Bex1 protein is upregulated in regenerating muscle and interacts with calmodulin, a Ca2+-binding protein involved in cell cycle regulation. Following cardiotoxin-induced injury the regenerating muscle of Bex1 knock-out mice exhibits prolonged cell proliferation and delayed cell differentiation compared to wild-type mice. To gain insight into this process, we compared the regenerating myogenic morphologies of Bex1 knock-out and wild-type mice at several time points. Bex1-positive cells were identified by double immunofluorescence staining. These studies demonstrated that a population of cells that are Bex1-positive after injury are c-Met/basal lamina-positive and Mac-1-negative indicating that they are derived from at least a subset of myogenic progenitor/satellite cells but not invading immune cells. In addition, in regenerating muscle, Bex1 co-localizes with calmodulin in the cytoplasm of the late myoblast or early myotube stage of myogenesis. These results suggest that Bex1 participates in muscle regeneration through the regulation of satellite cell proliferation and differentiation by its interaction with calmodulin. Current studies of Bex1 may provide a new molecular tool for the identification of activated satellite cell and open the way to new or improved therapeutic regimens against progressive muscular atrophy.
Animals ; Calmodulin ; Cell Cycle ; Cell Differentiation ; Cell Proliferation ; Cytoplasm ; Fluorescent Antibody Technique ; Mice ; Mice, Knockout ; Muscle Development ; Muscle Fibers, Skeletal ; Muscles ; Muscular Atrophy, Spinal ; Myoblasts ; Regeneration ; Satellite Cells, Skeletal Muscle

Bex1 protein is upregulated in regenerating muscle and interacts with calmodulin, a Ca2+-binding protein involved in cell cycle regulation. Following cardiotoxin-induced injury the regenerating muscle of Bex1 knock-out mice exhibits prolonged cell proliferation and delayed cell differentiation compared to wild-type mice. To gain insight into this process, we compared the regenerating myogenic morphologies of Bex1 knock-out and wild-type mice at several time points. Bex1-positive cells were identified by double immunofluorescence staining. These studies demonstrated that a population of cells that are Bex1-positive after injury are c-Met/basal lamina-positive and Mac-1-negative indicating that they are derived from at least a subset of myogenic progenitor/satellite cells but not invading immune cells. In addition, in regenerating muscle, Bex1 co-localizes with calmodulin in the cytoplasm of the late myoblast or early myotube stage of myogenesis. These results suggest that Bex1 participates in muscle regeneration through the regulation of satellite cell proliferation and differentiation by its interaction with calmodulin. Current studies of Bex1 may provide a new molecular tool for the identification of activated satellite cell and open the way to new or improved therapeutic regimens against progressive muscular atrophy.
Animals ; Calmodulin ; Cell Cycle ; Cell Differentiation ; Cell Proliferation ; Cytoplasm ; Fluorescent Antibody Technique ; Mice ; Mice, Knockout ; Muscle Development ; Muscle Fibers, Skeletal ; Muscles ; Muscular Atrophy, Spinal ; Myoblasts ; Regeneration ; Satellite Cells, Skeletal Muscle

BACKGROUND: Muscle is a target of immunological injury in several muscle diseases, such as idiopathic inflammatory myopathy. However, it is also a target for gene therapy. Therefore, it is important to understand the immunological capabilities of muscle cells. To assess as to whether muscle cells are actively involved in the inflamed muscle tissue, a human skeletal muscle cell line was tested for the expression of several cytokines and chemokine at the mRNA level. METHODS: A human skeletal muscle cell line (SKM14) had been developed by a retroviral vector encoding v-myc transfection into a 12-week-old human fetal skeletal muscle tissue characterized by the immunostaining of several musclespecific markers. Human skeletal myoblasts of this cell line were tested for their capacity to express different cytokines (IL-1beta, -6, -10, -12, -15, and TNF-alpha) and chemokine (IL-8) mRNA levels at the basal state and in the presence of TNF-alpha(10 ng/ml). RESULTS: The SKM14 cell line was confirmed to be able to express various cytokines constitutively (IL-6, -8, -12, -15, and TNF-alpha) and in the presence of TNF-alpha(IL-1beta, -6, -8, -10, -12, -15, and TNF-alpha). CONCLUSIONS: Our results suggest that muscle cells may play a role as immunocompetent cells.
Cell Line* ; Cytokines* ; Genetic Therapy ; Humans* ; Muscle Cells ; Muscle, Skeletal* ; Myoblasts ; Myoblasts, Skeletal ; Myositis ; RNA, Messenger* ; Transfection ; Zidovudine

