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This review identifies some key concepts of muscle regeneration, viewed from perspectives of classical and modern research. Early insights noted the pattern and sequence of regeneration across species was similar, regardless of the type of injury, and differed from epimorphic limb regeneration. While potential benefits of exercise for tissue repair was debated, regeneration was not presumed to deliver functional restoration, especially after ischemia–reperfusion injury; muscle could develop fibrosis and ectopic bone and fat. Standard protocols and tools were identified as necessary for tracking injury and outcomes. Current concepts vastly extend early insights. Myogenic regeneration occurs within the environment of muscle tissue. Intercellular cross-talk generates an interactive system of cellular networks that with the extracellular matrix and local, regional, and systemic influences, forms the larger gestalt of the satellite cell niche. Regenerative potential and adaptive plasticity are overlain by epigenetically regionalized responsiveness and contributions by myogenic, endothelial, and fibroadipogenic progenitors and inflammatory and metabolic processes. Muscle architecture is a living portrait of functional regulatory hierarchies, while cellular dynamics, physical activity, and muscle–tendon–bone biomechanics arbitrate regeneration. The scope of ongoing research—from molecules and exosomes to morphology and physiology—reveals compelling new concepts in muscle regeneration that will guide future discoveries for use in application to fitness, rehabilitation, and disease prevention and treatment. [ABSTRACT FROM AUTHOR]

Satellite cells (SCs), stem cells in skeletal muscle, are mitotically quiescent in adult mammals until activated for growth or regeneration. In mouse muscle, SCs are activated by nitric oxide (NO), hepatocyte growth factor (HGF) and the mechanically induced NO-HGF signaling cascade. Here, the SC population on fibers from the adult, ectothermic zebrafish and SC responsiveness to activating stimuli were assessed using the model system of isolated fibers cultured at 27 and 21°C. SCs were identified by immunostaining for the HGF receptor, c-met, and activation was determined using bromodeoxyuridine uptake in culture or in vivo. In dose-response studies, SC activation was increased by treatment with the NO-donor drug isosorbide dinitrate (1 mmol l-1) or HGF (10 ng ml-1) to maximum activation at lower concentrations of both than in previous studies of mouse fibers. HGF-induced activation was blocked by anti-c-met antibody, and reduced by culture at 21°C. The effect of cyclical stretch (3 h at 4 cycles per minute) increased activation and was blocked by nitric oxide synthase inhibition and reduced by culture at 21°C. The number of c-met+ SCs per fiber increased rapidly (by 3 h) after stretching. The character of signaling in SC activation on zebrafish fibers, in particular temperature-dependent responses to HGF and stretch, gives new insights into the influence of ectothermy on regulation of muscle growth in teleosts and suggests the use of the single-fiber model system to explore the basis of fiber hyperplasia and the conservation of regulatory pathways between species. [ABSTRACT FROM AUTHOR]

Satellite cells (SC), stem cells in skeletal muscle, are mitotically quiescent in adult mammals until activated for growth or regeneration. In mouse muscle, SC are activated by nitric oxide (NO), hepatocyte growth factor (HGF) and the mechanically induced NO-HGF signaling cascade. Here, the SC population on fibers from the adult, ectothermic zebrafish and SC responsiveness to activating stimuli were assessed using the model system of isolated fibers cultured at 27°C and 21°C. SC were identified by immunostaining for the HGF receptor, c-met, and activation was determined using bromodeoxyuridine uptake in culture or in vivo. In dose-response studies, SC activation was increased by treatment with the NO-donor drug isosorbide dinitrate (1mM) or HGF (10 ng/mL) to maximum activation at lower and higher concentrations, respectively, than in previous studies of mouse fibers. HGF-induced activation was blocked by anti-c-met antibody, and reduced by culture at 21°C. The effect of cyclical stretch (3 hr at 4 cycles per min) increased activation and was blocked by NOS inhibition and reduced by culture at 21°C. The number of c-met+ SC/fiber increased rapidly (by 3 hr) after stretching. The character of signaling in SC activation on zebrafish fibers, in particular temperature-dependent responses to HGF and stretch gives new insights into the influence of ectothermy on regulation of muscle growth in teleosts and suggests use of the single-fiber model system to explore the basis of fiber hyperplasia and the conservation of regulatory pathways between species. [ABSTRACT FROM AUTHOR]

