OBJECTIVETo study the effects of skeletal muscle satellite cells implanted into infarcted myocardium on the volume of remnant myocytes.

METHODSThirty-six adult mongrel canines were divided randomly into implantation group and control group. In the implantation group, skeletal muscle satellite cells taken from the gluteus maximus muscles of the dogs were cultured, proliferated and labeled with 4',6-diamidino-2-phenylindone (DAPI) in vitro. In both groups, a model of acute myocardial infarction was established in every dog. In the implantation group, each dog was injected with M199 solution containing autologous skeletal muscle satellite cells. The dogs in the control group received M199 solution without skeletal muscle satellite cells. The dogs of both groups were killed 2, 4 and 8 weeks after implantation (six dogs in a separate group each time). Both infarcted myocardium and normal myocytes distal from the infracted regions isolated were observed under optical and fluorescent microscope. Their volumes were determined using a confocal microscopy image analysis system and analyzed using SAS. A P < 0.05 was considered significant.

RESULTSA portion of the implanted cells differentiated into muscle fiber with striations and were connected with intercalated discs. Cross-sectional area and cell volume were increased in normal myocardium. Hypertrophy of remnant myocytes in the infarcted site after skeletal muscle cell implantation was much more evident than in the control group. Cross-sectional area, cell area and cell volume differed significantly from those of the control group (P < 0.05). Hypertrophy of the cells occurred predominantly in terms of width and thickness, whereas cell length remained unchanged.

CONCLUSIONSkeletal muscle satellite cells implanted into infarct myocardium, could induce the hypertrophy of remnant myocyte cells in the infarcted site and could also aid in the recovery of the contractile force of the infarcted myocardium.

Animals ; Cell Size ; Dogs ; Myocardial Infarction ; pathology ; Myocardium ; pathology ; Myocytes, Cardiac ; cytology ; Random Allocation ; Satellite Cells, Skeletal Muscle ; cytology ; physiology

OBJECTIVETo study the effect of different access routes on autologous satellite cell implantation to stimulate myocardial regeneration.

METHODSSatellite cells were procured from skeletal muscle (gluteus max) of adult mongrel canine, cultured, proliferated and labeled with 4', 6-diamidino-2-phenylindone (DAPI) in vitro. The cells were autologously implanted into the site of acute myocardial infarction by local injection or perfusion through the ligated distal left anterior descending coronary artery. Specimens were harvested 2, 4 and 8 weeks later for histological study.

RESULTSThe labeling efficiency of satellite cells with DAPI was close to 100%. Fluorescent cells were found at the infarcted zone, papillary muscle and local injection site. Some of these cells had progressively differentiated into striated muscle fibers connected to intercalated discs. The infant cells appeared different from the mature myocardium under an electron microscope. Satellite cells implanted by perfusion through the coronary artery were arranged in order of consistency with host myocardial fibers. The satellite cells, implanted by local injection, were found growing in a disordered way.

CONCLUSIONSatellite cells, implanted by coronary artery perfusion, can progressively differentiate into striated muscle fibers, arranging in order and disseminating over the infarcted zone. This approach seems more favorable for the recovery of myocardial contractile function than that of local injection.

Animals ; Cell Differentiation ; physiology ; Dogs ; Myocardial Infarction ; pathology ; therapy ; Myocardium ; cytology ; Regeneration ; Satellite Cells, Skeletal Muscle ; cytology ; transplantation ; Transplantation, Autologous

The dielectric spectroscopy of the skeletal muscle cells is approached by extracellular perfusion, using curve fitting and numerical calculation with nonlinear Cole-Cole equation, and the dielectric parameter of skeletal muscle cells is compared after perfusion, The results show: (1) the permittivy at low frequency (epsilon1) and the conductivity at hight frequency (kappa(h)) reduces in keeping with extracellular perfusate; (2) the effect of extracellular perfusion on the characteristic frequency (f(c1),f(c2)) is small; (3) the extracellular perfusion mostly influences the peaks of deltaepsilon" and tgdelta for the skeletal muscle cells.
Animals ; Bufo bufo ; Electric Conductivity ; Electricity ; Electrophysiology ; Extracellular Fluid ; physiology ; Muscle, Skeletal ; cytology ; physiology ; Spectrum Analysis ; methods

