Exercise Promotes Cardiac And Skeletal Muscle Health:The Role And The Underlying Mechanisms Of Branched-chain Amino Acid Catabolism

Physical inactivity is suggested as one of the major risk factors for numerous non-communicable diseases（NCD）,including tumor,cardiovascular diseases,diabetes and neuropsychical diseases etc.Regular exercise is implicated in decreasing morbidity and mortality of NCD.Exercise-enhanced mitochondrial metabolism plays a key role in exercise-promoted health.Branched-chain amino acids（BCAA）are necessary amino acids with functional R groups in branched chains,consisting of valine,leucine and isoleucine.Evidence has shown that BCAA metabolism defect was implicated in progress of many diseases including Maple Syrup Urine Disease,diabetes and cardiovascular diseases etc.However,study showed that BCAA supplementation to healthy animals promotes the mitochondrial biogenesis and extends the life expectancy.The most important is the ability to metabolize BCAA.Both animal and human studies have found that BCAA supplementation can improve exercise ability and relieve exercise fatigue.In addition,prolonged exercise increases the catabolism of BCAA in skeletal muscle.However,it is still unclear what the specific effect of exercise on BCAA catabolism in different tissues,and role of BCAA catabolism in the exercise-induced health,and what the specific mechanisms are.Objective1.To investigate the effect of exercise on BCAA catabolism in heart and other tissues and whether exercise play a cardioprotective role by regulating BCAA catabolism.2.To clarify the mechanism by which aerobic exercise exerts cardioprotective effects by regulating BCAA catabolism.3.To investigate whether aerobic exercise can promote skeletal muscle health by regulating BCAA catabolism.Methods and Results1.To investigate the effect of long-term aerobic exercise on human metabolism,16 athletes and 16 non-athletes were recruited in the early stage,and the serum of the subjects was collected for non-targeted metabolome detection and bioinformatics analysis.The results showed that long-term aerobic exercise had a more significant effect on the levels of circulating amino acids,and BCAA metabolism was significantly affected.It is found using ELISA kit that aerobic exercise can increase the level of BCAA in human serum.2.To investigate the effect of aerobic exercise on BCAA metabolism in mice,4-week-old C57 mice were used for experiments.They were divided into an exercise group（EXE）and a control group（SED）.The exercise group underwent 8-week swimming training（swimming for 90 min each time,5 times a week）,while the control group was fed quietly for 8 weeks.The results showed that,compared with the SED group,the BCKD enzyme activities in the heart,liver and skeletal muscle of the EXE group increased,the PP2Cm protein level increased,and the p-BCKDHA protein level decreased,indicating that aerobic exercise promotes BCAA catabolism in the heart,liver and skeletal muscle.3.To investigate the effect of promoting BCAA metabolism on myocardial infarction（MI） injury,8-week-old C57 mice were used for experiments,and they were divided into 6 groups:mice with sham procedure（Sham）;mice with myocardial infarction procedure（MI）;mice with sham operation and BCAA supplementation（Sham+BCAA）;mice with myocardial infarction and BCAA supplementation（MI+BCAA）;mice with sham operation and BT2 supplementation（Sham+BT2）;mice with myocardial infarction and BT2 supplementation（MI+BT2）.BT2 is a drug that promotes BCAA metabolism.AfterMI surgery,mice were given BCAA mixture（1.5 mg/g/d）or BT2（40 mg/kg/day）through gavage for 4 weeks.Cardiac function,degree of myocardial fibrosis,degree of myocardial apoptosis and blood vessel density were then detected.The results showed that compared with the MI group,the BCAA+MI group had worse cardiac function,more obvious cardiac fibrosis,lower blood vessel density,and more severe apoptosis.Compared with the MI group,the BT2+MI group had better cardiac function,lower heart weight to body weight ratio,less cardiac fibrosis,higher blood vessel density,and less apoptosis.4.To investigate whether aerobic exercise exerts cardioprotective effect by regulating BCAA catabolism,heart specific Ppm1k knockout mouse(Ppm1k^CKO)was constructed.Ppm1k is a soluble protein located in the mitochondrial matrix,which is a dephosphatase that activates BCKDH.Ppm1k knockout mice was used as model mice with abnormal BCAA catabolism,and the cumulative concentration of BCAA in the blood of the model mice increases.The 4-week-old control mice Ppm1k^flox/flox and Ppm1k^CKO mice were subjected to sham surgery or MI treatment for 4 weeks after swimming training for 8 weeks.Then we detect cardiac function,myocardial fibrosis,myocardial apoptosis and blood vessel density.The results showed that exercise can improve the cardiac function of Ppm1k^flox/flox mice in the control group after MI,and decrease the ratio of heart to body weight and lung to body weight.At the same time,exercise reduced the area of collagen fibers in Ppm1k^flox/flox mice,decreased the number of apoptotic cardiomyocytes,and increased their vascular density.In contrast,exercise did not improve cardiac function,myocardial infarction size,myocardial fibrosis,vascular density and myocardial cell apoptosis in Ppm1k^CKO mice after MI injury.These results above suggest that BCAA metabolism plays an important role in exercise-induced cardioprotection.5.To elucidate the mechanism of exercise-induced cardioprotective effects through regulating BCAA catabolism,primary cardiomyocytes of rat neonatal mice were isolated.Under hypoxic conditions,the accumulation of BCAA activates the m TOR signaling pathway,and promoting BCAA metabolism inhibits the m TOR signaling pathway.Inhibition of m TOR under hypoxic conditions reduces cardiomyocyte apoptosis exacerbated by BCAA accumulation,and inhibition of m TOR reduces myocardial hypertrophy exacerbated by BCAA accumulation under conditions of adding phenylephrine（PE）.Activation of m TOR aggravated cardiomyocyte apoptosis in the Ad-Ppm1k group,and activation of m TOR in the presence of PE aggravated the pathological cardiac hypertrophy in the Ad-Ppm1k group.These results suggest that aerobic exercise exerts cardioprotective effects by promoting BCAA catabolism and reducing m TOR and downstream molecular phosphorylation.6.To investigate whether aerobic exercise promotes skeletal muscle health by promoting skeletal muscle BCAA metabolism,skeletal muscle-specific Ppm1k knockout mice(Ppm1k^SMKO)were constructed.Firstly,skeletal muscle-specific knockout of Ppm1k reduced exercise endurance of skeletal muscle,and its mitochondrial morphology and function were abnormal.Compared with the control group,the mitochondrial density of the Ppm1k^SMKO group was reduced,with obvious vacuoles,abnormal morphology,and decreased expression of mitochondrial synthesis-related molecules.This suggests that BCAA metabolism may play an important role in skeletal muscle health.The exercise ability of the Ppm1k^SMKO group after exercise,including grip,time-to-failure distance,and time to turn the bar,did not change significantly.Exercise also failed to increase the cross-sectional area of skeletal muscle fibers in Ppm1k^SMKO mice,and had no significant effect on muscle fiber types.The above results suggest that exercise may improve skeletal muscle health such as exercise capacity by promoting BCAA metabolism in skeletal muscle.ConclusionsThis study found that aerobic exercise promote the catabolism of BCAA in different tissues,including heart and skeletal muscle.And BCAA catabolism is involved in the exercise-induced cardioprotective effect and the improvement of exercise capacity.Furthermore,it was elucidated that aerobic exercise plays a cardioprotective role through promoting cardiac BCAA metabolism that inactivates m TOR signaling.