The Regulation Of Mitochondrial Motility And Dynamics By H₂O₂ And Ca²⁺ In Skeletal Muscle

Objective: The dynamic changes of mitochondria morphology and distribution have a profound influence on mitochondrial function and cell energy metabolism. The research into the mechanism of mitochondria movement and distribution is still in its early stages. The understanding of the change of mitochondrial motility and regulation mechanism of mitochondrial motility by H2O2 and Ca2+ in skeletal muscle is currently very limited. The purpose of this study is to investigate the expression of mitochondrial motility-related protein Miro1, mitochondrial fusion protein Mfn2, mitochondrial fission protein Drp1 and changes in the characteristics of H2O2 concentration and mitochondrial function during an acute bout of prolonged exercise and to discuss the relationship of the protein expressions and physiological significance. Through in vitro experiments, further studies of mitochondria characteristic and the regulation mechanism from moderate oxidatie stress caused by exogenous H2O2 had been studied. The roles of H2O2 and Ca2 + in regulating dynamic changes of mitochondria morphology and distribution will be explored from new perspective. New target and potential theoretical basis for the further study of sports exercise fatigue, metabolic disease, neurodegenerative diseases and aging will be provided.Methods: C57 BL/6 mice underwent a moderate intensity treadmill run with 0°and at the speed of 13m/min, and they were randomly divided into 5 groups: resting group(R),exercise groups running for 30 minutes (E30), 60 minutes (E60), 90 minutes (E90) and 120 minutes (E120), respectively. By using Oxytherm liquid oxygen electrode and Chemiluminescence Apparatus, respiratory control ratio and ATP synthesis activity in isolated mitochondria of skeletal muscle were measured respectively. H2O2 concentration of skeletal muscle was also measured by using a UV-VIS spectrophotometer. The western blotting and Real-time Fluorescence Quantitative PCR methods were utilized to determine the contents of Miro1, Mfn2, Drp1 proteins and genes respectively. The immunofluorescence method was selected to observe the cellular distribution of Miro1 in skeletal muscle tissue. In order to investigate the gene and protein expressions of Miro1, Mfn2, Drp1 during an acute bout of prolonged exercise and to analysis the relationship of the gene and protein expressions, the abovementioned methods have been used. C2C12 myoblast models of mild oxidative stress induced by exogenous H2O2 were established. By using Oxytherm liquid oxygen electrode and fluorospectrophotometer, oxygen consumption rate and ATP content of C2C12 myoblast were detected respectively. The western blotting method was used to determine the contents of Miro1, mfn2, drp1 proteins in C2C12 myoblast. The abovementioned methods have been used to investigate whether H2O2 regulated mitochondrial dynamics proteins acted as signal molecules and its potential physiological significance. To explore the roles of H2O2 and Ca2+ in regulating mitochondrial motility in myoblast, dynamic changes of cellular free calcium ion concentration and H2O2 were detected by laser confocal microscopy. Results:Changes of mitochondrial motility and dynamics during an acute bout of prolonged exercise in skeletal muscleThis is the first discovery: Miro1 mRNA and protein contents were significantly increased during 120 minutes of exercise, as compared with the resting group. Mfn2 mRNA and protein contents were significant decreased in groups E60～E120; Drpl mRNA expression increased in groups E60～E120. As well as expression changes of Miro1 gene and protein precede Mfn2 and Drp1. H2O2 contents of skeletal muscle were progressively increased during 120 minutes of exercise. ATP synthesis activity elevated at 30 minutes and returned to the resting level thereafter. As compared with the resting group, respiration rate of states 3 significantly increased in groups E30 and E60, respiration rate of states 4 significantly increased in group E60, whereas respiratory control ratio without remarkable changed during 120 minutes of exercise.Ca2+ regulated mitochondrial motility in myoblastMitochondria were highly active in C2C12 myoblast, displayed two kinds of mitochondrial motility mode–movement and local swinging. 