The development of skeletal muscle is a highly regulated, multi-step process in which pluripotent mesodermal cells give rise to myoblasts that subsequently withdraw from the cell cycle and differentiate into myotubes as well as myofibers. The plasticity of myoblasts plays a critical role in maintaining skeletal muscle structure and function by myoblast activation, migration, adhesion, membrane reorganization, nuclear fusion, finally forming myotubes/myofibers. Our studies demonstrate that the local hypoxic microenvironment, a great diversity of regulatory factors such as IL-6 superfamily factors (IL-6, LIF, CNTF) and TGF-beta1 could regulate the myoblast plasticity. The aim of this paper is to review the previous studies focused on the regulation of myoblast plasticity and its mechanism in our laboratory. Knowledge about the microenvironment or factors involved in regulating the myoblast plasticity will help develop the prevention and cure measures of skeletal muscle diseases.
Cell Differentiation ; Cellular Microenvironment ; Humans ; Hypoxia ; Muscle Fibers, Skeletal ; cytology ; Muscle, Skeletal ; cytology ; Myoblasts ; cytology

OBJECTIVE: Caveolae are the microdomain of the plasma membrane that have been implicated in signal transduction and caveolin is a principal component of the caveolae. Caveolin-3, a family of caveolin related protein, is expressed only in muscle tissue. Here we examined the expression of caveolin-3 in the course of myobalst differentiation and within the muscle tissue. METHOD: L6 cell, rat skeletal myoblast, was cultured in the low mitogen medium and caveolin-3 expression was observed both by immunocytochemistry and western blot analysis. Localization of caveolin-3 within the muscle tissue was investigated and compared to that of dystrophin. RESULTS: While caveolin-3 was not expressed in the proliferating myolast, caveolin-3 was expressed in the differentiated myoblast. Caveolin-3 and dystrophin were co-expressed in the membrane of muscle tissue and integrated density of caveolin-3 was elevated in the area of muscle injury. In the Duchenne muscular dystrophy, caveolin-3 was expressed in the membrane of muscle tissue, but dystrophin was not. CONCLUSION: Caveolin-3 was induced during the myobalst differentiation and its expression was increased during the muscle regeneration. Caveolin-3 was physically associated with dystrophin as a complex, but not absolutely required for the biogenesis of dystrophin complex.
Animals ; Organelle Biogenesis ; Blotting, Western ; Caveolae ; Caveolin 3* ; Cell Membrane ; Dystrophin ; Humans ; Immunohistochemistry ; Membranes ; Muscle Cells* ; Muscle, Skeletal ; Muscular Dystrophy, Duchenne ; Myoblasts ; Myoblasts, Skeletal ; Rats ; Regeneration ; Signal Transduction

Duchenne muscular dystrophy (DMD) is a fatal, genetic neuromuscular disorders that manifests as progressive muscle wasting. Although there has been enormous progress in the studies of the molecular mechanism of muscular dystrophy, there is still no cure. Cell-based therapy is a promiseful option. This review will focus on the present status of cell-based therapy. Myoblast transfer therapy is hindered by minimal distribution of cells after injection, immune rejection, and poor cell survival. The drawback of bone marrow-derived stem cell transplantation is the low efficiency of transdifferentiation. Compared with them, the injection of postnatal muscle-derived stem cells (MDSC) results in a superior regeneration of dystrophin-expressing myofibers.
Animals ; Bone Marrow Cells ; cytology ; Humans ; Muscle, Skeletal ; cytology ; Muscular Dystrophy, Duchenne ; therapy ; Myoblasts, Skeletal ; transplantation ; Stem Cell Transplantation ; methods

OBJECTIVES: To isolate and culture of human muscle cells by manipulating culture conditions. METHODS: Muscle samples were obtained during total hip replacement or bedside muscle biopsy. We isolated myoblasts from freshly obtained human muscle tissue by trypsin and collagenase digesion. RESULTS: Selective isolation of myoblasts was identified through histologic examination by light-microscope. We identified monoc]onal antibodies(Leu-19) as molecular markers in human skeletal muscle were expressed. CONCLUSIONS: This study demonstrated that it had been possible to culture morphologically and immunelogically identifiable myoblasts isolated from human skeletal muscle tissue.
Arthroplasty, Replacement, Hip ; Biopsy ; Collagenases ; Humans* ; Muscle Cells* ; Muscle, Skeletal ; Myoblasts ; Trypsin

OBJECTIVETo test the effects of autologous skeletal myoblast transplantation on the hemodynamics in dogs with coronary microembolization-induced chronic heart failure (CHF).

