Mitochondrial Protein HSG Is A Primary Mediator of Oxidative Apoptotic Death

程序性细胞死亡，或称细胞凋亡，对大多数器官的发育以及成人组织的动态平衡和重塑都很重要。然而，终末分化的心肌细胞的不可阻挡的丧失是心力衰竭的关键原因，心力衰竭是目前发达国家的主要死亡原因。在这里，我们证明了线粒体蛋白HSG(也称为mitofusin-2)是氧化应激介导的心肌细胞凋亡的主要决定因素。心肌梗死通过活性氧簇(ROS)依赖机制在体内显著增加内源性HSG的表达和心肌细胞的凋亡。同样，过氧化氢的氧化应激同时增加培养的大鼠心肌细胞HSG的表达和细胞的凋亡。此外，腺病毒基因转移介导的HSG过表达足以抑制基础和血清刺激的Akt激活，并触发强大的心肌细胞凋亡。HSG诱导的心肌细胞凋亡可通过抑制caspase-9而不是caspase-8，以及通过表达固有活性的PI3K突变体来激活Akt或线粒体抗凋亡蛋白Bclxl而被完全消除，这表明HSG通过抑制PI3K-Akt细胞生存信号和由此激活主要的线粒体凋亡途径来促进心肌细胞的凋亡。重要的是，siRNA介导的HSG沉默保护细胞免受氧化应激诱导的细胞凋亡。目前的结果表明，心肌HSG通过抑制细胞存活信号Akt，激活线粒体细胞死亡途径，成为心肌细胞对缺血和氧化应激反应中细胞凋亡的主要决定因素。重要的是，心肌HSG表达增加是氧化应激诱导心肌细胞凋亡的必要条件和充分条件，提示HSG失控可能是心力衰竭的一个重要致病因素和一个有前途的治疗靶点。 此外，在血管平滑肌细胞(VSMCs)中，HSG作为一种强大的细胞增殖抑制因子发挥作用(Chen等人。自然细胞生物学，6：872-883,2004)。当HSG过表达时，它会触发VSMC的凋亡(Guo等人，Circ.2007年出版)。首先，多种死亡诱导刺激，包括过氧化氢氧化应激，星形孢子素抑制PKC，Forsklin激活PKA，以及血清剥夺，同时增加MFN-2的表达并诱导VSMC凋亡。其次，HSG过表达还可诱导培养和球囊损伤大鼠颈动脉VSMCs的凋亡，从而有助于HSG介导的血管成形术后新生内膜的形成，而HSG沉默可保护VSMC免受H_2O_2或HSG过表达诱导的细胞凋亡，提示HSG上调是氧化应激介导的VSMC凋亡的必要条件和充分条件。最后，HSG的促凋亡作用不依赖于其在线粒体融合中的作用，而是通过激活线粒体的凋亡途径来实现的。HSG同时具有抗增殖和促凋亡作用，是治疗心血管增生性疾病的潜在靶点，因为VSMCs的过度生长是动脉粥样硬化和血管成形术后再狭窄的关键病因学因素。

Programmed cell death, or apoptosis, is important for the development of most organs also for adult tissue homeostasis and remodeling. However, an inexorable loss of terminally differentiated heart muscle cells is a crucial causal factor for heart failure, the current leading cause of death in developed countries. Here, we demonstrate that HSG (also named mitofusin-2), a mitochondria protein, is a major determinant of oxidative stress-mediated cardiomyocyte apoptosis. Myocardial infarction profoundly elevates endogenous HSG expression and myocyte apoptosis in vivo via a reactive oxygen species (ROS)-dependent mechanism. Similarly, oxidative stress with H2O2 concurrently increases HSG expression and apoptosis in cultured rat cardiomyocytes. Furthermore, adenoviral gene transfer-mediated overexpression of HSG is sufficient to suppress both basal and serum-stimulated Akt activation, and triggers robust cardiomyocyte apoptosis. The HSG-induced apoptosis is fully abrogated by inhibition of caspase-9 but not caspase-8, and by expression of a constitutively active PI3K mutant to activate Akt or a mitochondrial antiapoptotic protein, Bcl-xL, indicating that HSG promotes cardiomyocytes apoptosis via inhibition of the PI3K-Akt cell survival signaling and the resultant activation of the primary mitochondrial apoptotic pathway. Importantly, siRNA-mediated HSG silencing protects cells against oxidative stress-induced apoptosis. The present results indicate that cardiac HSG functions as a major determinant of heart muscle cell apoptosis in response to ischemia and oxidative stress, via inhibiting the primary cell survival signal, Akt, resulting in activation of the mitochondrial cell death pathway. Importantly, increased cardiac HSG expression is both necessary and sufficient for oxidative stress-induced heart muscle cell apoptosis, suggesting that HSG deregulation may be a crucial pathogenic element and a promising therapeutic target for heart failure. Additionally, in vascular smooth muscle cells (VSMCs), HSG functions as a powerful cell proliferation suppressor (Chen et al. Nature Cell Biology, 6:872-883, 2004). When overexpressed, HSG triggers VSMC apoptosis (Guo et al., Circ. Res. 2007, in press). First, multiple death-inducing stimuli, including oxidative stress with H2O2, inhibition of PKC with staurosporine, activation of PKA with forskolin, and serum deprivation, concurrently elevated Mfn-2 expression and induced VSMC apoptosis. Second, overexpression of HSG triggered apoptosis of VSMCs in culture and in balloon-injured rat carotid arteries as well, thus contributing to HSG-mediated prevention of neointima formation after angioplasty, while HSG silencing protected VSMCs against H2O2 or HSG overexpression induced apoptosis, indicating upregulation of HSG is necessary and sufficient for oxidative stress mediated VSMC apoptosis. Finally, HSG proapoptotic effect was independent of its role in mitochondrial fusion, but mediated by activation of the mitochondrial apoptotic pathway. The concurrent antiproliferative and proapoptotic effects mark HSG as a potential therapeutic target in treating cardiovascular proliferative disorders, because overgrowth of VSMCs is a pivotal etiological factor in the development of atherosclerosis and restenosis after angioplasty.