Abstract: Age-related muscle atrophy or sarcopenia results in progressive loss of muscle function and satellite cells in aging muscle are increasingly refractory to activation that could mitigate atrophy. We know that nitric oxide release triggered by mechanical stretch of skeletal muscle, initiates satellite cell activation in vitro in single fiber, single cell and whole-muscle cultures, and in vivo in animals. This study examined muscle cell activation using tritiated-thymidine incorporation into the DNA of muscle cells in cultured muscles from female mice between 6 weeks and 18 months-of-age. Experiments examined age-related changes in activation by mechanical stretch and/or NO treatments (with the substrate of nitric oxide synthase (l-arginine), a nitric oxide donor (isosorbide dinitrate) and/or nitric oxide synthase inhibition). Activation without stretch was highest at 8 months. Stretching muscles by 10% more than doubled activation in muscles at 6 weeks of age and only a 20% stretch similarly activated cells in cultured 6-month-old muscles. Only treatment with ISDN in combination with a 20% stretch activated cell proliferation in muscles from 8-month-old mice. A nitric-oxide donor drug rescued muscle satellite cells in adult, 8-month-old mice from being refractory to mechanical stretch, apparently by overcoming an ineffective release of nitric oxide during stretch. Results suggest that treatment with nitric oxide has the potential to enhance the effectiveness of exercise in preventing the onset of age-related muscle atrophy in adult muscle. [Copyright &y& Elsevier]

Knowledge of the events underlying satellite cell activation and the counterpart maintenance of quiescence is essential for planning therapies that will promote the growth and regeneration of skeletal muscle in healthy, disease and aging. By modeling those events of satellite cell activation in studies of single muscle fibers or muscles in culture, the roles of mechanical stretching and nitric oxide are becoming understood. Recent studies demonstrated that stretch-induced activation is very rapid and exhibits some features of satellite cell heterogeneity. As well, gene expression studies showed that expression of the c-met receptor gene rises rapidly after stretching muscles in culture compared to those without stretch. This change in gene expression during activation, and the maintenance of quiescence in both normal and dystrophic muscles are dependent on NO, as they are blocked by inhibition of nitric oxide synthase (NOS). Mechanical, contractile activity is the defining feature of muscle function. Therefore, ongoing studies of stretch effects in satellite cell activation and quiescence in quiescent fiber and muscle cultures provides appropriate models by which to explore the regulatory steps in muscle in vivo under many conditions related to disease, repair, rehabilitation, growth and the prevention or treatment of atrophy. [ABSTRACT FROM AUTHOR]

Satellite cells, muscle precursor cells in skeletal muscle, are normally quiescent and become activated by disease or injury. A lack of dystrophin and changes in the expression or activity of neuronal nitric oxide synthase (NOS-I) affect the timing of activation in vivo. Nitric oxide synthase inhibition delays muscle repair in normal mice, and worsens muscular dystrophy in the mdx mouse, a genetic homologue of Duchenne muscular dystrophy. However, the potential role of activation and repair events mediated by nitric oxide in determining the outcome of steroid or other treatments for muscular dystrophy is not clear. We tested the hypothesis that the extent of repair in dystrophic muscles of mdx mice is partly dependent on NOS-Iμ expression and activity. Myotube formation in regenerating muscle was promoted by deflazacort treatment of mdx dystrophic mice (P<0.05), and improved by combination with the nitric oxide synthase substrate, l-arginine, especially in the diaphragm. NOS-Iμ mRNA expression and activity were present in satellite cells and very new myotubes of regenerating and dystrophic muscle. Deflazacort treatment resulted in increased NOS-Iμ expression in regenerating muscles in a strong and specific correlation with myf5 expression (r=0.95, P<0.01), a marker for muscle repair. Nitric oxide synthase inhibition prevented the deflazacort-induced rise in NOS-Iμ and myf5 expression in the diaphragm without affecting the diameter of non-regenerating fibres. These in vivo studies suggest that gains in NOS-Iμ expression and nitric oxide synthase activity in satellite cells can increase the extent and speed of repair, even in the absence of dystrophin in muscle fibres. NOS-Iμ may be a useful therapeutic target to augment the effects of steroidal or other treatments of muscular dystrophy. [Copyright &y& Elsevier]