METHODSCHF models were successfully induced in 19 dogs and divided into ASMT group (n=9) and control group (n=10). The myoblasts were injected into the embolized region in the 9 dogs of the ASMT group, and saline was injected in the control dogs, and the hemodynamics of the dogs were evaluated 5 weeks after the injections.

RESULTCompared with saline injection, ASMT significantly increased dP/dtmax, MAP and LVSP (P<0.05) and decreased LVEDP (P<0.05) 5 weeks after myoblast transplantation. Desmin and Brd-U immunofluorescent staining showed myoblast survival at the injected sites in the dogs.

CONCLUSIONASMT provides mild improvements in the hemodynamics of dogs with CHF.

Animals ; Chronic Disease ; Dogs ; Female ; Heart Failure ; physiopathology ; therapy ; Hemodynamics ; Male ; Myoblasts, Skeletal ; transplantation ; Transplantation, Autologous

OBJECTIVE: The phenomenon of fibroblast overgrowth is one of the major problems encountered during long-term culture such as myoblast culture. The first goal of the study is to determine the effects of proline analogue and cytosine arabinoside to reduce fibroblasts in myoblast culture. The second goal is to investigate whether the chemicals influence the growth and differentiation of myoblast. METHOD: Muscle tissues were obtained from legs of healthy men, and then fibroblasts and myoblasts were isolated and cultured. Those mixed cells were divided into three groups; control group, proline analogue (cis-hydroxyproline) treated group and cytosine arabinoside (araC) treated group. We evaluated the effectiveness of cis-hydroxyproline and araC on selective removal of fibroblasts in culture. We have also determined if cis-hydroxyproline and araC could alter differentiation of myoblast in each group. RESULTS: The treatment with araC was effective to eliminate fibroblasts comparing to the control group (p<0.05) while there was no statistically significant difference between proline analogue and control group (p>0.05). Myoblasts of all three groups were differentiated into myotube. CONCLUSION: Using araC, we could reduce a number of fibroblasts in myoblast culture where contamination and subsequent overgrowth with fibroblasts remained a problem.
Cytarabine* ; Cytosine* ; Fibroblasts* ; Humans ; Leg ; Male ; Muscle Fibers, Skeletal ; Myoblasts* ; Proline*

Severe muscle injury is hard to heal and always results in a poor prognosis. Recent studies found that extracellular vesicle-based therapy has promising prospects for regeneration medicine, however, whether extracellular vesicles have therapeutic effects on severe muscle injury is still unknown. Herein, we extracted apoptotic extracellular vesicles derived from mesenchymal stem cells (MSCs-ApoEVs) to treat cardiotoxin induced tibialis anterior (TA) injury and found that MSCs-ApoEVs promoted muscles regeneration and increased the proportion of multinucleated cells. Besides that, we also found that apoptosis was synchronized during myoblasts fusion and MSCs-ApoEVs promoted the apoptosis ratio as well as the fusion index of myoblasts. Furthermore, we revealed that MSCs-ApoEVs increased the relative level of creatine during myoblasts fusion, which was released via activated Pannexin 1 channel. Moreover, we also found that activated Pannexin 1 channel was highly expressed on the membrane of myoblasts-derived ApoEVs (Myo-ApoEVs) instead of apoptotic myoblasts, and creatine was the pivotal metabolite involved in myoblasts fusion. Collectively, our findings firstly revealed that MSCs-ApoEVs can promote muscle regeneration and elucidated that the new function of ApoEVs as passing inter-cell messages through releasing metabolites from activated Pannexin 1 channel, which will provide new evidence for extracellular vesicles-based therapy as well as improving the understanding of new functions of extracellular vesicles.
Creatine/metabolism* ; Extracellular Vesicles ; Muscle, Skeletal/metabolism* ; Myoblasts/metabolism* ; Regeneration ; Connexins/metabolism*