involved a three-phase process: the emergence of spontaneous synaptic transmission (P1), the acquisition of evoked potentials of weak efficacy (P2), and the establishment of functional reinnervation (P3). Differential synaptic efficacy at muscle contacts was recapitulated in cell culture. Differences in motoneuronal presynaptic properties (i.e., quantal content) were the basis of disparate neuromuscular synapse function, suggesting a role for retrograde target influences. We propose a homeostatic model of molluscan motor system regeneration. This model has three restoration events: (1) transient central synaptogenesis during axonal outgrowth, (2) intermotoneuronal inhibitory synaptogenesis during initial neuromuscular synapse formation, and (3) target-dependent regulation of neuromuscular junction formation., Introduction Regeneration is the process of repairing damaged tissues or replacing lost body parts after injury. The mechanisms that underlie regeneration vary considerably among animal groups and across a wide [...]

Injury of skeletal muscle is followed by muscle regeneration in which new muscle tissue is formed from the proliferating mononuclear myoblasts, and by systemic response to stress that exposes proliferating myoblasts to increased glucocorticoid (GC) concentration. Because of its various causes, hypoxia is a frequent condition affecting skeletal muscle, and therefore both processes, which importantly determine the outcome of the injury, often proceed under hypoxic conditions. It is therefore important to identify and characterize in proliferating human myoblasts: 1) response to hypoxia which is generally organized by hypoxia-inducible factor-1[alpha] (HIF-1[alpha]); 2) response to GCs which is mediated through the isoforms of glucocorticoid receptors (GRs) and 11[beta]-hydroxysteroid dehydrogenases (11[beta]-HSDs), and 3) the response to GCs under the hypoxic conditions and the influence of this combination on the factors controlling myoblast proliferation. Using real-time PCR, Western blotting, and HIF-1[alpha] small-interfering RNA silencing, we demonstrated that cultured human myoblasts possess both, the HIF-1[alpha]-based response to hypoxia, and the GC response system composed of GR[alpha] and types 1 and 2 11[beta]-HSDs. However, using combined dexamethasone and hypoxia treatments, we demonstrated that these two systems operate practically without mutual interactions. A seemingly surprising separation of the two systems that both organize response to hypoxic stress can be explained on the evolutionary basis: the phylogenetically older HIF-1[alpha] response is a protection at the cellular level, whereas the GC stress response protects the organism as a whole. This necessitates actions, like downregulation of IL-6 secretion and vascular endothelial growth factor, that might not be of direct benefit for the affected myoblasts. hypoxia-inducible factor-[alpha]; antisense version of hypoxia-inducible factor; vascular endothelial growth factor; glucocorticoid receptors; 11[beta]-hydroxysteroid dehydrogenases doi: 10.1152/ajpregu.00133.2010

Cardiac and skeletal muscle development and maintenance require complex interactions between DNA-binding proteins and chromatin remodeling factors. We previously reported that Smyd1, a muscle-restricted histone methyltransferase, is essential for cardiogenesis and functions with a network of cardiac regulatory proteins. Here we show that the muscle-specific transcription factor skNAC is the major binding partner for Smyd1 in the developing heart. Targeted deletion of skNAC in mice resulted in partial embryonic lethality by embryonic day 12.5, with ventricular hypoplasia and decreased cardiomyocyte proliferation that were similar but less severe than in Smyd1 mutants. Expression of Irx4, a ventricle-specific transcription factor down-regulated in hearts lacking Smyd1, also depended on the presence of skNAC. Viable [skNAC.sup.-/-] adult mice had reduced postnatal skeletal muscle growth and impaired regenerative capacity after cardiotoxin-induced injury. Satellite cells isolated from [skNAC.sup.-/-] mice had impaired survival compared with wild-type littermate satellite cells. Our results indicate that skNAC plays a critical role in ventricular cardiomyocyte expansion and regulates postnatal skeletal muscle growth and regeneration in mice. doi/ 10.1073/pnas.1013493107

BACKGROUND: The incidence of low birth weights is increased in offspring of women who are exposed to high concentrations of arsenic in drinking water compared with other women. We hypothesized [...]

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.sup.+] 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.sup.+]/[CD206.sup.+] 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.sup.+]/[CD206.sup.+] 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. skeletal muscle; macrophage; neutrophil; muscular dystrophy; chemokines doi: 10.1152/ajpregu.00735.2009.