Satellite stem cell activation is the process by which quiescent precursor cells resident on muscle fibers are recruited to cycle and move. Two processes are reported to affect satellite cell activation. In vivo, nitric oxide (NO) produced by NO synthase in fibers (NOS-Iμ) promotes activation. In cell cultures, hepatocyte growth factor (HGF) is the major activating factor isolated from crushed muscle extract (CME). In this study we hypothesized that distinct and possibly related events were mediated by NO and HGF during activation. Intact fibers were cultured in the presence of bromodeoxyuridine (BrdU) to label DNA synthesis over 48 h. Experiments were designed to test the effects of CME, HGF, a NOS substrate l-arginine, and the NOS inhibitor l-NAME on activation, determined as the number of BrdU-positive satellite cells per fiber. Activation was increased significantly by CME, HGF, and l-arginine. l-Arginine increased activation in a dose–response manner. CME-induced activation was reduced significantly by NOS inhibition. Exposure to marcaine (10 min) caused reversible membrane damage without hypercontraction, as shown by characterizing the sarcolemmal integrity. The resulting decrease in satellite cell activation could be overcome by exogenous HGF. Results support the hypothesis that NO is involved in recruiting to cycle those satellite cells resident on fibers. Separate assessments of resident and free muscle cells showed that HGF and NO also participate in mobilizing satellite cells. Since HGF counteracted NOS inhibition and marcaine-induced membrane damage, data suggest that NO may mediate early steps in activation and precede HGF-mediated events. [Copyright &y& Elsevier]

The activity of satellite cells during myogenesis, development, or skeletal muscle regeneration is strongly modelled using cultures of single muscle fibers. However, there are variations in reported features of gene or protein expression as examined with single-fiber cultures. Here, we examined the potential differences in activation of satellite cells on normal mouse muscle fibers produced during a standard isolation protocol, with or without agitation during collagenase digestion. Activation was detected in satellite cells on fibers after 24 and 48 h of culture in basal growth medium using immunodetection of the incorporation of bromodeoxyuridine (BrdU) into DNA and quantification of the number of BrdU-positive cells per fiber. After 24 and 48 h in culture under nonactivating conditions, the number of activated (BrdU+) satellite cells was greater on fibers that had received gentle agitation during collagenase digestion than on those that were subject to digestion without agitation during isolation. The findings are interpreted to mean that at least some of the variation among published reports may derive from the application of various methods of fiber isolation. The information should be useful for maintaining satellite cell quiescence during studies of the regulatory steps that lead to satellite cell activation. [ABSTRACT FROM AUTHOR]