10mmol/L CaCl2 induced a decrease in mitochondrial motility, meanwhile [Ca2+]i elevation evoked by addition of CaCl2. 5mmol/L EGTA promoted mitochondrial motility, at the same time [Ca2+]i decreased.Effect of H2O2 on mitochondrial motility and dynamics and mitochondrial function in myoblast1 Myoblast stimulated by various concentrations of H2O2 showed different characteristics:cell survival rates of myoblast stimulated by low concentration (1μmol/L~100μmol/L )H2O2 had no obvious changes, but when stimulated by higher concentrations of H2O2(1mmol/L or 10mmol/L)showed significantly low cell survival. The results showed that there were no obvious toxicity effects but mildly oxidatie stress caused by low concentration of H2O2, however significant oxidative damage induced by 10mM H2O2. Miro1 mRNA and protein contents were increased by 10μmol/L H2O2. Drp1 mRNA and protein contents were increased and then decreased when treated with H2O2. Mfn2 mRNA and protein contents were decreased then tend to increased after treated with H2O2. The results also showed that expression changes of Miro1 precede Mfn2 and Drp1, Ca2+ involved in expression changes of Miro1 induced by H2O2. 2 Various concentrations of H2O2 had bidirectional roles in mitochondrial motility in myoblast. 1μmol/L～1mmol/L H2O2 promoted mitochondrial motility, however 10mmol/L H2O2 inhibited mitochondrial motility.3 The rates of respiration of myoblast cells were transient reversibly inhibited when stimulated by low concentrations(100nmol/L~1mmol/L)of H2O2 and with the increasing concentration, inhibition time prolonged. 10mM H2O2 strongly inhibited cellular respiration. 100μmol/L～10mmol/L H2O2 decreased the ATP content of cellular levels, nevertheless there were no significantly effects when myoblasts were treated with 1μmol/L or 10μmol/L H2O2.Regulation mechanism of mitochondrial motility induced by H2O2 in myoblast 10μmol/L H2O2 promoted mitochondrial motility as well as decreased [Ca2+]i in myoblast. Preliminary treated with EGTA obviously inhibited the promotion of mitochondrial motility and transient decrease in [Ca2+]i due to H2O2 alone. Myoblasts that were treated with 5μmol/L Thapsigargin in advance significantly increased [Ca2+]i , at the same time obviously decreased mitochondrial motility. After that [Ca2+]i was slightly decreased and mitochondrial motility had no obviously increased by 10μmol/L H2O2 in myoblast than stimulated with H2O2 alone.Conclusions:1 Mitochondrial motility increased during 120 minutes of exercise in skeletal muscle, at the same time mitochondrial fusion suppressed tend to fission. The up-regulated expressions of mitochondrial motility related protein Miro1 might be the base of mitochondrial fusion-fission. It might have promoted mitochondrial network structure reconstitution thus facilitating mitochondrial respiratory and ATP synthesis for matching the energy demand during exercise.2 Mitochondria are highly active in C2C12 myoblast, displayed two kinds of mitochondrial motility mode–movement and local swinging. Ca2+ regulated mitochondrial motility in myoblast: mitochondrial motility decreased as well as [Ca2+]i improved, while mitochondrial motility increased meanwhile [Ca2+]i suppressed.3 Various concentrations of H2O2 had bidirectional role in mitochondrial motility in myoblast. At lower concentration of H2O2 promoted mitochondrial motility, however high concentration of H2O2 inhibited mitochondrial motility. Mild oxidatie stress caused by H2O2 in myoblasty. Ca2+ involved in the changes of mitochondrial motility induced by H2O2. And it might be the basis of mitochondrial fusion-fission for promoting mitochondrial network structure reconstitution. During this process mitochondrial fusion suppressed and tend to fission, soon afterwards mitochondrial network structure changed again and tend to fusion might be due to adaptation response of myoblast. 4 H2O2 promoted mitochondrial motility as well as decreased [Ca2+]i in myoblast. Sarco-endoplasmic reticulum Ca2+ pump uptake involved in [Ca2+]i reduced by H2O2. And extracellular Ca2+ influx involved in change of [Ca2+]i and mitochondrial motility stimulated by H2O2.