The bone morphogenetic protein (BMP) pathway is known to be involved in limb myogenesis during development, but whether it is involved in postnatal muscle regeneration is unclear. We have found that adult inhibitor of differentiation (Id)-mutant ([Id1.sup.+/-][Id3.sup.-/-]) mice display delayed and reduced skeletal muscle regeneration after injury compared with either wild-type littermates or Id3-null mice. Immunoblotting of wild-type muscle lysates revealed that, not only were Id1 and Id3 highly upregulated within 24 h after injury, but other upstream components of the BMP pathway were as well, including the BMP receptor type II and phosphorylated Smad1/5/8 (pSmad1/5/8). Inhibition of BMP signaling in injured skeletal muscle by Noggin injection reduced pSmad1/5/8, Id1, and Id3 protein levels. The mouse myoblast-derived cell line C2C12 also expressed Id1, Id3, BMP receptor type II, and pSmad1/5/8 during proliferation, but all were reduced upon differentiation into myotubes. In addition, these cells secreted mature BMP-4, and BMP signaling could be inhibited with exogenous Noggin, causing a reduction in pSmad1/5/8, Id1, and Id3 levels. Confocal immunofluorescence microscopy revealed that activated [Pax7.sup.+] myoblasts coexpressed nuclear pSmad1/5/8, Id1, and Id3 in injured mouse skeletal muscle sections. Although we did not observe differences in the numbers of quiescent [Pax7.sup.+] satellite cells in adult uninjured hindlimb muscles, we did observe a significant reduction in the number of proliferating [Pax7.sup.+] cells in the Id-mutant mice after muscle injury compared with either wild-type or Id3-null mice. These data suggest a model in which BMP signaling regulates Id1 and Id3 in muscle satellite cells, which directs their proper proliferation before terminal myogenic differentiation after skeletal muscle injury in postnatal animals. bone morphogenetic protein signaling; inhibitor of differentiation-1; inhibitor of differentiation-3; Pax7; satellite cells doi:10.1152/ajpcell.00388.2009.

Recent studies indicate that mammals, including humans, maintain some capacity to renew cardiomyocytes throughout postnatal life (1,2). Yet, there is little or no significant cardiac muscle regeneration after an injury such as acute myocardial infarction (3). By contrast, zebrafish efficiently regenerate lost cardiac muscle, providing a model for understanding how natural heart regeneration may be blocked or enhanced (4,5). In the absence of lineage-tracing technology applicable to adult zebrafish, the cellular origins of newly regenerated cardiac muscle have remained unclear. Using new genetic fate-mapping approaches, here we identify a population of cardiomyocytes that become activated after resection of the ventricular apex and contribute prominently to cardiac muscle regeneration. Through the use of a transgenic reporter strain, we found that cardiomyocytes throughout the subepicardial ventricular layer trigger expression of the embryonic cardiogenesis gene gata4 within a week of trauma, before expression localizes to proliferating cardiomyocytes surrounding and within the injury site. Cre-recombinase-based lineage-tracing of cells expressing gata4 before evident regeneration, or of cells expressing the contractile gene cmlc2 before injury, each labelled most cardiac muscle in the ensuing regenerate. By optical voltage mapping of surface myocardium in whole ventricles, we found that electrical conduction is re-established between existing and regenerated cardiomyocytes between 2 and 4 weeks post-injury. After injury and prolonged fibroblast growth factor receptor inhibition to arrest cardiac regeneration and enable scar formation, experimental release of the signalling block led to gata4 expression and morphological improvement of the injured ventricular wall without loss of scar tissue. Our results indicate that electrically coupled cardiac muscle regenerates after resection injury, primarily through activation and expansion of cardiomyocyte populations. These findings have implications for promoting regeneration of the injured human heart., After removal of the apex of their cardiac ventricle, zebrafish replace the resected myocardium (4,5). Although these events involve cardiomyocyte hyperplasia, it is unclear whether proliferating cardiomyocytes derive from existing [...]

Sphingosine l-phosphate (S1P) is a bioactive lipid known to control cell growth that was recently shown to act as atrophic factor for skeletal muscle, reducing the progress of denervation atrophy. The aim of this work was to investigate whether S1P is involved in skeletal muscle fiber recovery (regeneration) after myotoxic injury induced by bupivacaine. The postnatal ability of skeletal muscle to grow and regenerate is dependent on resident stem cells called satellite cells. Immunofluorescence analysis demonstrated that S1P-specific receptors S1[P.sub.1] and S1[P.sub.3] are expressed by quiescent satellite cells. Soleus muscles undergoing regeneration following injury induced by intramuscular injection of bupivacaine exhibited enhanced expression of S1[P.sub.1] receptor, while S1[P.sub.3] expression progressively decreased to adult levels. S1[P.sub.2] receptor was absent in quiescent cells but was transiently expressed in the early regenerating phases only. Administration of SIP (50 [micro]M) at the moment of myotoxic injury caused a significant increase of the mean crosssectional area of regenerating fibers in both rat and mouse. In separate experiments designed to test the trophic effects of S1P, neutralization of endogenous circulating SIP by intraperitoneal administration of anti-S1P antibody attenuated fiber growth. Use of selective modulators of S1P receptors indicated that S1[P.sub.1] receptor negatively and S1[P.sub.3] receptor positively modulate the early phases of regeneration, whereas S1[P.sub.2] receptor appears to be less important. The present results show that SIP signaling participates in the regenerative processes of skeletal muscle. sphingosine 1-phosphate receptors; satellite cells doi:10.1152/ajpcell.00072.2009