Age-related sarcopenia reduces the size, strength, and function of muscle, and the diameter of muscle fibers. It also disrupts the dystrophin-glycoprotein complex, dislocating nitric oxide synthase 1 (NOS-1) and reducing sarcolemmal integrity. This study of quadriceps muscle in 18-mo-old mice showed that NO-donor treatment with isosorbide dinitrate (I) for 6 wk, in combination with voluntary exercise for 3 wk, increased muscle mass by 25% and stimulated cell proliferation. The resulting fiber hypertrophy was accompanied by a lower ratio of protein:DNA, consistent with myogenic-cell hyperplasia. Treatment enhanced the ratio of NOS-1:β-dystroglycan in correlation with fiber diameter, improved sarcolemmal integrity, and increased vascular density after an increase in vascular endothelial growth factor protein at 3 wk. Results demonstrate that age-related muscle refractoriness to exercise can be overcome with NO-donor treatment. Since activation of muscle stem cells and vascular perfusion are limiting factors in the maintenance, regeneration, and growth of aged muscle, results suggest the feasibility of using NO-donor drugs to combat atrophy and muscle ischemia. Improved function and quality of life from the NO-amplified effects of exercise may be useful in aging and other conditions such as disuse, insulin resistance, or microgravity. [ABSTRACT FROM AUTHOR]

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 earlydifferentiation 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. [ABSTRACT FROM AUTHOR]

Background: Rotator-cuff injury (RCI) is common and painful; even after surgery, joint stability and function may not recover. Relative contributions to atrophy from disuse, fibrosis, denervation, and satellite-cell responsiveness to activating stimuli are not known. Methods and Findings: Potential contributions of denervation and disrupted satellite cell responses to growth signals were examined in supraspinatus (SS) and control (ipsilateral deltoid) muscles biopsied from participants with RCI (N = 27). Biopsies were prepared for explant culture (to study satellite cell activity), immunostained to localize Pax7, BrdU, and Semaphorin 3A in satellite cells, sectioning to study blood vessel density, and western blotting to measure the fetal (γ) subunit of acetylcholine receptor (γ-AchR). Principal component analysis (PCA) for 35 parameters extracted components identified variables that contributed most to variability in the dataset. γ-AchR was higher in SS than control, indicating denervation. Satellite cells in SS had a low baseline level of activity (Pax7+ cells labelled in S-phase) versus control; only satellite cells in SS showed increased proliferative activity after nitric oxide-donor treatment. Interestingly, satellite cell localization of Semaphorin 3A, a neuro-chemorepellent, was greater in SS (consistent with fiber denervation) than control muscle at baseline. PCAs extracted components including fiber atrophy, satellite cell activity, fibrosis, atrogin-1, smoking status, vascular density, γAchR, and the time between symptoms and surgery. Use of deltoid as a control for SS was supported by PCA findings since “muscle” was not extracted as a variable in the first two principal components. SS muscle in RCI is therefore atrophic, denervated, and fibrotic, and has satellite cells that respond to activating stimuli. Conclusions: Since SS satellite cells can be activated in culture, a NO-donor drug combined with stretching could promote muscle growth and improve functional outcome after RCI. PCAs suggest indices including satellite cell responsiveness, atrogin-1, atrophy, and innervation may predict surgical outcome. [ABSTRACT FROM AUTHOR]

A healthy skeletal muscle mass is essential in attenuating the complications of obesity. Importantly, healthy muscle function is maintained through adequate repair following overuse and injury. The purpose of this study was to investigate the impact of diet-induced obesity ( DIO) on skeletal muscle repair and the functionality of the muscle satellite cell ( SC) population. Male C57 BL/6J mice were fed a standard chow or high-fat diet (60% kcal fat; DIO) for 8 weeks. Muscles from DIO mice subjected to cardiotoxin injury displayed attenuated muscle regeneration, as indicated by prolonged necrosis, delayed expression of MyoD and Myogenin, elevated collagen content, and persistent embryonic myosin heavy chain expression. While no significant differences in SC content were observed, SCs from DIO muscles did not activate normally nor did they respond to exogenous hepatocyte growth factor ( HGF) despite similar receptor ( cMet) density. Furthermore, HGF release from crushed muscle was significantly less than that from muscles of chow fed mice. This study demonstrates that deficits in muscle repair are present in DIO, and the impairments in the functionality of the muscle SC population as a result of altered HGF/c-met signaling are contributors to the delayed regeneration. [ABSTRACT FROM AUTHOR]

