Isolation,Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.     

Evaluation of Skeletal and Cardiac Muscle Function after Chronic Administration of Thymosin β-4 in the Dystrophin Deficient Mouse

Thymosin beta-4 (Tβ4) is a ubiquitous protein with many properties relating to cell proliferation and differentiation that promotes wound healing and modulates inflammatory mediators. We studied the effects of chronic administration of Tβ4 on the skeletal and cardiac muscle of dystrophin deficient mdx mice, the mouse model of Duchenne muscular dystrophy. Female wild type (C57BL10/ScSnJ) and mdx mice, 8–10 weeks old, were treated with 150 µg of Tβ4 twice a week for 6 months. To promote muscle pathology, mice were exercised for 30 minutes twice a week. Skeletal and cardiac muscle function were assessed via grip strength and high frequency echocardiography. Localization of Tβ4 and amount of fibrosis were quantified using immunohistochemistry and Gomori''s tri-chrome staining, respectively. Mdx mice treated with Tβ4 showed a significant increase in skeletal muscle regenerating fibers compared to untreated mdx mice. Tβ4 stained exclusively in the regenerating fibers of mdx mice. Although untreated mdx mice had significantly decreased skeletal muscle strength compared to untreated wild type, there were no significant improvements in mdx mice after treatment. Systolic cardiac function, measured as percent shortening fraction, was decreased in untreated mdx mice compared to untreated wild type and there was no significant difference after treatment in mdx mice. Skeletal and cardiac muscle fibrosis were also significantly increased in untreated mdx mice compared to wild type, but there was no significant improvement in treated mdx mice. In exercised dystrophin deficient mice, chronic administration of Tβ4 increased the number of regenerating fibers in skeletal muscle and could have a potential role in treatment of skeletal muscle disease in Duchenne muscular dystrophy.     

Oxygen delivery to skeletal muscle fibers: effects of microvascular unit structure and control mechanisms

The number of perfused capillaries in skeletal muscle varies with muscle activation. With increasing activation, muscle fibers are recruited as motor units consisting of widely dispersed fibers, whereas capillaries are recruited as groups called microvascular units (MVUs) that supply several adjacent fibers. In this study, a theoretical model was used to examine the consequences of this spatial mismatch between the functional units of muscle activation and capillary perfusion. Diffusive oxygen transport was simulated in cross sections of skeletal muscle, including several MVUs and fibers from several motor units. Four alternative hypothetical mechanisms controlling capillary perfusion were considered. First, all capillaries adjacent to active fibers are perfused. Second, all MVUs containing capillaries adjacent to active fibers are perfused. Third, each MVU is perfused whenever oxygen levels at its feed arteriole fall below a threshold value. Fourth, each MVU is perfused whenever the average oxygen level at its capillaries falls below a threshold value. For each mechanism, the dependence of the fraction of perfused capillaries on the level of muscle activation was predicted. Comparison of the results led to the following conclusions. Control of perfusion by MVUs increases the fraction of perfused capillaries relative to control by individual capillaries. Control by arteriolar oxygen sensing leads to poor control of tissue oxygenation at high levels of muscle activation. Control of MVU perfusion by capillary oxygen sensing permits adequate tissue oxygenation over the full range of activation without resulting in perfusion of all MVUs containing capillaries adjacent to active fibers.     

Isoform switching in myofibrillar and excitation-contraction coupling proteins contributes to diminished contractile function in regenerating rat soleus muscle.

Postnatal development of skeletal muscle occurs through the progressive transformation of diverse biochemical, metabolic, morphological, and functional characteristics from the embryonic to the adult phenotype. Since muscle regeneration recapitulates postnatal development of muscle fiber, it offers an appropriate experimental model to investigate the existing relationships between diverse muscle functions and the expression of key protein isoforms, particularly at the single-fiber level. This study was carried out in regenerating soleus muscle 14 days after injury. At this intermediate stage, the regenerating muscle exhibited a recovery of mass greater than its force generation capacity. The lower specific tension of regenerating muscle suggested intrinsic defective excitation-contraction coupling and/or contractility processes. The presence of developmental isoforms of both the voltage-gated Ca(2+) channel (alpha(1)C) and of ryanodine receptor 3, paralleled by an abnormal caffeine contracture development, confirms the immature excitation-contraction coupling of the regenerating muscle. The defective Ca(2+) handling could also be confirmed by the lower sarcoplasmic reticulum caffeine sensitivity of regenerating single fibers. Also, regenerating single fibers revealed a lower maximal specific tension, which was associated with the residual presence of embryonic myosin heavy chains. Moreover, the fibers showed a reduced Ca(2+) sensitivity of myofibrillar proteins, particularly those simultaneously expressing the slow and fast isoforms of troponin C. The present results indicate that the expression of developmental proteins determines the incomplete functional recovery of regenerating soleus.     

