Exercise Enhances Myogenic Differentiation In Aging Satellite Cells Through Mitochondrial Remodeling

Objective:Satellite cells myogenic differentiation has been shown to play a key role in sacopenia. Oxidative stress has been shown to contribute to age-associated decrease in satellite cells differentiation. It has been shown that moderate-intensity exercise training reduces oxidative stress in aged satellite cells, which may be related to increase in myogenesis ability, but the functional relevance and the molecular mechanisms remain elusive. During myogenic differentiation the short mitochondria of myoblasts change into the extensively elongated network observed in myotubes. In addition, morphofunctional analysis of mitochondria suggests a numerical increase of mitochondrial cristae from the undifferentiated to differentiated condition during myogenic differentiation, which suggesting that mitochondrial remodeling may have a role in myogenesis. In fact, mitochondria are not only able to remodel their structure and fuction in order to meet their energetic needs better, but they also able to drectly participate in the process of differentiatin. In previous works, we have shown that redox signaling involved in exercise induced mitochondrial dynamic remodeling. In this project, we will study the role of ROS in regulation of exercise on myogenic differentiation in aged satellite cells.1) To investigate the relationship among ROS, mitochondrial remodeling (including mitocondrial enengy metaboliasm, biogenesis, crisae remodeling, fusion and fisson), myogenesis performation, and relative signaling molecule during myogenic differentiation in satellite cells were prepaired from aged rats and exercise training rats.2) To observe the temporal relations among ROS, mitochondrial remodeling, myogenesis performation, and relative signaling molecule during C2C12myoblast differentiation.3) To examine the potential mechanisms involed in different dose of H2O2regulation of mTOR signaling and mitochondrial remodeling during myogenic differentiation.Methods:1) C57BL/6mice were randomly divided into four groups:young normal group, young exercise traning group, aged normal group and aged exercise training group. The exercise training animals were exercised on a motor-driven rodent treadmill for12weeks. HE staining was employed to identify the change of muscle atrophy. Purified satellite cells were obtained by two step enzymatic digestion method and primary culture technique. Differentiation was induced by changing the growth medium to differentiation medium composed of2%horse serum, and was observed by inverted microscopy. At24h of differentiation, cytoplasmic ROS and mitochondrial ROS generation were stained with DCF. The mitochndral mambrane potential were stained with JC-1. The mitochondrial respiration parameters were carried out on intact and permeabilized cells using an Oxygraph-2K. The expression of MyHCⅠ,MyHCⅡa,MyHCⅡx mRNA and MyHC II b mRNA were measured with RT-PCR. And the MyHC,OOXⅣ,Mfn1,OPA1,Drp1,MnSOD,PGC-1α, AMPK,p-AMPK (Thr172),mTOR,p-mTOR (Ser2448) and p21protein expression were measured with western-blot.2) C2C12myoblast were indced myogenic differentiation using2%horse serum. The parameters of ROS, mitochondrial remodeling, myogenesis performation, and relative signaling molecule were measured at Oh,3h,6h,9h,12h,24h of differentiation.3) To definite concentration of low and high concentration of H2O2, set up the model of the differentiation of exogenous gradient H2O2on C2C12cells, and detect the expression of MyHC by PCR and detecting cells survival by MTT assay. Separate C2C12myoblasts into five groups: control group, low concentration of H2O2, high concentration of H2O2, low concentration of H2O2+rapamycin, and high concentration of H2O2+leucine. At24h of differentiation, the parameters of ROS, mitochondrial remodeling, myogenesis performation, and relative signaling molecule were measured.Results:1. Effect of exercise on mitochondrial ROS generation and mitochondrial remodeling during aged muscle satellite cells differentiationMuscle satellite cells were isolated from four group rat skeletal muscle with a high degree of myogenic purity. Satellite cells isolated from aged rats exhibited decreased differentiation into myotubes and demonstrated decreased MyHC I, MyHCⅡa, MyHC Ⅱx mRNA and MyHC protein compared with young rats. Mitochondrial respiration, membrane potential, the amount of ATP, and mitochondrial remodeling protein (MnSOD,COXⅣ,Mfn1,L-OPA1,S-OPA1,Drp1,Tfam) expression were lower in satellite cells isolated from aged rats than from young