Abstract: Resident myogenic stem cells, satellite cells, up-regulate a secreted multi-functional modulator, semaphorin 3A (Sema3A), exclusively at the early-differentiation phase in response to muscle-crush injury and treatment with hepatocyte growth factor (HGF) or basic fibroblast growth factor (FGF2). Here, we add evidence that the Sema3A expression and secretion induced by the growth factors is significantly higher in primary cultures from adult rat soleus than from the fast-twitch extensor digitorum longus (EDL) muscle. The higher Sema3A response, revealed by quantitative PCR and Western blotting of cell lysates and conditioned media, may account for the higher myogenin expression of soleus muscle satellite cells early in differentiation since addition of recombinant Sema3A stimulates myogenin expression in cultures. These experiments also showed that mRNA expression of plexin A2, which together with neuropilins, constitutes Sema3A composite-receptors, was higher in satellite cells from soleus than EDL with no difference in plexin A1 and A3 and neuropilin-1 and 2 levels. These comparative studies, therefore, highlight a possible Sema3A-plexin A2-myogenin signaling axis that may ensure promoting early differentiation by soleus muscle satellite cells. [Copyright &y& Elsevier]

Successful regeneration and remodeling of neuromuscular junctions are critical for restoring functional capacities and properties of skeletal muscle after damage, and axon-guidance molecules may be involved in the signaling that regulates such restoration. Recently, we found that early-differentiated satellite cells up-regulate a secreted neural chemorepellent Sema3 A upon in vivo muscle-crush injury. The study also revealed that Sema3 A expression is up-regulated in primary satellite-cell cultures in response to hepatocyte growth factor ( HGF) and basic fibroblast growth factor ( FGF2) and is prevented by transforming growth factor ( TGF)-β2, 3. In order to verify the physiological significance of this regulation in vitro, the present study was designed to estimate the time-course of extracellular HGF, FGF2 and TGF-β3 concentrations after crush-injury of Gastrocnemius muscle in the rat lower hind-limb, using a combination of a non-homogenization/non-spin extraction of extracellular wound fluids and enhanced chemiluminescence- Western blotting analyses. Results clearly demonstrated that active HGF and FGF2 are prevalent in 2-8 days post-crush, whereas active TGF-β3 increases after 12 days, providing a better understanding of the time-coordinated levels of HGF, FGF2 and TGF-β3 that drive regulation of Sema3 A expression during regenerative intramuscular moto-neuritogenesis. [ABSTRACT FROM AUTHOR]

When skeletal muscle is stretched or injured, satellite cells, resident myogenic stem cells positioned beneath the basal lamina of mature muscle fibers, are activated to enter the cell cycle. This signaling pathway is a cascade of events including calcium-calmodulin formation, nitric oxide (NO) radical production by NO synthase, matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the receptor c-met, as demonstrated by assays of primary cultures and in vivo experiments. Here, we add evidence that two ion channels, the mechanosensitive cation channel (MS channel) and the long-lasting-type voltage-gated calcium-ion channel (L-VGC channel), mediate the influx of extracellular calcium ions in response to cyclic stretch in satellite cell cultures. When applied to 1-h stretch cultures with individual inhibitors for MS and L-VGC channels (GsMTx-4 and nifedipine, respectively) or with a less specific inhibitor (gadolinium chloride, Gd), satellite cell activation and upstream HGF release were abolished, as revealed by bromodeoxyuridine-incorporation assays and Western blotting of conditioned media, respectively. The inhibition was dose dependent with a maximum at 0.1 μM (GsMTx-4), 10 μM (nifedipine), or 100 μM (Gd) and canceled by addition of HGF to the culture media; a potent inhibitor for transient-type VGC channels (NNC55-0396, 100 μM) did not show any significant inhibitory effect. The stretch response was also abolished when calcium-chelator EGTA (1.8 mM) was added to the medium, indicating the significance of extracellular free calcium ions in our present activation model. Finally, cation/calcium channel dependencies were further documented by calcium-imaging analyses on stretched cells; results clearly demonstrated that calcium ion influx was abolished by GsMTx-4, nifedipine, and EGTA. Therefore, these results provide an additional insight that calcium ions may flow in through L-VGC channels by possible coupling with adjacent MS channel gating that promotes the local depolarization of cell membranes to initiate the satellite cell activation cascade. [ABSTRACT FROM AUTHOR]