Embryonic-like myosin heavy chains in regenerating chicken muscle

An antibody to chicken ventricular myosin was found to cross-react by enzyme immunoassay with myosin heavy chains from embryonic chicken pectorials, but not with adult skeletal myosins. This antibody, which was previously shown to label cultured muscle cells from embryonic pectoralis (Cantini et al., J cell biol 85 (1981) 903), was used to investigate by indirect immunofluorescence the reactivity of chicken skeletal muscle cells differentiating in vivo during embryonic development and muscle regeneration. Muscle fibers in 11-day old chick embryonic pectoralis and anterior latissimus dorsi muscles showed a differential reactivity with this antibody. Labelled fibers progressively decreasgd in number during subsequent stages and disappeared completely around hatching. Only rare small muscle fibers, some of which had the shape and location typical of satellite elements, were labelled in adult chicken muscle. A cold injury was produced with dry ice in the fast pectoralis and the slow anterior latissimys dorsi muscles of young chickens. Two days after injury a number of labelled cells was first seen in the intermediate region between the outer necrotic area and the underlying uninjured muscle. These muscle cells rapidly increased in number and size, thin myotubes were seen after 3 days and by 4–5 days a superficial layer of brightly stained newly formed muscle fibers was observed at the site of the injury. Between one and two weeks after the lesion the intensity of staining of regenerated fibers progressively decreased as their size further increased. These findings indicate that an embryonic type of myosin heavy chain is transitorily expressed during muscle regeneration.     

The in vivo differentiation of murine neuroblastoma

C1300 neuroblastoma was implanted with regenerating skeletal muscle to study the role of tissue interactions during tumor cell differentiation. Combined tumor-muscle implants, placed subcutaneously or within diffusion chambers were compared with control tumors implanted without muscle. Neuroblastoma implanted with injured muscle undergoes a partial neuronal differentiation. The tumor cells lose their normal round cell configuration and develop numerous cytoplasmic processes. Accompanying these outward changes are an increased content of microtubules in the neuritic processes; the appearance of glial-like processes containing abundant microfilaments; and the occurrence of growth vesicles identical to those of the growth cones of normal neurites. Although the implants usually contain large numbers of regenerated myofibers, tumor cell differentiation is not dependent upon the presence of these newly formed fibers. Tumor differentiation occurs equally well on the surfaces of degenerating muscle fragments, fibrin deposits and on the membrane surfaces of the diffusion chambers. These observations suggest that non-specific cell surface phenomena, rather than neuromuscular interactions were primarily responsible for the tumor cell differentiation in vivo.     

Endogenous interferon-gamma is required for efficient skeletal muscle regeneration

The inflammatory response is thought to play important roles in tissue healing. The hypothesis of this study was that the inflammatory cytokine interferon (IFN)-gamma is produced endogenously following skeletal muscle injury and promotes efficient healing. We show that IFN-gamma is expressed at both mRNA and protein levels in skeletal muscle following injury, and that the time course of IFN-gamma expression correlated with the accumulation of macrophages, T-cells, and natural killer cells, as well as myoblasts, in damaged muscle. Cells of each type were isolated from injured muscle, and IFN-gamma expression was detected in each cell type. We also demonstrate that administration of an IFN-gamma receptor blocking antibody to wild-type mice impaired induction of interferon response factor-1, reduced cell proliferation, and decreased formation of regenerating fibers. IFN-gamma null mice showed similarly impaired muscle healing associated with impaired macrophage function and development of fibrosis. In vitro studies demonstrated that IFN-gamma and its receptor are expressed in the C2C12 muscle cell line, and that the IFN-gamma receptor blocking antibody reduced proliferation and fusion of these muscle cells. In summary, our results indicate that IFN-gamma promotes muscle healing, in part, by stimulating formation of new muscle fibers.     

