The Role Of NRG1 In Intermittent Exercise To Improve Normal And Ischemic Cardiac Cardiomyocyte Contractile Function And Its Mechanism

Objectives:This research aims to discuss the effect of Aerobic interval training(AIT) on cardiomyocytes calcium transient and contractility of both normal and myocardial infarction(MI) induced ischemic heart, then explore the role of Neuregulinl(NRG1) and its downstream signaling pathway in the effect, so that provide experimental evidence for prevention and treatment of ischemic heart disease.Materials and Methods:40 male sprague-dawley rats,3 months old, were randomly divided into 5 groups: Control group(C), AIT group(CE), Sham-operated group(S), sedentary MI group(MI) and MI with AIT group(ME). Rats in C group were fed in a sedentary lifestyle, meanwhile rats in CE group were subjected to 8-week treadmill running as follows, one week adaptive training (10m/min×30min/d) and subsequently 8-week aerobic interval training:starting with 10m/min×10min warming-up, then reran the rats with 2 intensities:15m/min×6min and 25m/min×4min,60min/d,5d/wk. The MI model was established by ligation of the left anterior descending coronary artery, however the Sham-operation was established by threading only without ligation, the rats in ME group started training 1 week after MI operation.At the end of the training, the cardiomyocytes of all the groups were isolated by Langendorff perfusion system, and part of cardiomyocytes in C group were stimulated by recombinated NRG1(C+NRG1 group). lonoptix video-based motion edge-detection system was performed to test cardiomyocytes calcium transient and contractility. Peak twitch amplitude(PTA), SL shortening% and ±dl/dtmax were used to evaluate contractility, Ratio amplitude, [Ca2+]i amplitude, Departure velocity, Return velocity, Time to peak(TTP) and Time to baseline(TTB) were used to evaluate calcium transient.Besides, another 48 male sprague-dawley rats,3 months old, were randomly divided into 6 groups:Control group(C), AIT group(CE), Sham-operated group(S), sedentary MI group(MI), MI with AIT group(ME) and ME with NRG1 receptor antagonist group(MA). Rats in S, MI and ME were operated as above, rats in MA were injected with NRG1 receptor antagonist AG1478, once every 2 days. At the end of the training, hemodynamic measurement was preformed to evaluate cardiac function; Western Blot was used to test the expression level of NRG1 and its downstream proteins; RT-qPCR was used to test the expression level of serca2a mRNA.Results:(1) AIT could significantly upregulate the expression of NRG1 protein in heart.(2) AIT could significantly enhance cardiomyocyte calcium transient, and had the same effect as NRG1 Stimulation. AIT or NRG1 stimulation could improve Ratio amplitude, [Ca2+]1 amplitude and velocity of calcium transient, shorten TTP and TTB, so that improved calcium handling and enhanced transient.(3) AIT could notably improve contractility of cardiomyocyte, and had the same effect as NRG1 Stimulation. AIT or NRG1 stimulation could increased SL Shortening% and±dl/dtmax.(4) AIT and NRG1 stimulation had the same effect on calcium transient and contractility, menawhile AIT could significantly upregulate the expression of NRG1 protein in heart. It was assumed that AIT maybe enhance calcium transient and contractility through a NRG1 pathway.(5) AIT could significantly enhance cardiomyocyte calcium transient after MI. Infarction-induced ischemic injury could decrease Ratio amplitude, [Ca2+]i amplitude and velocity of calcium transient, prolong TTP and TTB. AIT after infarction could increase Ratio amplitude, [Ca2+]i amplitude and velocity of calcium transient, shorten TTP and TTB. The result indicated that calcium handling disorder happened after Infarction-induced ischemic injury however AIT acted as a protective role.(6) AIT could significantly improve cardiomyocyte contractility after MI. PTA, SL Shortening% and ±dl/dtmax decreased obviously after infarction. But AIT could enhance PTA, SL Shortening% and ±dl/dtmax notably.(7) AIT had a important effect on activating NRG1-PI3K-Akt-eNOS-PKG-PLN pathway. The expression of NRG1, pPI3K, pAkt, peNOS, PKG and pPLN were inhibited in infarcted heart. However, the expression of NRG1, pPI3K, pAkt, peNOS, PKG and pPLN were significantly upregulated after 8-weeks AIT. But AG1478 could inactivate expression of pAkt, peNOS, PKG and pPLN. NRG1 and its downstream pathway was inhibited by infarction-induced ischemic injury, and AIT had a significant influence in activating NRG1-PI3K-Akt-eNOS-PKG-PLN pathway, but the influence could be attenuated by AG1478.(8) The gene and protein expression of SERCA2a also were downregulated after MI, and AIT could significantly upregulate the expression of serca2a mRNA and SERCA2a protein in infarcted heart, however AG1478 could decrease the expression of SERCA2a. So AIT improved expression of SERCA2a being deeply associated with NRG1.(9) LVSP and ±dp/dtmax decreased and LVEDP increased after MI. AIT significantly reduced LVEDP, improved LVSP and ±dp/dtmax. However AG1478 could reduce LVSP and ±dp/dtmax significantly. Prove that infarction-induced ischemic injury could attenuated seriously heart function, and AIT could improve impaired heart function, even the protective effect was tightly associated with NRG1.(10) AIT could significantly activated NRG1-PI3K-Akt-eNOS-PKG-PLN pathway, so that upregulated expression of SERCA2a in infarcted heart. Heart function improved by AIT had a close association with NRG1. It was turned out that the activation of NRG1-PI3K-Akt-eNOS-PKG-PLN-SERCA2a pathway maybe was one of the reasons why AIT could improve heart function after MI.Conclusion:1. AIT and NRG1 stimulation had the same effect on enhancing cardiomyocyte calcium transient and contractility,and AIT could significantly upregulate expression of NRG1 in heart. It turned out that calcium transient and contractility improved by AIT was tightly associated with NRG1.2. AIT significantly improved calcium transient and contractility in cardiomyocyte of MI rats, and significantly activated the NRG1-PI3K-Akt-eNOS-PKG-PLN pathway, which could be inhibited by AG1478. AIT significantly increased MI myocardial SERCA2a gene and protein expression and AG1478 significantly reduced SERCA2a protein expression. The results showed that, AIT significantly restored SERCA2a expression in MI heart, and NRG1 was closely related.3. AIT improved heart function in MI rats, AG1478 could significantly reduce the effect. It is demonstrated that NRG1 played a critical role in MI rat heart function improved by AIT, and activation of NRG1-PI3K-Akt-eNOS-PKG-PLN-SERCA2a pathway may be one of the mechanisms of AIT to improve cardiac function after MI.