In order to elucidate muscle functional changes by acute reloading, contractile and fatigue properties of the rat soleus muscle were investigated at three weeks of hindlimb suspension and the following 1 hr, 5 hr, 1 d, and 2 weeks of reloading. Compared to age-matched controls, three weeks of unloading caused significant changes in myofibrillar alignments, muscle mass relative to body mass (-43%), normalized tension (-35%), shortening velocity (+143%), and response times. Further significant changes were not observed during early reloading, because the transitional reverse process was gradual rather than abrupt. Although most of the muscle properties returned to the control level after two weeks of reloading, full recovery of the tissue would require more than the two-week period. Delayed recovery due to factors such as myofibrillar arrangement and fatigue resistance was apparent, which should be considered for rehabilitation after a long-term spaceflight or bed-rest.
Animals ; Hindlimb Suspension ; Lactic Acid/metabolism ; Microscopy, Electron ; Muscle Contraction/*physiology ; Muscle Fatigue/*physiology ; Muscle, Skeletal/cytology/*physiology ; Myofibrils/ultrastructure ; Rats ; Rats, Sprague-Dawley ; Research Support, Non-U.S. Gov't ; Weight-Bearing/physiology

Myostatin, a member of the TGF-beta family, negatively regulates skeletal muscle development. Mutation of myostatin activity leads to increases muscle growth and carcass lean yield. The bovine myostatin mutation cDNA was amplified by polymerase chain reaction, and then sub-cloned into the expression vector pET-30a( + ) to form the expression plasmid pET30a (+)-action/ Myostatin. The recombinant plasmid was transformed into E. coli BL21. The overexpression product of pET30a (+)-action/ Myostatin was been showed in vitro. Sheep skeletal muscle cell were cultured with the purified myostatin mutation C-terminal peptide. The results of this study suggest that had a powerful activity to stimulate the hyperplasia and proliferation of sheep muscle cells and shows high biochemical activity.
Animals ; Cattle ; Cell Proliferation ; Cells, Cultured ; Cloning, Molecular ; Genetic Vectors ; genetics ; Muscle Development ; genetics ; physiology ; Muscle, Skeletal ; cytology ; metabolism ; Mutation ; Myostatin ; genetics ; metabolism ; Peptides ; genetics ; metabolism ; Sheep

OBJECTIVETo evaluate the compositional variation of fibrous callus in the fracture site and the joint cavity and joint cartilage after being transplanted in the muscle pouch.

METHODSThirty 2 month old New Zealand white rabbits (weighing 1-1.5 kg) were randomly divided into two groups: a callus transplantation group (Group A, n=15) and a cartilage transplantation group (Group B, n=15). In Group A, closed radius fracture was made and the autologous fibrous callus was transplanted in the right knee joint cavity at 12 days postoperatively. In Group B, the right knee joint cartilage of the animals was transplanted in the autologous back muscle pouches under anesthesia. Then all the animals were killed by overdose anesthetic 3 weeks after transplantation. And the transplanted fibrous callus, the healed bones in the fracture sites and the transplanted joint cartilage were obtained for assessment of compositional variation.

RESULTSPure fibrous composition was found in the callus at the fracture sites in Group A at 12 days postoperatively. And for 11 out of the 15 animals, the fibrous callus was transformed into cartilaginous tissues after 3 weeks of transplantation, but the fibrous callus was absent in the other 4 animals. The fibrous calluses at the original site and the fracture locus were differentiated into bony tissues. Bony tissue transformation was found in the transplanted joint cartilages in the muscle pouch of all the animals in Group B.

CONCLUSIONSThe fracture sites or joint cavity may facilitate callus differentiation in different ways: the former is helpful for osteogenesis while the latter for the development and maintenance of cartilages, and the muscle pouch is inclined to induce the osteogenic phenotype for cartilages.