Disruption of DAG1 in differentiated skeletal muscle reveals a role for dystroglycan in muscle regeneration

Striated muscle-specific disruption of the dystroglycan (DAG1) gene results in loss of the dystrophin-glycoprotein complex in differentiated muscle and a remarkably mild muscular dystrophy with hypertrophy and without tissue fibrosis. We find that satellite cells, expressing dystroglycan, support continued efficient regeneration of skeletal muscle along with transient expression of dystroglycan in regenerating muscle fibers. We demonstrate a similar phenomenon of reexpression of functional dystroglycan in regenerating muscle fibers in a mild form of human muscular dystrophy caused by disruption of posttranslational dystroglycan processing. Thus, maintenance of regenerative capacity by satellite cells expressing dystroglycan is likely responsible for mild disease progression in mice and possibly humans. Therefore, inadequate repair of skeletal muscle by satellite cells represents an important mechanism affecting the pathogenesis of muscular dystrophy.     

The urokinase-type plasminogen activator receptor is not required for skeletal muscle inflammation or regeneration

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.     

MicroRNA-206 is highly expressed in newly formed muscle fibers: implications regarding potential for muscle regeneration and maturation in muscular dystrophy

miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMD(J) dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMD(J) TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.     

Isoforms of troponin during regeneration of chicken skeletal muscle fibers after cold injury

Summary The regeneration of skeletal muscle fibers of the adult chicken was examined after a focal injury brought about with a liquid-nitrogen cooled brass rod. Immunofluorescence microscopy with antibodies specific for troponin (TN) components (T, I, and C) from adult chicken breast and ventricular muscles showed the presence of different fiber types in both the anterior and posterior latissimus dorsi muscles. New fibers produced in the regions adjacent to the site of injury in both muscles exhibited the same immunoreactivities as those previously seen in embryonic skeletal muscles. As differentiation proceeded, regenerating cells lost their embryonic antigenicities and recovered their characteristic adult reactivities. These results indicate that, during regeneration from cold injury, skeletal muscles apparently pass again through an embryonic stage during which they synthesize embryonic-like TN isoforms.     

A myosin isoform repressed in hypertrophied ALD muscle of the chicken reappears during regeneration following cold injury

A library of monoclonal antibodies specific for myosin heavy chain (HC) was used to study myosin expression in regenerating fibers. The response to cold injury of slow skeletal ALD muscle previously induced to eliminate SM1 myosin by weight overload was compared to that of its contralateral control. Native gel electrophoresis combined with immunoblotting demonstrated that slow SM1 myosin HC eliminated from hypertrophic muscle reappeared both at the site of active regeneration and unexpectedly, also distal to the site of injury. The regeneration response of hypertrophied muscles was similar to that of the controls. In addition to SM1 myosin HC, ventricular-like and embryonic/fast isoforms were also expressed in both muscles during the early stages of regeneration and disappeared as the muscle fibers matured. These observations demonstrate that regenerating slow muscle fibers reexpress myosins' characteristic of developing muscle irrespective of the myosin phenotype prior to injury. The reappearance of repressed myosin HC in the hypertrophied ALD muscle is consistent with the presence of newly differentiated myonuclei.     

Hematopoietic contribution to skeletal muscle regeneration in acid alpha-glucosidase knockout mice.

Recent studies have shown that cells from bone marrow (BM) can give rise to differentiated skeletal muscle fibers. However, the mechanisms and identities of the cell types involved remain unknown. We performed BM transplantation in acid alpha-glucosidase (GAA) knockout mice, a model of glycogen storage disease type II, and our observations suggested that the BM cells contribute to skeletal muscle fiber formation. Furthermore, we showed that most CD45+:Sca1+ cells have a donor character in regenerating muscle of recipient mice. Based on these findings, CD45+:Sca1+ cells were sorted from regenerating muscles. The cell number was increased with granulocyte colony-stimulating factor after cardiotoxin injury, and the cells were transplanted directly into the tibialis anterior (TA) muscles of GAA knockout mice. Sections of the TA muscles stained with anti-laminin-alpha2 antibody showed that the number of CD45+:Sca1+ cells contributing to muscle fiber formation and glycogen levels were decreased in transplanted muscles. Our results indicated that hematopoietic stem cells, such as CD45+:Sca1+ cells, are involved in skeletal muscle regeneration.     

