心肌细胞IP3R在小鼠心脏发育与心肌重塑过程中的作用

Calcium and calcium dependent signaling pathways play an essential role in cardiac development and normal physiology. Disturbed calcium handling in cardiomyocytes is one of major pathogenesis of cardiac developmental defects and cardiac diseases. IP3 receptor is one of two calcium release channels locating on the membrane of endoplasmic reticulum, which has three distinct subtypes in mammals. It has been well recognized that IP3R2 is the predominant IP3R subtype expressed in cardiac cells. However, our study published recently showed that loss of IP3R2 doesn't affect cardiac development and remodeling. In fact, we further found that all three IP3R subtypes are co-expressed in cardiac cells and their expression are all dramatically increased during cardiac hypertrophy induced by pressure overload. Thus we hypothesized that differnet IP3R subtype in cardiac cells function redundantly. In this proposal, we are planning to generate cardiac-specific IP3R triple knockout mouse in which IP3R mediated calcium signals will be genetically and specifically ablated in cardiac cells. Furthermore, we will investigate the effects of IP3R triple deletion on cardiac development, adult cardiac physiology, cardiac hypertrophy, and heart failure. We also aim to explore how IP3R mediated calcium release regulates the generation of different types of calcium signals in embryonic and adult cardiac cells, and how it regulates cardiac physiology and pathology via CaMKII-HDAC and Calcineurin-NFAT signaling pathways.

钙信号及钙信号转导通路在心脏发育与心脏正常生理过程中均发挥重要作用。钙信号紊乱是心脏发育异常与发生心肌病的重要原因。三磷酸肌醇受体（IP3R）是内质网膜钙释放通道之一，在哺乳动物中有三种亚型，在心肌细胞中IP3R2一直以来被认为主要亚型。然而在我们前期工作中，我们发现单敲除IP3R2并不影响心脏发育与心肌重塑，与此同时我们还发现三种亚型均在心肌细胞中表达并在心肌肥大过程中表达均上升，因此推测心肌细胞IP3R三亚型相互之间功能共冗。本项目拟建立心肌细胞特异性IP3R三敲除小鼠，从而实现真正意义上地消除心肌细胞中IP3R介导的钙释放，并进一步观察IP3R三敲除对胚胎心脏发育、成年心脏功能、心肌肥大与心衰过程中的影响；同时探讨IP3R介导的钙释放是如何参与不同时期心肌细胞钙信号的产生以及如何通过CaMKII-HDAC与Calcineurin-NFAT信号转导通路影响心脏生理与病理过程。

钙信号及钙信号转导通路在心脏发育与心脏正常生理过程中均发挥重要作用。钙信号紊乱是心脏发育异常与发生心肌病的重要原因。在心肌细胞中，引起细胞收缩的Ca2+主要来源于肌质网（内质网）由RyR介导的钙释放，但是心肌细胞中也存在IP3R。在胚胎发育过程中，IP3R比RyR的表达更早。同时，IP3R1/IP3R2双敲除与IP3R1/ IP3R3双敲除均会引起小鼠胚胎发育异常与死亡，提示IP3R不同亚型之间存在功能共冗，但是IP3R双敲除导致小鼠胚胎发育异常的分子机制尚不清楚。此外，在成年小鼠心脏中，IP3R2被认为是主要的IP3R亚型，然而在小鼠中敲除IP3R2并不会影响小鼠正常的心脏功能，也不影响负荷过载引起的心肌肥厚。那么这是否是因为在成年小鼠心脏中其他两种IP3R起到补偿功能呢？为了解答这些科学问题，本项目拟通过构建细胞特异性的IP3R敲除小鼠来（1）确定心肌细胞IP3R介导钙释放在心脏胚胎发育过程中的作用；（2）确定心肌细胞IP3R介导钙信号在成年心脏生理与病理过程中的作用。我们利用TnT-Cre、Tie2-Cre、SM22-Cre以及Mesp1-Cre在心肌细胞、内皮细胞、平滑肌细胞乃至前中胚层细胞中构建了IP3R双敲除或三敲除小鼠，但是我们发现这些细胞特异性敲除并不会引起小鼠胚胎死亡与心脏发育异常。然而我们发现Mox2-Cre介导的IP3R双敲除会引起胚胎发育异常，说明IP3R对于小鼠胚胎发育的调节作用发生在胚胎发育的最早期；此外，我们还发现心肌细胞IP3R三敲除并不影响成年小鼠正常生理条件下的心脏形态与功能，也并不影响负荷过载或者异丙肾上腺素引起的心肌肥厚过程，但是对于血管紧张素II诱导的心肌肥厚过程有一定的抑制作用，说明IP3R介导的钙信号在某些特定刺激诱导的心肌重塑过程中起调节作用。