Therapeutic vascularization remains a significant challenge in regenerative medicine applications. Whether the goal is to induce vascular growth in ischemic tissue or scale up tissue-engineered constructs, the ability to induce the growth of patent, stable vasculature is a critical obstacle. We engineered polyethylene glycol--based bioartificial hydrogel matrices presenting protease-degradable sites, cell-adhesion motifs, and growth factors to induce the growth of vasculature in vivo. Compared to injection of soluble VEGF,'these matrices delivered sustained in vivo levels of VEGF over 2 weeks as the matrix degraded. When implanted subcutaneously in rats, degradable constructs containing VEGF and arginine-glycine-aspartic acid tripeptide induced a significant number of vessels to grow into the implant at 2 weeks with increasing vessel density at 4 weeks. The mechanism of enhanced vascularization is likely cell-demanded release of VEGF, as the hydrogels may degrade substantially within 2 weeks. In a mouse model of hind-limb ischemia, delivery of these matrices resulted in significantly increased rate of reperfusion. These results support the application of engineered bioartificial matrices to promote vascularization for directed regenerative therapies. biomaterial | hydrogel | ischemia | PEG | VEGF doi/10.1073/pnas.0905447107

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. alginate | insulin-like growth factor-1 | tissue engineering | VEGF | satellite cells doi/10.1073/pnas.0903875106

Ge Y, Wu A, Warnes C, Liu J, Zhang C, Kawasome H, Terada N, Boppart MD, Schoenherr CJ, Chen J. mTOR regulates skeletal muscle regeneration in vivo through kinase-dependent and kinase-independent mechanisms. Am J Physiol Cell Physiol 297: C1434-C1444, 2009. First published September 30, 2009; doi: 10.1152/ajpcell.00248.2009.--Rapamycin- sensitive signaling is required for skeletal muscle differentiation and remodeling. In cultured myoblasts, the mammalian target of rapamycin (roTOR) has been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of roTOR kinase activity. However, the kinase-independent function of roTOR remains controversial, and no in vivo studies have examined those roTOR myogenic mechanisms previously identified in vitro. In this study, we find that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of roTOR function, we have generated and characterized transgenic mice expressing two mutants of roTOR under the control of human skeletal actin (HSA) promoter: rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI). Our results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in wild-type muscles but partially resistant to rapamycin in both RR-and RR/KI-mTOR muscles, consistent with roTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of roTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, roTOR regulates muscle regeneration through kinase-independent and kinase-dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively. rapamycin; rapamycin-resistant; s6kl; mice; myogenesis doi: 10.1152/ajpcell.00248.2009

Macrophages play an essential role in the resolution of tissue damage through removal of necrotic cells, thus paving the way for tissue regeneration. Macrophages also directly support the formation of new tissue to replace the injury, through their acquisition of an anti-inflammatory, or M2, phenotype, characterized by a gene expression program that includes IL-10, the IL-13 receptor, and arginase 1. We report that deletion of two CREB-binding sites from the Cebpb promoter abrogates Cebpb induction upon macrophage activation. This blocks the downstream induction of M2-specific Msr1, II10,II13ra, and Arg-1 genes, whereas the inflammatory (M1) genes II1, I16, Tnfa, and II12 are not affected. Mice carrying the mutated Cebpb promoter ([beta][DELTA]Cre) remove necrotic tissue from injured muscle, but exhibit severe defects in muscle fiber regeneration. Conditional deletion of the Cebpb gene in muscle cells does not affect regeneration, showing that the C/EBP[beta] cascade leading to muscle repair is muscle-extrinsic. While/[beta][DELTA]Cre macrophages efficiently infiltrate injured muscle they fail to upregulate Cebpb, leading to decreased Arg-1 expression. CREB-mediated induction of Cebpb expression is therefore required in infiltrating macrophages for upregulation of M2-specific genes and muscle regeneration, providing a direct genetic link between these two processes. macrophage polarization | muscle regeneration | transcription doi/ 10.1073/pnas.0908641106