Cardiac troponin T in developing, regenerating and denervated rat skeletal muscle

Fetal rat skeletal muscles express a troponin T (TnT) isoform similar to the TnT isoform expressed in the embryonic heart with respect to electrophoretic mobility and immunoreactivity with cardiac TnT-specific monoclonal antibodies. Immunoblotting analyses reveal that both the embryonic and the adult isoforms of cardiac TnT are transiently expressed during the neonatal stages. In addition, other TnT species, different from both cardiac TnTs and from the TnT isoforms expressed in adult muscles, are present in skeletal muscles during the first two postnatal weeks. By immunocytochemistry, cardiac TnT is detectable at the somitic stage and throughout embryonic and fetal development, and disappears during the first weeks after birth, persisting exclusively in the bag fibers of the muscle spindles. Cardiac TnT is re-expressed in regenerating muscle fibers following a cold injury and in mature muscle fibers after denervation. Developmental regulation of this TnT variant is not coordinated with that of the embryonic myosin heavy chain with respect to timing of disappearance and cellular distribution. No obligatory correlation between the two proteins is likewise found in regenerating and denervated muscles.     

An early post‐traumatic reaction of lymph‐heart striated muscle fibers in adult frog Rana temporaria during the first postoperative week: An electron microscopic and autoradiographic study

According to the current opinion, lymph‐heart striated muscle represents a specialized type of skeletal muscles in frogs. Here, we studied muscle fibers in mechanically damaged lymph hearts during the first postoperative week using electron‐microscopic autoradiography. We present evidence that both, the satellite cells and pre‐existing muscle fibers bordering the site of injury, contribute directly to the lymph‐heart muscle regeneration. Several muscle fibers located in the vicinity of the damaged area displayed features of nuclear and sarcoplasmic activation. We also observed ultrastructural changes indicating activation of a few satellite cells, namely decondensation of chromatin, enlargement of nuclei and nucleoli, appearance of free ribosomes and rough endoplasmic reticulum tubules in the cytoplasm. Electron‐microscopic autoradiography showed that 4 h after single ³H‐thymidine administration on the seventh day after injury not only the activated satellite cells, but also some nuclei of myofibers bordering the injured zone are labeled. We showed that both, the myonuclei of fibers displaying the signs of degenerative/reparative processes in the sarcoplasm and the myonuclei of the fibers enriched with highly organized myofibrils, can re‐enter into the S‐phase. Our results indicate that the nuclei of lymph‐heart myofibers can reactivate DNA synthesis during regenerative myogenesis, unlike the situation in regenerating frog skeletal muscle where myogenic cells do not synthesize DNA at the onset of myofibrillogenesis. J. Morphol. 276:1525–1534, 2015. © 2015 Wiley Periodicals, Inc.     

Voluntary running induces fiber type-specific angiogenesis in mouse skeletal muscle

Adult skeletal muscle undergoes adaptation in response to endurance exercise, including fast-to-slow fiber type transformation and enhanced angiogenesis. The purpose of this study was to determine the temporal and spatial changes in fiber type composition and capillary density in a mouse model of endurance training. Long-term voluntary running (4 wk) in C57BL/6 mice resulted in an approximately twofold increase in capillary density and capillary-to-fiber ratio in plantaris muscle as measured by indirect immunofluorescence with an antibody against the endothelial cell marker CD31 (466 ± 16 capillaries/mm² and 0.95 ± 0.04 capillaries/fiber in sedentary control mice vs. 909 ± 55 capillaries/mm² and 1.70 ± 0.04 capillaries/fiber in trained mice, respectively; P < 0.001). A significant increase in capillary-to-fiber ratio was present at day 7 with increased concentration of vascular endothelial growth factor (VEGF) in the muscle, before a significant increase in percentage of type IIa myofibers, suggesting that exercise-induced angiogenesis occurs first, followed by fiber type transformation. Further analysis with simultaneous staining of endothelial cells and isoforms of myosin heavy chains (MHCs) showed that the increase in capillary contact manifested transiently in type IIb + IId/x fibers at the time (day 7) of significant increase in total capillary density. These findings suggest that endurance training induces angiogenesis in a subpopulation of type IIb + IId/x fibers before switching to type IIa fibers. adaptation; capillary density; endothelial cells; fiber type transformation; vascular endothelial growth factor     

Emerin increase in regenerating muscle fibers

The fate of emerin during skeletal muscle regeneration was investigated in an animal model by means of crush injury. Immunofluorescence, immunoblotting and mRNA analysis demonstrated that emerin level is increased in regenerating rat muscle fibers with respect to normal mature myofibers. This finding suggests an involvement of emerin during the muscle fiber regeneration process, in analogy with its reported involvement in muscle cell differentiation in vitro. The impairment of skeletal muscle physiological regeneration or reorganization could be a possible pathogenetic mechanism for Emery Dreifuss muscular dystrophy.