胚胎心室肌细胞增龄性变化中Rem蛋白对钙离子通道和微管蛋白的调控机制研究

Calcuim is key point in progress of heart failure and effects excitation-contraction coupling. The contractile function of ventricular myocytes is related with microtubulin. Microtubulin relies on G protein to transmit signal. It is not clear the mechanism of inducing heart failure between calcuim and microtubulin. The gene which controled the growth of cardiac myocytes in embryo is re-expressed in earlier period of heart failure. Because the adult cardiac myocytes do not like the embryonic cardiac myocytes to keep growing up, the adult cardiac myocytes appear the hypertrophy or even heart failure. Previous studies represented calcuim channel current was regulated by calcuim channel β subunit in ventricular myocytes of embryo 18 days.Rem, one of G protein is highest expression in the heart.Rem was specificity to binding calcuim channel β subunit and significantly depressed calcuim internal and external flow in embryonic ventricular myocytes. It is hypothesis that calcuim channel and microtubulin should be regulated by Rem. How to regulate among them. We attempt to investigate the mechanism in aging related changes of embryonic ventricular myocytes via patch clamp, calcuim imaging,laser confocal technic and molecular biology. It is identified that the regulated mechanism of calcuim channel current following aging related changes of embryonic ventricular myocytes. Thereby, it is revealed the mechanism of cell electrophysiology in the progress of heart failure.

Ca2+在心力衰竭发生过程中起关键作用，影响心肌细胞的兴奋收缩耦联效应，心肌的收缩功能与心肌细胞骨架微管蛋白有关，而微管蛋白又依赖G蛋白传递信号。Ca2+与微管蛋白诱发心力衰竭的机制目前仍不清楚。我们前期的工作发现：妊娠18天胚胎心室肌细胞Ca2+通道的β受体（Cavβ）主要调控Ca2+内流； G蛋白中的Rem与 Cavβ结合具有特异性；Rem显著降低胚胎心室肌细胞Ca2+的转运。据此提出假设Rem、Ca2+通道和微管蛋白三者之间存在必然联系。我们将通过膜片钳、Ca2+成像、激光扫描共聚焦等技术研究胚胎不同生长阶段心室肌细胞中Rem对Ca2+通道和微管蛋白的影响，证实Rem主要参与调控Ca2+通道电流以及细胞骨架微管蛋白的信息传递。通过研究胚胎心室肌细胞增龄性变化中细胞膜Ca2+电流，认识心肌细胞生长过程中随细胞骨架改变Ca2+通道的调控机制，从而揭示心力衰竭发生过程中细胞电生理机制。

Ca2+在心力衰竭发生过程中起关键作用，影响心肌细胞的兴奋收缩耦联效应。本研究通过膜片钳、Ca2+成像、激光扫描共聚焦和分子生物学等技术研究胚胎和成年心室肌细胞中Rem对Ca2+通道和微管蛋白的影响。研究结果发现胚胎期处于不同生长阶段的心室肌细胞膜有T型和L型Ca2+通道，出生后心室肌细胞膜只有L型Ca2+通道表达。胚胎心室肌细胞表现得自律性主要决定于T型Ca2+电流。尽管胚胎心室肌细胞肌质网Ca2+通道（IP3R1、IP3R2和RyR2）转录水平呈增龄性变化，因肌质网发育不健全参与心肌细胞兴奋收缩耦联作用有限。心室肌细胞转录因子Nkx2.5、GATA4和Tbx5转录水平在胚胎时期呈现高水平，出生后至成年期明显降低与细胞骨架微管呈现负相关，提示这些转录因子与微管合成相关。G蛋白和相关因子cAMP、PKA、PKC和CaMKⅡ参与心室肌细胞的自身调节。G蛋白对L型Ca2+通道蛋白有特异性调控作用。Rem作为G蛋白质一种在心肌细胞中有较高的表达，并与Cavβ相结合影响Ca2+通道功能，阻断Ca2+电流；对T型Ca2+通道和Ca2+电流无影响。Rem和Cavβ的结合需要cAMP、PKA、PKC和CaMKⅡ调控。Rem和细胞骨架微管蛋白表达无内在联系。心室肌细胞Rem蛋白、细胞骨架微管蛋白和Ca2+通道蛋白随胚胎心室肌细胞增龄无量效关系，Rem过表达影响Ca2+通道，但须β亚单位参与绑定。综合研究结果提示胚胎心室肌细胞增龄性变化中Rem蛋白对Ca2+通道和微管蛋白通过自身调节实现，G蛋白Rem通过Ca2+通道影响Ca2+内流，过程受相关因子cAMP、PKA、PKC和CaMKⅡ的调节，表现为心肌细胞兴奋收缩耦联的影响。对心肌细胞兴奋收缩耦联的影响反射性作用细胞骨架微管蛋白，这一作用受细胞转录因子Nkx2.5、GATA4和Tbx5转录水平调节，随着胚胎心室肌细胞增龄心肌细胞的发育成熟这一作用逐渐减弱。当成年个体开始发生心力衰竭时，成年心肌因无法像胚胎时期心肌那样生长发育，心肌出现肥大以致心力衰竭。本研究结果对了解心肌肥大乃至心力衰竭早期的发生发展机制提供了理论依据,通过干预心肌细胞Rem蛋白、Ca2+通道和微管蛋白自身调节的不同环节，来选择新的治疗靶点。从而影响心力衰竭的发生和发展。