Regenerative coordination and remodeling of the intramuscular motoneuron network and neuromuscular connections are critical for restoring skeletal muscle function and physiological properties. The regulatory mechanisms of such coordination remain unclear, although both attractive and repulsive axon guidance molecules may be involved in the signaling pathway. Here we show that expression of a neural secreted chemorepellent semaphorin 3A (Sema3A) is remarkably upregulated in satellite cells of resident myogenic stem cells that are positioned beneath the basal lamina of mature muscle fibers, when treated with hepatocyte growth factor (HGF), established as an essential cue in muscle fiber growth and regeneration. When satellite cells were treated with HGF in primary cultures of cells or muscle fibers, Sema3A message and protein were upregulated as revealed by reverse transcription-polymerase chain reaction and immunochemical studies. Other growth factors had no inductive effect except for a slight effect of epidermal growth factor treatment. Sema3A upregulation was HGF dose dependent with a maximum (about 7- to 8-fold units relative to the control) at 10-25 ng/ml and occurred exclusively at the early-differentiation stage, as characterized by the level of myogenin expression and proliferation (bromodeoxyuridine incorporation) of the cells. Neutralizing antibody to the HGF-specific receptor, c-met, did not abolish the HGF response, indicating that c-met may not mediate the Sema3A expression signaling. Finally, in vivo Sema3A was upregulated in the differentiation phase of satellite cells isolated from muscle regenerating following crush injury. Overall, the data highlight a heretofore unexplored and active role for satellite cells as a key source of Sema3A expression triggered by HGF, hence suggesting that regenerative activity toward motor innervation may importantly reside in satellite cells and could be a crucial contributor during postnatal myogenesis. hepatocyte growth factor; motor neuron; muscle regeneration; semaphorin 3A

During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine., The salamander is a powerful regeneration model because it can reconstitute a fully functional limb after injury. Amputation anywhere between the shoulder and the hand triggers the formation of a [...]

When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production by NO synthase (NOS), matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extraeellular matrix, and presentation of HGF to the c-met receptor as demonstrated by a primary culture and in vivo assays. We now add evidence that calcium-calmodulin is involved in the satellite cell activation cascade in vitro. Conditioned medium from cultures that were treated with a calcium ionophore (A23187, ionomycin) for 2 h activated cultured satellite cells and contained active HGF, similar to the effect of mechanical stretch or NO donor treatments. The response was abolished by addition of calmodulin inhibitors (calmidazolium, W-13, W-12) or a NOS inhibitor [N.sup.G]-nitro-L-arginine methyl ester hydrochloride but not by its less inactive enantiomer [N.sup.G]-nitro-Darginine methyl ester hydrochloride. Satellite cells were also shown to express functional calmodulin protein having a calcium-binding activity at 12 h postplating, which is the time at which the calcium ionophore was added in this study and the stretch treatment was applied in our previous experiments. Therefore, results from these experiments provide an additional insight that calcium-calmodulin mediates HGF release from the matrix and that this step in the activation pathway is upstream from NO synthesis. muscle regeneration; stretch-activation

In the adult skeletal muscle, various kinds of trauma promote proliferation of satellite cells that differentiate into myoblasts forming new myofibers or to repair the damaged one. The aim of present work was to perform a comparative spatial and temporal analysis of connexin (Cx) 37, Cx39, Cx40, Cx43, and Cx45 expression in the adult regenerating skeletal muscle in response to crush injury. Within 24 h from injury, Cx37 expression was upregulated in the endothelial cells of blood vessels, and, 5 days after injury, Cx37-expressing cells were found inside the area of lesion and formed clusters generating new blood vessels with endothelial cells expressing Cx37. Three days after injury, Cx39 mRNA was selectively expressed in myogenin-positive cells, forming rows of closely apposed cell nuclei fusing in myotubes. Cx40 mRNA-labeled cells were observed within 24 h from injury in the endothelium of blood vessels, and, 5 days after lesion, Cx40-labeled cells were found inside the area of lesion-forming rows of myogenin-positive, closely apposed cells coexpressing Cx39. Within 24 h from lesion, both Cx43 and Cx45 mRNAs were upregulated in individual cells, and some of them were positive for M-cadherin. Three days after injury, a large number of both Cx43 and Cx45 mRNA-labeled and myogenin-positive cells were found inside the area of lesion. Taken together, these results show that at least four Cxs, out of five expressed in regenerating skeletal muscle, can be differentially involved in communication of myogenic cells during the process of cell proliferation, aggregation, and fusion to form new myotubes or to repair damaged myofibers. connexin 37; connexin 39; connexin 40; connexin 43; connexin 45; myogenic cells; muscle regeneration

Keywords: dystrophin, sarcoglycan, dysferlin, lamin A/C, nitric oxide synthase Abstract To withstand the rigors of contraction, muscle fibers have specialized protein complexes that buffer against mechanical stress and a multifaceted repair system that is rapidly activated after injury. Genetic studies first identified the mechanosensory signaling network that connects the structural elements of muscle and, more recently, have identified repair elements of muscle. Defects in the genes encoding the components of these systems lead to muscular dystrophy, a family of genetic disorders characterized by progressive muscle wasting. Although the age of onset, affected muscles, and severity vary considerably, all muscular dystrophies are characterized by muscle necrosis that overtakes the regenerative capacity of muscle. The resulting replacement of muscle by fatty and fibrous tissue leaves muscle increasingly weak and nonfunctional. This review discusses the cellular mechanisms that are primarily and secondarily disrupted in muscular dystrophy, focusing on membrane degeneration, muscle regeneration, and the repair of muscle.

