运动作为心肌肥厚预适应因子通过调节lncRNA Mhrt发挥心肌保护作用

Recently we first provided experimental evidence that preconditioning by prohypertrophic factors increases the resistance of the heart to subsequent hypertrophic stress and delays progression from hypertrophy to heart failure, indicting the existence of hypertrophic preconditioning phenomenon, implying that induction of hypertrophic preconditioning has the potential to become a new approach to protect cardioprotection for patients with pressure overload. Further studies on searching and confirmation of new and easy-operating hypertrophic precondition factors and clarifying the potential mechanismswould be beneficial for clinical prevention and therapy of heart failure. It is known that physical exercise can induce physiological myocardial hypertrophy and also benefit patients with heart failure, but it is unclear whether physical exercise is a hypertrophic precondition factor to exert effects of antihypertrophy and anti-heart failure in hearts subjected to prolonged subsequent pathological hypertrophic stress. Our preliminary experimental evidence shows that physical exercise in mice upregulates long noncoding RNA(lncRNA) myosin-heavy-chain-associated-RNA transcripts (lncRNA Mhrt). Previous report have demonstrated that activation of lncRNA Mhrt can attenuate pathological hypertrophy and improve heart failure. In this study, we hypothesize that physical exercise would act as a hypertrophic preconditioning factor to render the heart resistant to subsequent hypertrophic stress as well as slowing progression to heart failure mediated by lncRNA Mhrt-involved signal pathway. We plan to perform cardiovascular physiologic and molecular biological experiments in mice subjected to physical excercise and subsequent pressure overload. This project contains 3 research parts: (1) To clarify whether physical exercise is a hypertrophic preconditioning factor; (2) If so, what are the changes on lncRNA Mhrt-regulated signal pathway (lncRNA Mhrt-Brg1–Hdac–Parp, class I Hdac members are known to induce hypertrophy); (3)Effects of loss and gain function of lncRNA Mhrt on hypertrophic precondtioning. We believe such studies will be of clinical significance to optimize hypertrophic preconditioning for clinical application.

我们首次实验证实了心肌肥厚预适应现象，即短暂的病理性肥厚刺激后可通过诱导肥厚消退因子抵抗后续的持续肥厚刺激从而产生抗肥厚及延缓心衰进程的效应。病理性肥厚刺激作为预适应因子不便临床推广，需要寻找生理性的肥厚预适应因子。已知运动可引起生理性心肌肥厚且对心功有保护作用，但不清楚能否作为心肌肥厚预适应因子。我们的预实验发现小鼠运动可上调肌球蛋白重链相关RNA转录子(lncRNA Mhrt)，而后者被认为有抗心肌肥厚和抗心衰的作用，因此我们假设运动可能通过lncRNA Mhrt介导的信号通路发挥心肌肥厚预适应的作用，从而保护心脏抵抗病理性肥厚刺激所致的心肌肥厚和心衰。本项目拟包括三个研究部分：（1）运动是否为肥厚预适应因子；（2）如果是，运动后lncRNA Mhrt-Brg1–Hdac–Parp介导的信号通路是否有变化；（3）过表达和敲低lncRNA Mhrt验证该lncRN对肥厚预适应的影响。

本研究中我们假设运动引起的生理性心肌肥厚消退后仍具有抗病理性心肌肥厚的作用，即存在抗肥厚记忆，运动通过上调lncRNA 779抑制brg1/HDAC2/Akt/GSK3beta病理性心肌肥厚信号通路。采用成年小鼠进行游泳运动诱导生理性心肌肥厚、小鼠压力负荷模型、分离大鼠乳鼠以及小鼠乳鼠心肌细胞进行了实验；直接心肌内注射腺相关病毒lncRNA mhrt779或sh lncRNA mhrt779，上调心肌内或敲低lncRNA 779的表达水平，观察不同表达水平lncRNA 779与运动抗肥厚作用、心功能和心肌肥厚之间的关系。用CHRIP-MS确定lncRNA mhrt79的结合蛋白brg1，探讨其作用机制，以期为运动肥厚预适应的心脏保护作用提供依据。在此基础上，我们还进行了较多的心肌再生、糖尿病心肌病和心衰等扩展研究，共取得8项研究成果：在权威SCI杂志发表论著6篇，申请国家发明专利2项（公示中）。以下为研究内容摘要：.背景：运动可以引起生理性心肌肥厚（PMH），据报道，前运动员比非运动对照组的存活时间长5-6年，这表明运动引起的PMH消退后会有益处。我们以前曾报道过，病理性心肌肥厚的消退后仍会产生抗肥厚作用。因此，我们假设PMH消退后也存在抗肥厚性记忆，它对随后的病理性肥厚应激具有抵抗力。方法和结果：C57BL/6小鼠接受21天的游泳训练以引起PMH，PMH在运动终止后1周消退。然PMH消退的小鼠（运动预处理组，EP）和久坐的小鼠分别接受横行主动脉缩窄（TAC）或假手术。TAC 1或4周后与静坐组相比，EP组的心肌肥厚增加减少，Nppa和Myh7基因的表达降低。TAC术后4周，与久坐小鼠相比，EP小鼠的肺充血程度更低，左室（LV）内径和舒张期末压力较小，LV射血分数更大，LV压力变化率最大。 LncRNA Mhrt779被筛选为EP组中明显上调的LncRNA之一。 Mhrt779的沉默和过表达分别减弱和增强了EP对患有TAC的小鼠和经血管紧张素II处理的心肌细胞中EP的抗肥厚作用。质谱和蛋白质印迹表明，Mhrt779可以结合brg1/HDAC2形成的复合物并抑制Akt和GSK3β的磷酸化。结论：运动引起的心肌肥厚预适应通过上调Mhrt779，抑制压力超负荷引起的HDAC2 / p-Akt /p-GSK3β信号通路活化介导的病理性心肌肥厚，从而延缓