加味补阳还五汤调控压力超负荷致心力衰竭大鼠心肌钙释放/钙渗漏的分子机制研究

Calcium handling is critically involved in myocardial contraction and relaxation. Calcium leak seems to play a central role in altered calcium handling in heart failure. It is involved in modulation of calcium release or leak by phosphorylation of RyR2 channel. On the other hand, sorcin, or FKBPs could modulate calcium leak. The present study addresse effects of improved Yang supplementing Five Returning Decoction in the pressure overload animal model by abdominal aortic coarctation. Hemodynamics and ventriclar remodeling parameters will measured.To explore the relationships between the loading status of the SR and functional state of the Ca2+ leak mechanism, we examine the effects of changes in SR Ca2+ content on spontaneous Ca2+ sparks in saponin-permeabilized adult ventricular myocytes. SR Ca2+ content was manipulated by pharmacologically altering the capacities of either Ca2+ uptake or leak. Ca2+ transient were recorded using a Zeiss microscope and Fura-2/AM and were quantified considering missed events. SR Ca2+ leak was assessed by application of caffeine. We also measure phosphorylation and dephosphorylation status of ryanodine receptor after Qi-Benefiting, Blood-Activating Therapy. We will investigate not only the protein levels of sorcin and FKBP12.6, but also the interaction of sorcin or FKBP with cardiac RyR. Then we want to explore the cellular and molecular mechanism of regulation of ryanodine receptor in Qi-Benefiting,Blood-Activating therapy.

国内外研究发现心肌钙释放功能障碍-舒张期钙渗漏是心力衰竭的特征事件，与RyR2通道过度磷酸化及通道调节蛋白功能下调密切相关。本课题组以往研究发现加味补阳还五汤能够通过上调SERCA2a活性及蛋白表达改善心力衰竭大鼠心肌钙摄取作用。在此基础上，本研究拟采用腹主动脉缩窄建立压力超负荷大鼠心力衰竭模型，通过心脏超声和压力-容积技术测定大鼠心脏功能参数，采用钙离子成像联合可视化动缘技术同步检测加味补阳还五汤对成年大鼠心肌细胞钙瞬变及舒缩功能的影响，并在亚细胞水平-肌浆网囊泡测定钙渗漏比率。进一步围绕RyR2通道过度磷酸化（p-RyR2表达，PKA、CaMKII、PP1和PP2A活性）探讨加味补阳还五汤抑制心肌钙渗漏的作用靶点。最后检测通道调节蛋白Sorcin、FKBP12.6表达，免疫荧光双标确定Sorcin/FKBP12.6与RyR2共定位情况，阐明加味补阳还五汤调控心力衰竭钙渗漏的分子机制。

心肌钙释放功能障碍-舒张期钙渗漏是心力衰竭的特征事件，与RyR2通道过度磷酸化及通道调节蛋白功能下调密切相关。本项目在前期工作的基础上，从多个层面和多个环节探讨了加味补阳还五汤调控心肌肌浆网钙释放及钙渗漏的作用机制。本项目发现加味补阳还五汤能够明显改善压力过载导致成年小鼠心肌功能不全，降低小鼠心肌细胞钙瞬变及钙渗漏，初步阐明了加味补阳还五汤调控心力衰竭钙渗漏的分子机制。其机制与RyR2通道过度磷酸化（与p-RyR2高表达，PKA、CaMKII、PP1和PP2A活性上调密切相关），与RyR2通道调节蛋白Sorcin、FKBP12.6低表达以及Sorcin/FKBP12.6与之共定位减弱相关。课题组完成了预期目标并取得了多方面的研究成果。发表标注论文12篇，其中SCI收录7篇，培养研究生5名。