Adult skeletal muscle robustly regenerates throughout an organism's life, but as the muscle ages, its ability to repair diminishes and eventually fails (1,2). Previous work suggests that the regenerative potential [...]

We report an age-dependent increase in nonimmunohematopoietic cells ([CD45.sup.neg]) in regenerating muscle characterized by high stem-cell antigen (Sca-1) expression. In aged regenerating muscle, only 14% of these [CD45.sup.neg][Sca-1.sup.pos] cells express MyoD, whereas 82% of [CD45.sup.neg][Sca-1.sup.pos] cells are [MyoD.sup.pos] in young adult muscle. In vitro, [CD45.sup.neg][MyoD.sup.neg][Sca-1.sup.pos] cells overexpress fibrosis-promoting genes, potentially controlled by Wnt2. The cells are proliferative, nonmyogenic, and nonadipogenic, and arise in clonally derived myoblast cultures from aged mice. [MyoD.sup.neg] [Sca-1.sup.pos] nonmyogenic cells also emerge in C2C12 myoblast cultures at late passage. Both in vitro and in vivo studies suggest that [MyoD.sup.neg][Sca-1.sup.pos] cells from aged muscle are more susceptible to apoptosis than myoblasts, which may contribute to depletion of the satellite cell pool. Thus, with age, a subset of myoblasts takes on an altered phenotype, which is marked by high Sca-1 expression. These cells do not participate in muscle regeneration, and instead may contribute to muscle fibrosis in aged muscle. Key Words: Sca-1--Skeletal muscle regeneration--Muscle stem cells--Fibrosis--Aging.

Skeletal muscle expresses high levels of integrin-linked kinase (ILK), predominantly at myotendinous junctions (MTJs) and costameres. ILK binds the cytoplasmic domain of [beta]1 integrin and mediates phosphorylation of protein kinase B (PKB)/Akt, which in turn plays a central role during skeletal muscle regeneration. We show that mice with a skeletal muscle-restricted deletion of ILK develop a mild progressive muscular dystrophy mainly restricted to the MTJs with detachment of basement membranes and accumulation of extracellular matrix. Endurance exercise training enhances the defects at MTJs, leads to disturbed subsarcolemmal myofiber architecture, and abrogates phosphorylation of Ser473 as well as phosphorylation of Thr308 of PKB/Akt. The reduction in PKB/Akt activation is accompanied by an impaired insulin-like growth factor 1 receptor (IGF-1R) activation. Coimmunoprecipitation experiments reveal that the [beta]1 integrin subunit is associated with the IGF-1R in muscle cells. Our data identify the [beta]1 integrin-lLK complex as an important component of IGF-1R/insulin receptor substrate signaling to PKB/Akt during mechanical stress in skeletal muscle.

Calcineurin activation ameliorates the dystrophic pathology of hindlimb muscles in mdx mice and decreases their susceptibility to contraction damage. In mdx mice, the diaphragm is more severely affected than hindlimb muscles and more representative of Duchenne muscular dystrophy. The constitutively active calcineurin A[alpha] transgene (CnA[alpha]) was overexpressed in skeletal muscles of mdx (mdx can[[alpha].sup.*]) mice to test whether muscle morphology and function would be improved. Contractile function of diaphragm strips and extensor digitorum longus and soleus muscles from adult mdx CnA[[alpha].sup.*] and mdx mice was examined in vitro. Hindlimb muscles from mdx CnA[[alpha].sup.*] mice had a prolonged twitch time course and were more resistant to fatigue. Because of a slower phenotype and a decrease in fiber cross-sectional area, normalized force was lower in fast- and slow-twitch muscles of mdx CnA[[alpha].sup.*] than mdx mice. In the diaphragm, despite a slower phenotype and a ~35% reduction in fiber size, normalized force was preserved. This was likely mediated by the reduction in the area of the diaphragm undergoing degeneration (i.e., mononuclear cell and connective and adipose tissue infiltration). The proportion of centrally nucleated fibers was reduced in mdx CnA[[alpha].sup.*] compared with mdx mice, indicative of improved myofiber viability. In hindlimb muscles of mdx mice, calcineurin activation increased expression of markers of regeneration, particularly developmental myosin heavy chain isoform and myocyte enhancer factor 2A. Thus activation of the calcineurin signal transduction pathway has potential to ameliorate the mdx pathophysiology, especially in the diaphragm, through its effects on muscle degeneration and regeneration and endurance capacity. muscle regeneration; muscular dystrophy; skeletal muscle; muscle contraction

