Relaxin经cGMP-Zyxin-nucAkt通路改善左室舒张功能的机制研究

Diastolic heart failure, also called Heart failure with preserved ejection fraction (HFpEF), accounts for nearly 50% of the heart failure population. And there is no drug has been proved to be effective in improving the prognosis of it. There are many mechanisms of diastolic dysfuntion, and change of cytosolic Ca2+ concentration during diastole is one of the most important mechanism. Phospholamban(PLB) and Sarco(endo)plasmic reticulum (SERCA2a) are the most important factors regulating Ca2+ concentration, and dephosphorylation of PLB and inactivation of SERCA2a will induce diastolic dysfunction. Therefore, drugs improving PLB phosphorylation and SERCA2a activity are important to improve diastolic function. It has been demonstrated that PLB is a downstream substrate of Akt pathway. Akt is an important kinase in cardiomyocytes. Activation of cytoplasmic Akt has been proved to induce hypertrophy of cardiomyocytes while nuclear-targeted activation of Akt has been demonstrated to be cardioprotective in cardiomyocytes without induction of hypertrophy. In our previous work, we have found that relaxin could improve diastolic function of pressure-overloaded rat induced by abdominal aorta constriction. Moreover, relaxin has been proved to activate nuclear-targeted Akt phosphorylation. However, the detail mechanism is still unknown. In the present study , we postulate that relaxin can improve diastolic function by activating cGMP-Zyxin-nuclear-targeted Akt pathway and finally increase PLB phosphorylation and SERCA2a activity. In the present study, pressure-overloaded rat model induced by abdominal aorta constriction will used and relaxin will be infused subcutaneously with osmotic minipump. UCG and hemodynamic monitoring will be used in evaluating diastolic funcion. Other experimental techniques such as confocal microscope, Western Blotting, co-immunoprecipitation et al will be used to demonstrated our postulation. Furtherly, to improve the postulation , KT5823 will be used to inhibit cGMP activity, siRNA for nuclear Zyxin will be used to knockdown nuclear Zyxin expression and LY 294002 be used to inhibit Akt phosphorylation.

舒张性心衰发病率高且缺乏有效改善预后的药物。心肌胞浆中Ca2+浓度调节异常是心肌舒张异常的关键，受磷蛋白（PLB）磷酸化和肌浆网钙泵ATP酶（SERCA2a）活性异常为其重要因素。研究证实PLB是Akt下游底物，核内Akt被cGMP-Zyxin途径激活并具有心血管保护作用。我们前期研究证实松弛素（RLX）能改善压力负荷大鼠舒张功能，并能靶向激活细胞核内Akt，但其机制尚不明确。本项目拟通过构建腹主动脉狭窄的压力负荷大鼠模型，经皮下渗透性微泵持续输注RLX，行心脏超声、Western Blot、免疫共沉淀等方法以及通过将RLX加入乳鼠心肌细胞并分别抑制cGMP、Zyxin、Akt磷酸化水平从大体、组织、细胞水平证实假说：RLX通过调节cGMP-Zyxin途径使细胞核内Akt磷酸化增加，进而上调PLB磷酸化水平及SERCA2a活性改善左室舒张功能，为RLX用于舒张性心衰的治疗提供理论基础。

射血分数代偿的心力衰竭(HFpEF)又称舒张性心衰，约占全部心衰患者的50%，其发病率高且缺乏有效改善预后的药物。松弛素（RLX）被证实有多重的心血管保护作用但是能否改善舒张功能目前尚不明确。心肌胞浆中Ca2+浓度调节异常是心肌舒张异常的关键，受磷蛋白（PLB）磷酸化和肌浆网钙泵ATP酶（SERCA2a）活性异常为其重要因素。Akt是心肌细胞中最重要的激酶之一，是连接细胞信号及下游反应的重要枢纽。激活细胞核内和胞浆内的Akt在心血管方面具有完全不同的作用。.本项目通过构建腹主动脉狭窄的压力负荷大鼠模型，经心肌注射过表达RLX的腺病毒载体（Ad-RLX），行心脏超声及有创血流动力学检测来评估心脏舒张功能，并通过Western Blot、RT-PCR、激光共聚焦等方法验证心肌细胞中Akt、PLB、SERCA2a磷酸化水平以及活性。同时用Ad-RLX转染乳鼠心肌细胞，通过上述方法检测RLX对细胞核内和胞浆内Akt、PLB等磷酸化水平的影响。.本研究结果证实：RLX能够改善压力负荷大鼠的舒张功能，其作用机制是通过使细胞核内Akt的Ser473 and Thr308位点磷酸化，而不激活胞浆内的Akt受体。进一步激活受磷蛋白（PLB）Ser16 and Thr17的位点磷酸化，从而增加SERCA2活性，使肌浆网钙内流增多。该研究为RLX用于舒张性心衰的治疗提供理论基础。