Exercise Preconditioning Improves Electrical Remodeling By Regulating The Expression Of Kv4.2,SERCA2a,NCX1,and Cx45 In Exhaustive Rats

Objective:Exhaustive exercise can lead to myocardial structural injury,cardiac dysfunction a nd various arrhythmias and even athletic sudden death.Exercise preconditioning can i mprove the tolerance of myocardial ischemia and hypoxia,improve the cellular and m olecular mechanisms of abnormal cardiac electrical activity,reduce ventricular electrical remodeling,and reduce the susceptibility to arrhythmia.Therefore,in this study,the EP rat model was established and one-time exhaustive exercise was regarded as injury factor.The effects of EP on QT,QTc and Tp-Te interval in exhaustive rats were dis cussed by observing the expression changes of the key proteins of electrical remodelin g voltage-gated potassium channel subfamily D members 2（Kv4.2）,sarcoplasmic retic ulum Ca²⁺-ATPase（SERCA2a）,sodium-calcium exchanger-1（NCX1）,and Connexin 45（Cx45）and the degree of myocardial fibrosis.Methods:Forty healthy male SD rats were randomly divided into 4 groups（n=10）:control group（Con）,exhaustive exercise group（EE）,exercise preconditioning group（EP）,an d exercise preconditioning+exhaustive exercise group（EP+EE）.The Con group did n ot exercise on the treadmill.The EE group kept running at the speed of 30～32 m/min on the treadmill with a slope of 5°until they were exhausted.The modeling method of EP group:the rats were given treadmill exercise at a speed of 26m/min（74.3±2.9%VO₂Max）on a treadmill with a slope of 5°for 6 days per week,and took a 5-minute break after15-minute exercise and the scheme lasted for 3 weeks.Rats in EP+EE group got EP training in the same way as the EP group,they kept running until exhausted on the next day after the end of EP training.After the model was establish ed,the 8-channel physiological recorder was used to recordⅡlead electrocardiogram o f rats in each group and Power Lab software was used to calculate relevant parameter s such as QT,QTc,Tp-Te interval.Myocardial injury markers were determined by enz yme-linked immunosorbent assay.The morphological and structural changes of myocar dium were observed by light microscopy after HE staining.The hyperplasia of collage n fiber secretion after Masson staining was observed by light microscopy and the area of collagen fiber in myocardial interstitium was quantitatively analyzed.The fluoresce nce signals distribution of key proteins of electrical remodeling（Kv4.2,SERCA2a,NC X1 and Cx45）were observed by immunofluorescence and their protein levels were det ected by Western Blot.Results:1.The influence of EP on cardiac mass index in exhaustive ratsCompared with the Con group,the heart mass index of the EE group was signifi cantly decreased,and the difference was statistically significant（P<0.05）.Compared with Con group,the heart mass index in EP group and EP+EE group was significantl y increased,and the difference was statistically significant（P<0.05）.Compared with the EE group,the body weight of the EP+EE group decreased significantly,and the difference was statistically significant（P<0.05）.Compared with the EE group,the B MI of the EP+EE group was significantly increased,and the difference was statisticall y significant（P<0.05）.2.The influence of EP on myocardial pathological changes in exhaustive ratsThe myocardial fibers of Con group were arranged neatly without cell edema and necrosis.Compared with Con group,myocardial fibers in EP group were thicker,inte rcellular connections were closer,and there was no necrosis or inflammatory infiltratio n.In EE group,myocardial fibers were disordered,seriously broken,and cells were s wollen and necrotic with a lot of inflammatory infiltration.Compared with EE group,EP+EE group had more orderly arrangement of myocardial fibers,and the degree of muscle fiber fracture and swelling necrosis was lighter,without obvious inflammatory i nfiltration.3.The influence of EP on myocardial collagen fiber hyperplasia in exhaustive rats.The myocardial structure of Con group was normal,and almost no collagen fibe r hyperplasia was found.Compared with Con group,cardiac fiber in EP group was th icker and collagen fiber was slightly hyperplasia.Myocardial fibers in EE group were seriously damaged,and the collagen fibers were proliferated in large numbers,which were interlaced with the myocardial fibers in a network pattern.Compared with the E E group,the EP+EE group had better arrangement of myocardial fibers,lighter breaka ge,and significantly reduced collagen fiber proliferation.The quantitative analysis resul ts of the left ventricular collagen fiber area of rats in each group by Image-Pro Plus6.0 software were as follows:compared with the Con group,the degree of myocardial fibrosis in each group was increased to varying degrees,and the increase was most obvious in the EE group,with statistical significance（P<0.05）.Compared with the EE group,the