The present study was designed to test the hypothesis that increasing physical activity by running exercise could favor the recovery of muscle mass after extensive injury and to determine the main molecular mechanisms involved. Left soleus muscles of female Wistar rats were degenerated by notexin injection before animals were assigned to either a sedentary group or an exercised group. Both regenerating and contralateral intact muscles from active and sedentary rats were removed 5, 7, 14, 21, 28 and 42 days after injury (n = 8 rats/group). Increasing contractile activity through running exercise during muscle regeneration ensured the full recovery of muscle mass and muscle cross-sectional area as soon as 21 days after injury, whereas muscle weight remained lower even 42 days postinjury in sedentary rats. Proliferator cell nuclear antigen and MyoD protein expression went on longer in active rats than in sedentary rats. Myogenin protein expression was higher in active animals than in sedentary animals 21 days postinjury. The Akt-mammalian target of rapamycin (roTOR) pathway was activated early during the regeneration process, with further increases of mTOR phosphorylation and its downstream ellectors, eukaryotic initiation factor-4E-binding protein-I and [p70.sup.s6k], in active rats compared with sedentary rats (days 7-14). The exerciseinduced increase in mTOR phosphorylation, independently of Akt, was associated with decreased levels of phosphorylated AMP-activated protein kinase. Taken together, these results provided evidence that increasing contractile activity during muscle regeneration ensured early and full recovery of muscle mass and suggested that these beneficial effects may be due to a longer proliferative step of myogenic cells and activation of mTOR signaling, independently of Akt, during the maturation step of muscle regeneration. muscle regeneration; myogenic regulatory factors; mitogen-activated protein kinases; mammalian target of rapamycin pathway; AMPactivated protein kinase

Little progress has been made toward the use of embryonic stem (ES) cells to study and isolate skeletal muscle progenitors. This is due to the paucity of paraxial mesoderm formation during embryoid body (EB) in vitro differentiation and to the lack of reliable identification and isolation criteria for skeletal muscle precursors. Here we show that expression of the transcription factor Pax3 during embryoid body differentiation enhances both paraxial mesoderm formation and the myogenic potential of the cells within this population. Transplantation of Pax3-induced cells results in teratomas, however, indicating the presence of residual undifferentiated cells. By sorting for the PDGF-[alpha] receptor, a marker of paraxial mesoderm, and for the absence of Flk-1, a marker of lateral plate mesoderm, we derive a cell population from differentiating ES cell cultures that has substantial muscle regeneration potential. Intramuscular and systemic transplantation of these cells into dystrophic mice results in extensive engraftment of adult myofibers with enhanced contractile function without the formation of teratomas. These data demonstrate the therapeutic potential of ES cells in muscular dystrophy., Pluripotent ES cells hold tremendous potential for cell-based therapies (1). Studies over the past decade have suggested that early steps in embryonic myogenesis take place during embryoid body differentiation of [...]

The purpose of this study was to compare squat strength gains and volume components when resting 2 minutes vs. 4 minutes between sets over multiple mesocycles. After the first squat 1 repetition maximum, 15 trained men were matched and randomly assigned to either a 2-minute (n = 7) or a 4-minute (n = 8) rest interval group. Each group performed the same training program, with the only difference being the length of the rest interval between sets. Subjects performed two squat workouts per week; one was labeled as Heavy and the other was labeled as Light. The squat workouts varied in the intensity, number of sets, and repetitions performed per set in a nonlinear periodized manner throughout each mesocycle. Differences in strength gains and volume components (the load utilized per set, the repetitions performed per set, the intensity per set, and the volume performed per workout) were compared between groups. Both groups demonstrated large strength gains; however, these differences were not significant between groups (P = 0.47). During all mesocycles, the 4-minute group demonstrated significantly higher total volumes for the Heavy workouts (P < 0.05). The findings of the present study indicate that large squat strength gains can be achieved with a minimum of 2 minutes' rest between sets, and little additional gains are derived from resting 4 minutes between sets. Therefore, athletes attempting to achieve specific volume goals may need longer rest intervals initially but may later adapt so that shorter rest intervals can be utilized without excessive fatigue, leaving additional time to focus on other conditioning priorities. KEY WORDS recovery, repetitions, fatigue, sets, intensity

Author(s): Akinobu Matsumoto [1]; Alessandra Pasut [1]; Masaki Matsumoto [2]; Riu Yamashita [3]; Jacqueline Fung [1]; Emanuele Monteleone [1, 4]; Alan Saghatelian [5]; Keiichi I. Nakayama [2]; John G. Clohessy [...]

