新的力学感受结构域：线粒体结合内质网膜介导压力超负荷心肌重塑的机制研究

Sustained pressure overload is known to trigger cardiac remodeling. Evidence points to a critical role of excessive endoplasmic reticulum stress (ERS) in the underlying pathophysiology. However, the mechanic biological mechanisms of ERS in pressure overload-induced cardiac remodeling remains unclear. The mitochondria-associated ER membrane (MAM) is a sub-domain of the ER membrane that facilitates cross-talk between the ER and mitochondria. The MAM contains some signaling and membrane-tethering proteins but also lipids including cholesterol, and is regarded as an intracellular detergent-resistant lipid raft-like domain. Our previous experiments demonstrated that, mechanical stretch that induced ERS increased the expression of calnexin, the specific marker chaperone located in MAM, in cultured neonatal cardiomyocytes. Therefore, the study is designed to test the hypothesis that MAM might function as a mechanosensory microdomain that could sense and transduce the mechanical stimulation, and the dynamic structure or functional modification of MAM might impair ER function, induce ERS and finally lead to cardiac remodeling. Mechanical stretch will be applied to the cultured neonatal rat cardiomyocytes to induce hypertrophic phenotype or apoptosis, and the expression of the MAM marker proteins, as well as ERS response will be detected. Mechanical stretch will also be applied to the cultured neonatal rat cardiac fibroblasts to induce activation and pheontype transformation, and the MAM dynamic changes, as well as ERS response will also be measured. Furthermore, cardiac hypertrophy and heart failure will be induced in rats by abdominal aortic constriction. Cardiac remodeling and ERS, as well as the expression of the MAM marker proteins would then be analyzed in rats failing hearts. Taken together, this study might contribute to increase our knowledge of the mechanisms underlying ERS and the pathogenesis of cardiac remodeling，and might be beneficial to future clinical therapy for cardiac remodeling induced by pressure overload.

内质网应激(ERS)参与了压力超负荷所致心肌重塑过程，但其力学生物学调控机制尚不清楚。内质网(ER)通过线粒体结合ER膜(MAM)与线粒体相连，而MAM是细胞内动态的脂筏样结构域。我们前期实验中观察到压力负荷使心肌细胞MAM特异分子伴侣calnexin表达改变，且这种改变与ERS变化一致。在本项目中我们提出"MAM是一种新的力学感受结构域，其结构功能因力学刺激而发生的动态改变将促发ERS，并导致心肌重塑"的假设。为此，本项目拟以MAM为研究对象，用不同的方法证实MAM是新的力学感受结构域，其结构功能因力学负荷刺激而发生的动态改变将导致心肌细胞或心肌成纤维细胞促发ERS，并导致心肌重塑。同时通过大鼠腹主动脉狭窄模型，观察由压力超负荷引起心肌肥厚或心力衰竭大鼠的心肌组织中MAM是否发生改变，以证实上述假设。从而为力学超负荷所致心肌重塑的机制提出全新观点；亦可望为其临床治疗提供新的靶标。

心肌重塑是多种心血管疾病发生发展过程中共有的病理特征，但其具体机制仍未阐明。超负荷条件下的力学牵张刺激是心肌重塑重要的始动因素之一。研究显示，内质网应激(endoplasmic reticulum stress，ER stress) 参与了压力超负荷导致的心肌重塑过程。内质网(ER)通过“线粒体结合内质网膜(mitochondria-associated endoplasmic reticulum membrane, MAM)”的结构域与线粒体外膜相连。因此，本研究的主要目的是证实MAM是一种新的力学感受结构域，并探索其结构功能是否因力学刺激而发生动态改变，同时促发ER stress，并导致心肌重塑。.本项目研究证实了：(1)心肌细胞肥大过程中，MAM感受力学刺激后，其结构域特异的蛋白(如calnexin, calreticulin, Sigma-1受体，PEMT，mitofusin2等)表达发生改变，使ER形态分布改变、引发ER stress，从而导致心肌细胞发生肥大或凋亡。(2) 心肌细胞MAM感受力学刺激后，通过分离线粒体、ER及MAM成分，观察到胞浆、ER腔内、MAM及线粒体内Ca2+浓度改变； ER-线粒体间接触程度改变，导致ER稳态失衡、引发ER stress，并导致心肌细胞肥大。(3) 对培养的心肌成纤维细胞加载牵张力学刺激，可诱导其发生活化表型转变。同时，其MAM结构域中Mitofusin2、自噬调节蛋白DFCP1等表达降低，导致ER稳态改变、引发ER stress及自噬流紊乱，从而介导了心肌成纤维细胞的活化过程。(4)在压力超负荷所致的心肌肥厚大鼠心肌组织中， MAM结构域特异蛋白calnexin、calreticulin、Sigma-1受体、Mitofusin2、DFCP1、Nogo-B等表达降低，而NADPH氧化酶4(NOX4)等表达增加，同时ER stress及相关UPR通路被激活。.通过上述的研究提示：MAM结构域是新的力学感受结构域，其结构功能因力学刺激而发生的动态改变将激活心肌细胞及心肌成纤维细胞中ER stress及UPR信号通路，从而导致心肌细胞发生肥大、凋亡，心肌成纤维细胞发生增殖活化。本研究明确了MAM介导的ER stress/UPR信号参与压力超负荷诱导的心肌重塑过程，为力学负荷诱导的心肌重塑机制研究和防治策略提供了新思路。