rats. PGC-la and mTOR pathway has been shown to be a key regulator for cell growth and mitochondrial metabolism. Herein, we also found decreased expression of mTOR, p-mTOR and PGC-la in satellite cells isolated from aged rats compared with young rats. In addition, p-AMPK was elevated and p21were decreased in aged rats. Training significantly increased mitochondrial enegy metabolism, mitochondrial remodeling, and myogenesis performation, whereas age-induced ROS production, Drpl and p-AMPK expression were attenuated in the trained rats. 2. Dynamic change of mitochondrial ROS generation and mitochondrial remodeling during C2C12myoblast differentiationAfter induction of differentiation for3h, ST3, RCR, ATP synthesis, membrane poteint, cytoplasmic ROS, mitochondrial ROS generation, MyHC, MnSOD, L-OPA1, Mfn1, PGC-1α, p-mTOR, p-AMPK and p21protein expression were progressively elevated. After induction f differentiation for6h, S-OPA1and mTOR protein expression were progressively elevated. After induction f differentiation for6h, Tfam protein expression was progressively elevated. Drp1protein expression was elevated on6and9h after induction f differentiation, but was decrease on12h, and com back to undifferented level on24h.3. Hydrogen peroxide regulating mitochondrial remolding during the differentiation of C2C12myoblastIn the model of the differentiation of exogenous gradient H2O2on C2C12cells,100μM of H2O2makes MyHC expression significantly increased and600μM of H2O2makes MyHC expression decreased by29%, and cell survival reduced by18%. And then try to ensure cell survival. It is identified that low concentration of H2O2is100μM and high concentration of H2O2is600p.M. We further observed after24hours differentiation of0,100,600μM H2O2on C2C12myoblasts. The results show that compared with control group (0μmol/L H2O2), low level ROS (100μmol/L H2O2) elevated mitochondrial mitocondrial enengy metaboliasm, biogenesis, crisae remodeling, fusion, and myogenesis performation. PGC-1α, Tfam, mTOR expression and activity were also elevated in this model. Rapamycin reversed low level ROS positive effect in mitochondrial remodeling and myogenic differentiation. Compared with control group (0μmol/L H2O2), high level ROS (600μmol/L H2O2) dismished mitochondrial mitocondrial enengy metaboliasm, biogenesis, crisae remodeling, fusion, and myogenesis performation. PGC-la, Tfam, mTOR expression and activity were also decreased in this model. Leucine reversed low level ROS inactive effect in mitochondrial remodeling and myogenic differentiation.Conclution:1. Endurance exercise training promoted mTOR and PGC-la level in aged muscle satellite cells, which in ture induced mitochondial adaptive remodeling, including mitochondrial crisae maturate, mitochondrial fusion, and mitchondrial biogenesis. As the result of mitochondrial adaptive remodeling, mitochondrial bioenergetic metabolism were elevated, which in turn enhanced myogenic differentiation through inactivation of AMPK and upregulation of p21. In addition, ROS generation was attenuated through upregulate Tfam and MnSOD.2. Mitochondrial ROS generation was progressively elevated during C2C12myoblast differentiation. The potential temporal relations of mitochondrial remodeling during myogenic differentiation may be as decribed below. At beginning of differentiation, mitochondrial crisae maturate were first work, and then mitochondrial fusion begin to form mitochondrial reticulum, and then mitochondrial fission begin to move and re-distribute mitochondria in cytoplasm. Mitochondrial fission may be quickly replaced by fusion in order to promoto energy metabolism. Mitochondrial biogenesis may work in later process of differentiation.3. Low level of H2O2can promoted mTOR and PGC-la level in differentiating C2C12myoblast, which in ture induced mitochondial adaptive remodeling, including mitochondrial crisae maturate, mitochondrial fusion, and mitchondrial biogenesis. As the result of mitochondrial adaptive remodeling, mitochondrial bioenergetic metabolism were elevated, which in turn enhanced myogenic differentiation through inactivation of AMPK and upregulation of p21. This phenomenon is similar to biological effect of exercise. High level of H2O2can limited mTOR and PGC-la level in differentiating C2C12myoblast, which in ture impaired mitochondial adaptive remodeling. As the result of mitochondrial abornomal remodeling, mitochondrial bioenergetic metabolism were decreased, which in turn delayed myogenic differentiation through activation of AMPK and down-regulation of p21. This phenomenon is similar to biological effect of aging.