Successful regeneration and remodeling of the intramuscular motoneuron network and neuromuscular connections are critical for restoring skeletal muscle function and physiological properties. The regulatory signals of such coordination remain unclear, although axon-guidance molecules may be involved. Recently, satellite cells, resident myogenic stem cells positioned beneath the basal lamina and at high density at the myoneural junction regions of mature fibers, were shown to upregulate a secreted neural chemorepellent semaphorin 3A (Sema3A) in response to in vivo muscle-crush injury. The initial report on that expression centered on the observation that hepatocyte growth factor (HGF), an essential cue in muscle fiber growth and regeneration, remarkably upregulates Sema3A expression in early differentiated satellite cells in vitro [Tatsumi et al., Am J Physiol Cell Physiol 297: C238-C252, 2009]. Here, we address regulatory effects of basic fibroblast growth factor (FGF2) and transforming growth factor (TGF)-βs on Sema3A expression in satellite cell cultures. When treated with FGF2, Sema3A message and protein were upregulated as revealed by reverse transcription-polymerase chain reaction and immunochemical studies. Sema3A upregulation by FGF2 was dose dependent with a maximum (8- to 1-fold relative to the control) at 2.5 ng/ml (150 pM) and occurred exclusively at the early differentiation stage. The response was highly comparable in dose response and timing to effects of HGF treatment, without any additive or synergistic effect from treatment with a combination of both potent upregulators. In contrast, TGF-β2 and -β3 potently decreased basal Sema3A expression; the maximum effect was at very low concentrations (40 and 8 pM, respectively) and completely cancelled the activities of FGF2 and HGF to upregulate Sema3A. These results therefore encourage the prospect that a time-coordinated increase in HGF, FGF2, and TGF-β ligands and their receptors promotes a programmed strategy for Sema3A expression that guarantees successful intramuscular motor reinnervation by delaying sprouting and reattachment of motoneuron terminals onto damaged muscle fibers early in regeneration pending restoration of muscle fiber contractile integrity. [ABSTRACT FROM AUTHOR]

In Duchenne muscular dystrophy (DMD), palliative glucocorticoid therapy can produce myopathy or calcification. Since increased nitric oxide synthase activity in dystrophic mice promotes regeneration, the outcome of two nitric oxide (NO) donor drugs, MyoNovin (M) and isosorbide dinitrate (I), on the effectiveness of the anti-inflammatory drug prednisone (P) in alleviating progression of dystrophy was tested. Dystrophic mdx mice were treated (18 days) as controls or with an NO donor ± P. Fiber permeability and DNA synthesis were labeled by Evans blue dye (EBD) and bromodeoxyuridine uptake, respectively. P decreased body weight gain, M increased quadriceps mass, and I increased heart mass. P increased fiber permeability (%EBD+ fibers) and calcification in diaphragm. Treatment with NO donors + P (M+P, I+P) reduced %EBD+ fibers and calcification vs. P alone. %EBD+ fibers in M+P diaphragm did not differ from control. NO donor treatment reduced proliferation and the population of c-met+ cells and accelerated fiber regeneration. Concurrent with P, NO donor treatment suppressed two important detrimental effects of P in mice, possibly by accelerating regeneration, rebalancing satellite cell quiescence and activation in dystrophy, and/or increasing perfusion. Results suggest that NO donors could improve current therapy for DMD. [ABSTRACT FROM AUTHOR]