线粒体/肌浆网钙耦联与心力衰竭后室性心律失常关系的研究

Chronic Heart failure (CHF) is considered to be end stage of different heart disease. Ventricular tachycardia (VT) is the most common arrhythmia in this stage. So far, researches had confirmed that the Calcium handling, defines as entire process of Ca2+ entry, store, release and reuptake in subcellular structures as Mitochondria and sarcoplasmic reticulum of cardiomyocytes, is central to the control of heart rhythm in failing heart. Further investigation showed that these two Organelles in charge of energy metabolism and local ion transfering system will self-modulation each other as a compensatory integrated biological entirety response to pathophysiological alteration. However, the role of Mitochondrion-sarcoplasmic reticulum crosstalk in arrhythmia has not been fully elucidated. .With their regular, we attempt to investigate mitochondrion-sarcoplasmic reticulum crosstalk on expression of crucial calcium transfering protein, in vivo and in vitro eletrophysiological characterization, calcium coupling situation like calcium concertration, calcium sparks, calcium transient, calcium content and leak and energy metabolism-related oxidized redox state simultaneously by western-blotting, patch clamp technique and confocal laser scanning technique.. This study will sought to clarify the role of the mitochondrion-sarcoplasmic reticulum coupling in mechanisms of ventricular arrhythmia during heart failure and then to explore new antiarrhythmic target.

心力衰竭（心衰）和室性心律失常可见于各种心脏疾病，尤其在终末期，且二者常同时出现。有证据表面心肌细胞钙离子调控异常可能是心衰和室性心律失常的共同的离子机制。本项目以线粒体/肌浆网钙耦联这一在心肌细胞钙调控中起重要作用的机制作为突破口，借助其调节剂，用分子生物学技术研究心衰后钙耦联异常对细胞钙转运关键蛋白的基因与蛋白表达的影响；用组织电生理学技术研究钙耦联异常对在体心脏、离体灌流心脏电生理学的影响；结合活细胞钙荧光检测技术、激光扫描共聚焦显微镜技术、膜片钳技术研究心衰时钙耦联特性、能量代谢相关氧化还原状态改变与单个心室肌细胞电生理学特性改变之间的关系。力图阐明线粒体/肌浆网钙耦联在心衰后室性心律失常发生机制中的作用以及干预肌浆网钙泵功能对心脏电生理学特性和心律失常的影响；为探索新的抗心律失常靶点和抗心律失常药物提供思路。

心肌细胞钙离子调控异常是心衰和室性心律失常的共同的离子机制。本研究通过建立心力衰竭模型观察线粒体/肌浆网钙耦联在心肌细胞钙调控及心律失常发生中可能的致心律失常作用结果发现：（1）线粒体钙单向转运体抑制剂Ru360可以在减少细胞内、肌浆网内钙浓度的同时抑制衰竭心肌细胞触发活动及在体动物室性心律失常发生率的同时；降低了衰竭心肌细胞L型钙电流密度及钠钙交换电流密度；此外，在该抑制剂干预下，衰竭心肌细胞内线粒体钙浓度降低、线粒体膜电位除级幅值降低，钙瞬变、肌浆网钙渗漏相对值也明显降低，该转运体功能增强剂Spermine则起到相反的作用；(2) Thapsigargin（肌浆网钙泵SERCA）抑制剂）可以升高肌浆网内钙离子浓度，导致与spermine类似的效应，这一效应在给予Ru360后明显减弱；同时给予2-APB（肌浆网IP3受体抑制剂）可以逆转通过调节线粒体内钙浓度逆转Thapsigargin起到相似的作用；（3）上述干预药物的电生理与效应不依赖于改变相关转运蛋白如线粒体钙单向转运体(MCU)、肌浆网钙释放通道(RyR)、肌浆网Ca2+离子依赖ATP酶 (SERCA)、钠钙交换蛋白、受磷蛋白、肌钙集蛋白等维持细胞钙稳态功能的重要大分子的蛋白表达水平；（4）这些研究结果显示，线粒体/肌浆网钙耦联是心衰后室性心律失常发生的重要机制之一，也是其重要干预靶点，调节线粒体钙转运功能继而促进衰竭心肌细胞钙稳态恢复可能是防治心衰后室性心律失常发生的新手段。