degree of myocardial fibrosis in the EP+EE group was significantly dec reased,with statistical significance（P<0.05）.4.The influence of EP on myocardial injury markers in exhaustive rats.Compared with Con group,the contents of CK-MB,CTn-I and MYO in EP+EE group and EE group were significantly increased,with the most significant increase in EE group（P<0.05）.Compared with EE group,the contents of CK-MB,CTn-I and MYO in EP+EE group were significantly decreased,and the differences were statistic ally significant（P<0.05）.5.The influence of EP on electrocardiogram changes in exhaustive rats.Compared with Con group,ST segment in EP+EE group and EE group was elev ated to different degrees（P<0.05）.Compared with EE group,the elevation of ST se gment in EP+EE group was significantly decreased（P<0.05）.Compared with Con g roup,HR in EP group was significantly decreased（P<0.05）;Compared with Con gr oup,HR in EP+EE group and EE group was significantly increased（P<0.05）;Com pared with EE group,HR in EP+EE group was significantly decreased（P<0.05）.Co mpared with Con group,the QRS duration of EP group,EP+EE group and EE group was prolonged to different degrees（P<0.05）.Compared with EE group,the QRS d uration of EP+EE group was significantly shortened（P<0.05）.Compared with the C on group,the QT and QTc interval of the EE group were significantly prolonged（55.36±2.36ms vs 80.25±10.10ms,140.89±7.58ms vs 217.58±29.92ms,P<0.05）.QT and QTc interval were significantly shortened in EP+EE group（64.15±7.29 ms vs 80.25±10.10 ms,165.55±14.74 vs 217.58±29.92 ms,P<0.05）.Compared with Con group,the interval of Tp-Te in EE group was significantly prolonged（28.90±0.76 m s vs 42.76±1.83 ms,P<0.05）.Compared with EE group,the interval of Tp-Te in EP+EE group was significantly shortened（35.67±0.67 ms vs 42.76±1.83 ms,P<0.05）.6.The influence of EP on expression and distribution of fluorescence signal of k ey factors of myocardial electrical remodeling in exhaustive rats.Immunofluorescence combined with confocal microscopy was used to observe the expression and distribution of fluorescence signals of Kv4.2,SERCA2a,NCX1 and Cx45 in left ventricular muscle tissue.Kv4.2,SERCA2a and NCX1 were uniformly distri buted in each group.In the Con group,Cx45 was less expressed in ventricular muscl e.Compared with the Con group,the expression of Kv4.2 in EP group,EP+EE group and EE group was decreased,and the decrease was most obvious in the EE group.Compared with the Con group,the expression levels of SERCA2a,NCX1 and Cx45were slightly increased in the EP group.Compared with Con group,SERCA2a,NCX1and Cx45 expressions were decreased in EP+EE group and EE group,and the decrea se was most obvious in EE group.Compared with EE group,the expression of Kv4.2and SERCA2a were significantly increased in EP+EE group.Compared with the EE group,the expressions of NCX1 and Cx45 were significantly decreased in the EP+EE group,and the distribution of Cx45 in the EP+EE group was more uniform and regu lar than that in the EE group.7.The influence of EP on expression of key factors of myocardial electrical remo deling in exhaustive rats.The expression levels of Kv4.2,SERCA2a,NCX1 and Cx45 in left ventricular m uscle of rats in each group were detected by Weltern Blot.Compared with Con group,the expression of Kv4.2 in EP group,EP+EE group and EE group was decreased,an d the decrease in EE group was the most significant,with statistical significance（P<0.05）.Compared with Con group,the expression levels of SERCA2a,NCX1 and Cx45 in EP group were slightly increased,with statistical significance（P<0.05）.Comp ared with Con group,the expression levels of SERCA2a,NCX1 and Cx45 were decre ased in EP+EE group and EE group,and the decrease was the most significant in EE group,with statistical significance（P<0.05）.Compared with the EE group,the expr ession levels of Kv4.2 and SERCA2a in the EP+EE group were significantly increased,with statistical significance（P<0.05）.Compared with the EE group,the expression levels of NCX1 and Cx45 in the EP+EE group were significantly decreased,with stati stical significance（P<0.05）.Conclusion:1.Exhaustive exercise can lead to myocardial structural damage and abnormal my ocardial electrical activity,which mainly manifested as prolonged QT,QTc and Tp-Te interval of electrocardiogram.2.Exhaustive exercise resulted in prolongation of QT,QTc and Tp-Te interval,w hich may be related to down-regulation of Kv4.2 and SERCA2a and up-regulation of NCX1 and Cx45.3.Exercise preconditioning can adapt the heart to exhaustive exercise induced hea rt injury and shorten the extension of QT,QTc and Tp-Te interval,thus maintain the homeostasis of cardiac electrophysiological characteristics.4.The mechanism of exercise preconditioning to maintain the homeostasis of myo cardial electrophysiological characteristics may be related to increasing the expression of Kv4.2 and SERCA2a and decreasing the expression of NCX1 and Cx45.