Stimulating the [beta]-adrenoceptor ([beta]-AR) signaling pathway can enhance the functional repair of skeletal muscle after injury, but long-term use of [beta]-AR agonists causes [beta]-AR downregulation, which may limit their therapeutic effectiveness. The aim was to examine [beta]-AR signaling during early regeneration in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles after bupivacaine injury and test the hypothesis that, during regeneration, [beta]-agonist administration does not cause [beta]-AR desensitization. Rats received either the [beta]-AR agonist fenoterol (1.4 mg x [kg.sup.-1] x [day.sup.-1] ip) or saline for 7 days postinjury. Fenoterol reduced [beta]-AR density in regenerating soleus muscles by 42%. Regenerating EDL muscles showed a threefold increase in [beta]-AR density, and, again, these values were 43% lower with fenoterol treatment. An amplified adenylate cyclase (AC) response to isoproterenol was observed in cell membrane fragments from EDL and soleus muscles 7 days postinjury. Fenoterol attenuated this increase in regenerating EDL muscles but not soleus muscles. [beta]-AR signaling mechanisms were assessed using AC stimulants (NaF, forskolin, and [Mn.sup.2+]). Although [beta]-agonist treatment reduces [beta]-AR density in regenerating muscles, these muscles can produce large cAMP responses relative to healthy (uninjured) muscles. Desensitization of [beta]-AR signaling in regenerating muscles is prevented by altered rates of [beta]-AR synthesis and/or degradation, changes in G protein populations and coupling efficiency, and altered AC activity. These mechanisms have important therapeutic implications for modulating [beta]-AR signaling to enhance muscle repair after injury. muscle regeneration; [beta]-adrenoceptor; muscle function; muscle injury; adenylate cyclase; G protein

The hypothesis of this study was the urokinase-type plasminogen activator receptor (uPAR) is required for accumulation of inflammatory cells in injured skeletal muscle and for efficient muscle regeneration. Expression of uPAR was elevated at 1 and 3 days after cardiotoxin-induced muscle injury in wild-type mice before returning to baseline levels. Neutrophil accumulation peaked 1 day postinjury in muscle from both wild-type (WT) and uPAR null mice, while macrophage accumulation peaked between 3 and 5 days postinjury, with no differences between strains. Histological analyses confirmed efficient muscle regeneration in both wild-type and uPAR null mice, with no difference between strains in the formation or growth of regenerating fibers, or recovery of normal morphology. Furthermore, in vitro experiments demonstrated that chemotaxis is not different between WT and uPAR null macrophages. Finally, fusion of cultured satellite cells into multinucleated myotubes was not different between cells isolated from WT and uPAR null mice. These results demonstrate that uPAR is not required for the accumulation of inflammatory cells or the regeneration of skeletal muscle following injury, suggesting uPA can act independently of uPAR to regulate events critical for muscle regeneration. muscle repair; macrophage; chemotaxis

The regulation of vascular endothelial growth factor (VEGF) levels and angiogenic events during skeletal muscle regeneration remains largely unknown. This study examined angiogenesis, VEGF levels, and muscle regeneration after cardiotoxin (CT)-induced injury in mice lacking the CC chemokine receptor 2 (CCR2). Muscle regeneration was significantly decreased in CCR2-/- mice as was the early accumulation of macrophages after injury. In both mouse strains, tissue VEGF was similar at baseline (no injections) and significantly decreased at day 3 post-CT. Tissue VEGF in wild-type (WT) mice was restored within 7 days postinjury but remained significantly reduced in CCR2-/- mice until day 21. Capillary density (capillaries/[mm.sup.2]) within regenerating muscle was maximal in WT mice at day 7 and double that of baseline muscle. In comparison, maximal capillary density in CCR2-/- mice occurred at 21 clays postinjury. Maximal capillary density developed concurrent with the restoration of tissue VEGF in both strains. A highly significant, inverse relationship existed between the size of regenerated muscle fibers and capillaries per square millimeter. Although this relationship was comparable in WT and CCR2-/- animals, there was a significant decrease in the magnitude of this response in the absence of CCR2, reflecting the observation that regenerated muscle fiber size in CCR2-/- mice was only 50% of baseline at 42 days postinjury, whereas WT mice had attained baseline fiber size by day 21. Thus CCR2-dependent events in injured skeletal muscle, including impaired macrophage recruitment, contribute to restoration of tissue VEGF levels and the dynamic processes of capillary formation and muscle regeneration. adipogenesis; angiogenesis; CCL2; muscle regeneration; monocyte/ macrophage

