Effects And Mechanisms Of Rapamycin On Myoblast Therapy During Skeletal Muscle Injury

Myoblasts are myofibril precursor cells in cytoplasm, which have the ability of self-renewal and promote muscle fiber regeneration. Myoblast therapy was used to treat Dunchenne’s Muscular Dystrophy and myocardial infarction in preclinical study, but the treatment effect is not ideal. It has been reported thatmyoblasts die rapidly due to immune response in host during clinical application after transplantation. Rapamycin (RAPA), as an immunosuppressive agent, has been used successfully in myoblast allotransplantation. However, the mechanisms of rapamycin on myoblast transplantation especially in poor nutritional microenvironment remain elusive. Firstly, we mimicked nutrition shortage microenvironment by serum starvation and evaluated the cell viability of rapamycin on myoblast C2C12cell by MTT assay. The aim was to realize myoblast myogenesis with starvation pre-treatment by rapamycin. We also further investigate the mechanism of RAPA on skeletal myogenesis by molecular and cell biology method under starvation conditions. Consifering these facts, we built soleus muscle injury model by Cardiotoxin and further investigated myoblast therapy by rapamycin during muscle regeneration.1. MTT was used to detect seven different concentrations (200ng/ml,150ng/ml,100ng/ml,50ng/ml,20ng/ml,10ng/ml和5ng/ml) of RAPA on myoblast myogenesis under normal or serum-starved conditon. Notably, except for200ng/ml of rapamycin, cell viability at differentiate state showed no significant difference compared with the control. Myoblast therapy was cell proliferation in vitro and back injected into injury area. Considering this fact, we therefore selected150ng/ml of rapamycin as an ideal maximum concentration of immunosuppressive agent to further investigate the mechanisms of myoblast myogenesis.2. We observed that the functions and mechanisms of myoblast myogenesis with starvation pretreatment by rapamycin. The results showed that:(1) Analyzed by flow cytometry with Annexin V-FITC/PI, RAPA can protect myoblast cells avoiding cell apoptosis;(2) By Q-PCR, we found that myogenic regulatory factors and myostatin antagonist follistatin showed significantly difference during myoblast myogenesis:a. For proliferate myoblast cells with or without starvation pre-treatment, The expression levels of MyoDl and follistation gene increased higher than control group, wherein MyoDl expressed extremely significant difference compared with control group (P<0.01). They both induced myogenic cell fate during skeletal myogenesis. b. For differentiate myoblast with or without starvation pre-treatment, cells showed sinificantly differences. To normal cells, rapamycin significantly repressed the expression of myogenin and MRF4gene. However, to serum-starved cells, myogenin and MRF4gene expresssion showed a relatively high level during cell differentiation. Expecially at72h of rapamycin, the expression level of MRF4gene, which plays a key role in myoblast terminal differentiation, showed significantly higher than control group (P<0.01). Meanwhile, follistation also expressed a significantly high level compared with the control group (P <0.01). The results were advantageous to induce skeletal myogenesis and reduce skeletal muscle fibrosis.(3) By semi-quantitave analysis, rapamycin showed no significant difference between MAPK/ERK and PI3K/Akt pathway in serum-starved or normal myoblast cells.(4) Analysis by Western-blot, we observed that protein MyoDl expressed significant high level in serum-starved cells than control group with rapamycin for48h (P <0.01). The finding showed same with the previous result by Q-PCR; However, protein myogenin showed a decreasing tendency while it was no significantly different than control group. Analyzed by signaling pathways during myoblast myogenesis, we found that the protein levels of ERK and Akt showed significantly different than control group (P <0.01). However, the protein ratio of pERK/ERK and pAkt/Akt showed no significant difference. These findings were consistence with the results of semi-quantiative analysis. Therefore, the concentration of rapamycin was a potential immunosuppressive agent for myoblast allotransplantation.3. In vitro wound-healing assay, we observed that150ng/ml of rapamycin was not applicable for cell migration. The finding showed same with the previous result of cell viability by MTT assay. It might due to rapamycin inhibit the mTOR pathway. Thus, RAPA could affect to some extent the results of myoblast allotransplantation in clinical trials.4. By cell fusion assay in vitro, we found that myotube number and diameter decreased significantly than control group by rapamycin in serum-starved cells (P<0.01). Moreover, the Fusion index, which was an important indicator for myoblast fusion, also decreased significantly in serum-starved cells (P<0.01). The findings indicated that rapamycin showed negative effect on cell fusion under nutrition shortage condition.5. Considering different attachment activity during cell isolation, muscle tissue was digested with dual-enzyme method. The cell monolayer was then seperated by percoll gradient centrifugation and cell preplate technology from neonatal mice. We further detected primary myoblast cell by Immunohistochemistry method according to the characteristic of protein desmin during myoblast cells. The results showed it was reliable and reasonable.6. We built muscle injury mode by Cardiotoxin. Considering previous findings in vitro, we then investigated myoblast allotransplantation in injury muscle. The results showed that rapamycin was applicable for myoblast therapy. It was consistence with the previous findings that rapamycin can protect cell viability and avoiding cell apoptosis during myoblast myogenesis in vitro.In summary, during nutrition shortage microenvironment, rapamycin can preserve cell viability and avoid cell apoptosis effectively during skeletal myogenesis. The findings explored the mechanism that rapamycin could potentially induce directional differentiation of myoblasts. However, the further findings indicated that RAPA was not applicable for cell migration and fusion. The results showed that rapamycin, as a low toxicity and potent immunosuppressive agent, should be used scientifically and rationally in future clinical trials of myoblast allotransplantation therapy.