Animals ; Bony Callus ; cytology ; transplantation ; Cartilage, Articular ; cytology ; transplantation ; Cell Differentiation ; Fracture Healing ; physiology ; Knee Joint ; Male ; Muscle, Skeletal ; Rabbits ; Radius Fractures ; physiopathology

Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.
Animals ; Cells, Cultured ; Gene Expression Regulation ; MEF2 Transcription Factors ; Mice ; Muscle, Skeletal ; metabolism ; Myoblasts ; cytology ; Myogenic Regulatory Factors ; genetics ; physiology ; Myostatin ; genetics ; physiology ; Promoter Regions, Genetic ; Swine

OBJECTIVETo study the effect of exercises of different intensities on rat skeletal muscle cell apoptosis.

METHODSRat models of exhaustive exercise-induced skeletal muscle injury was established using tread mill exercises at different speeds, and the skeletal muscle cell apoptosis was detected using propidium iodide (PI) staining and flow cytometry.

RESULTNo obvious gastrocnemius cell apoptosis was observed in rats with normal exercise (P>0.05), but the cell apoptosis index was statistically significant in moderate and exhaustive exercise groups (P<0.05), reaching the highest level in exhaustive exercise group after exercising. The gastrocnemius cell apoptosis index increased obviously on days 1 and 3 of exercise, and stabilized in moderate and exhaustive exercise groups.

CONCLUSIONExercises can evoke cell apoptosis and accelerate apoptotic cell clearance, and the imbalance between the two events during exercises may contribute to skeletal muscle injury.

Animals ; Apoptosis ; physiology ; Flow Cytometry ; Male ; Muscle, Skeletal ; cytology ; physiopathology ; Physical Conditioning, Animal ; physiology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Time Factors

OBJECTIVETo investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into myocytes and their expression of dystrophin/utrophin after transplantation in mdx mice.

METHODSBrdU-labeled fifth-passage rat MSCs were transplanted in mdx mice with previous total body gamma irradiation (7 Gy). At 4, 8, 12 and 16 weeks after the transplantation, the mice were sacrificed to detect dystrophin/BrdU and utrophin expressions in the gastrocnemius muscle using immunofluorescence assay, RT-PCR and Western blotting. Five normal C57 BL/6 mice and 5 mdx mice served as the positive and negative controls, respectively.

RESULTSFour weeks after MSC transplantation, less than 1% of the muscle fibers of the mdx mice expressed dystrophin, which increased to 15% at 16 weeks. Donor-derived nuclei were detected in both single and clusters of dystrophin-positive fibers. Some BrdU-positive nuclei were centrally located, and some peripherally within myofibers. Utrophin expression decreased over time after transplantation.

CONCLUSIONThe myofibers of mdx mice with MSC transplantation express dystrophin, which is derived partially from the transplanted MSCs. Dystrophin expression from the transplanted MSCs partially inhibits the upregulation of utrophin in mdx mouse muscle, showing a complementary relation between them.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Dystrophin ; genetics ; metabolism ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred mdx ; metabolism ; Muscle Fibers, Skeletal ; cytology ; metabolism ; Muscular Dystrophy, Animal ; metabolism ; therapy ; Rats ; Utrophin ; metabolism

OBJECTIVETo look for a gene delivery route to the treatment of Duchenne muscular dystrophy(DMD).

METHODSThe recombinant adeno-associated virus vector(rAAV) carrying a LacZ reporter gene was constructed. rAAVLacZ was delivered into the skeletal muscle tissue of C57/BL6 mice by intramuscular injection. Then an intraarterial delivery route was taken to reveal whether rAAVLacZ could transduce muscle tissue.

RESULTS(1) The LacZ gene was efficiently transduced and expressed persisting for 5 months after intramuscular injection. (2) The membrane of muscle and smooth muscle of vessel was widely transduced by intra-arterial delivery rAAVLacZ.

CONCLUSIONThese data provide the evidence that rAAVLacZ can efficiently transduce muscle for a long period. Improving intraarterial gene delivery will be promising means for rAAV-mediated gene therapy for generalized skeletal muscle of DMD.

Animals ; Cell Line ; Dependovirus ; genetics ; Embryo, Mammalian ; Gene Expression ; Genes, Reporter ; Genetic Vectors ; Humans ; Injections, Intramuscular ; Kidney ; cytology ; metabolism ; Lac Operon ; physiology ; Mice ; Mice, Inbred C57BL ; Muscle, Skeletal ; cytology ; metabolism ; Recombinant Proteins ; metabolism ; Transfection ; methods