Calcineurin signaling is essential for successful muscle regeneration. Although calcineurin inhibition compromises muscle repair, it is not known whether calcineurin activation can enhance muscle repair after injury. Tibialis anterior (TA) muscles from adult wild-type (WT) and transgenic mice overexpressing the constitutively active calcineurin-A[alpha] transgene under the control of the mitochondrial creatine kinase promoter (MCKCnA[alpha]*) were injected with the myotoxic snake venom Notexin to destroy all muscle fibers. The TA muscle of the contralateral limb served as the uninjured control. Muscle structure was assessed at 5 and 9 days postinjury, and muscle function was tested in situ at 9 days postinjury. Calcineurin stimulation enhanced muscle regeneration and altered levels of myoregulatory factors (MRFs). Recovery of myofiber size and force-producing capacity was hastened in injured muscles of MCK-CnA[alpha]* mice compared with control. Myogenin levels were greater 5 days postinjury and myocyte enhancer factor 2a (MEF2a) expression was greater 9 days postinjury in muscles of MCK-CnA[alpha]* mice compared with WT mice. Higher MEF2a expression in regenerating muscles of MCK-CnA[alpha]* mice 9 days postinjury may be related to an increase of slow fiber genes. Calcineurin activation in uninjured and injured TA muscles slowed muscle contractile properties, reduced fatigability, and enhanced force recovery after 4 min of intermittent maximal stimulation. Therefore, calcineurin activation can confer structural and functional benefits to regenerating skeletal muscles, which may be mediated in part by differential expression of MRFs. myotoxic injury; skeletal muscle; muscle function

Muscle atrophy contributes to morbidity and mortality in aging and chronic disease, emphasizing the need to gain understanding of the mechanisms involved in muscle atrophy and (re)growth. We hypothesized that the magnitude of muscle regrowth during recovery from atrophy determines whether myonuclear accretion and myogenic differentiation are required and that insulin-like growth factor (IGF)-I/Akt/glycogen synthase kinase (GSK)-3[3 signaling differs between regrowth responses. To address this hypothesis we subjected mice to hindlimb suspension (HS) to induce atrophy of soleus (-40%) and plantaris (-27%) muscle. Reloading-induced muscle regrowth was complete after 14 days and involved an increase in IGF-IEa mRNA expression that coincided with Akt phosphorylation in both muscles. In contrast, phosphorylation and inactivation of GSK-3[beta] were observed during soleus regrowth only. Furthermore, soleus but not plantaris regrowth involved muscle regeneration based on a transient increase in expression of histone 3.2 and myosin heavy chain-perinatal, which are markers of myoblast proliferation and differentiation, and a strong induction of muscle regulatory factor (MRF) expression. Experiments in cultured muscle cells showed that IGF-I-induced MRF expression is facilitated by inactivation of GSK-3[beta] and selectively occurs in the myoblast population. This study suggests that induction of IGF-I expression and Akt phosphorylation during recovery from muscle atrophy is independent of the magnitude of muscle regrowth. Moreover, our data demonstrate for the first time that the regenerative response characterized by myoblast proliferation, differentiation, and increased MRF expression in recovering muscle is associated with the magnitude of regrowth and may be regulated by inactivation of GSK-3[beta]. glycogen synthase kinase-3[beta]; Akt; muscle growth; muscle atrophy doi:10.1152/ajpcell.00504.2006.

Although p38 MAPK activation is essential for myogenesis, the upstream signaling mechanism that activates p38 during myogenesis remains undefined. We recently reported that p38 activation, myogenesis, and regeneration in cardiotoxin-injured soleus muscle are impaired in TNF-[alpha] receptor double-knockout (p[55.sup.-/-]p[75.sup.-/-]) mice. To fully evaluate the role of TNF-[alpha] in myogenic activation of p38, we tried to determine whether p38 activation in differentiating myoblasts requires autocrine TNF-[alpha], and whether forced activation of p38 rescues impaired myogenesis and regeneration in the p[55.sup.-/-]p[75.sup.-/-] soleus. We observed an increase of TNF-[alpha] release from C2C12 or mouse primary myoblasts placed in low-serum differentiation medium. A TNF-[alpha]-neutralizing antibody added to differentiation medium blocked p38 activation and suppressed differentiation markers myocyte enhancer factor (MEF)-2C, myogenin, p21, and myosin heavy chain in C2C12 myoblasts. Conversely, recombinant TNF-[alpha] added to differentiation medium stimulated myogenesis at 0.05 ng/ml while inhibited it at 0.5 and 5 ng/ml. In addition, differentiation medium-induced p38 activation and myogenesis were compromised in primary myoblasts prepared from p[55.sup.-/-]p[75.sup.-/-] mice. Increased TNF-[alpha] release was also seen in cardiotoxin-injured soleus over the course of regeneration. Forced activation of p38 via the constitutive activator of p38, MKK6bE, rescued impaired myogenesis and regeneration in the cardiotoxin-injured p[55.sup.-/-]p[75.sup.-/-] soleus. These results indicate that TNF-[alpha] regulates myogenesis and muscle regeneration as a key activator of p38. myocyte enhancer factor-2C; myogenin; p21; myosin heavy chain; Akt; tumor necrosis factor-[alpha]; mitogen-activated protein kinase doi: 10.1152/ajpcell.00486.2006.