ER stress: role in myocyte apoptosis and myocardial remodeling

描述(由申请人提供)： 心力衰竭是退伍军人发病和死亡的主要原因。交感神经活动增强是心力衰竭患者的中心特征。细胞凋亡导致的心肌细胞丢失在心力衰竭的发展过程中起着重要作用。刺激2-肾上腺素能受体(2-AR)可增加体外和体内心肌细胞的凋亡。刺激22-AR和抑制G蛋白(GI)可对抗2-AR刺激的细胞凋亡。最近，我们有证据表明，糖原合成酶激酶-32(GSK-32)的激活通过参与线粒体死亡途径，在2-AR诱导的细胞凋亡中起促凋亡作用。然而，在心肌细胞中，参与GSK-32激活和线粒体死亡途径的分子信号在很大程度上是未知的。内质网(心肌细胞中的内质网或肌浆网)是蛋白质折叠、钙存储和钙信号的主要场所。错误折叠蛋白的堆积和钙稳态的改变所引起的内质网应激可触发细胞凋亡。我们最近的初步数据表明，2-AR刺激在体外和体内都能在成人心肌细胞中诱导ER应激，表现在GRP-78(ER应激感受器)和Gadd153(ER应激诱导的转录因子)的表达增加，以及caspase-12(在ER应激诱导的细胞凋亡中起核心作用的蛋白酶)的激活。心肌梗死后心肌GRP-78和Gadd153表达增加，caspase-12活化。抑制GSK-32可抑制2-AR刺激的Gadd153水平的升高。众所周知的药理内质网应激源(布雷菲尔丁A、塔普西尔金和衣霉素)激活GSK-32并增加心肌细胞的凋亡。已知的保护细胞免受内质网应激的Salubrine，在体外抑制了2-AR刺激的心肌细胞凋亡的增加。小鼠输注/治疗丹参素可降低2-AR刺激和MI诱导的左心功能不全和心肌细胞凋亡的程度。这些观察结果导致了我们的中心假设，即2-AR刺激的内质网应激诱导激活GSK-32，GSK-32的激活通过增加内质网应激并参与线粒体死亡途径在2-AR刺激的细胞凋亡中起促凋亡作用。对特定目标1的研究主要集中在了解导致内质网应激和心肌细胞凋亡的近端信号通路。特定目的2将使用Gadd153基因敲除小鼠在体内研究内质网应激在2-AR刺激和MI后细胞凋亡和心肌重塑中的作用。具体目标3将使用药理学、腺病毒和siRNA策略来确定内质网应激在GSK-32激活中的作用，并深入了解激活的GSK-32增强内质网应激和激活线粒体死亡途径的机制。特异性目的4将利用心脏特异性过表达显性阴性GSK-32的转基因小鼠，研究GSK-32在2-AR刺激和MI后心肌细胞凋亡和心肌重塑中的作用。研究内质网应激在心肌细胞凋亡和心肌重塑中的作用可能为治疗心力衰竭提供新的治疗策略。 公共卫生相关性： 心力衰竭是退伍军人发病和死亡的主要原因。众所周知，交感神经活动增加会对心脏造成不良影响。最近的研究表明，2-肾上腺素能受体(2-AR)阻滞剂治疗可以提高慢性心力衰竭患者的存活率并减少住院时间。这为心力衰竭的治疗增加了2-AR阻滞剂。尽管存活率有所提高，但疾病过程仍然是影响慢性心脏病患者发病率和死亡率的主要因素。该项目的目标是了解心力衰竭期间交感神经活动增加所引发的细胞内信号。因此，研究内质网应激在2-AR刺激和心肌梗死后心肌细胞凋亡和心肌重构中的作用可能为心力衰竭的治疗提供新的治疗策略。

DESCRIPTION (provided by applicant): Heart failure represents a major cause of morbidity and mortality among veterans. Increased sympathetic activity is a central feature in patients with heart failure. Cardiac myocyte loss due to apoptosis plays an important role in the progression of heart failure. Stimulation of 2-adrenergic receptor (2-AR) increases apoptosis in cardiac myocytes in vitro and in vivo. Stimulation of 22-AR and inhibitory G-protein (Gi) protects against 2-AR-stimulated apoptosis. Recently, we provided evidence that activation of glycogen synthase kinase-32 (GSK-32) plays a pro-apoptotic role in 2-AR- stimulated apoptosis via the involvement of mitochondrial death pathway. However, molecular signals involved in the activation of GSK-32 and mitochondrial death pathway of apoptosis are largely unknown in cardiac myocytes. The endoplasmic reticulum (ER or sarcoplasmic reticulum in cardiac myocytes) is a principal site for protein folding, calcium storage and calcium signaling. ER stress induced by accumulation of misfolded proteins and alterations in calcium homeostasis can trigger apoptosis. Our recent preliminary data demonstrate that 2-AR stimulation induces ER-stress in vitro in adult cardiac myocytes and in vivo in the heart as evidenced by increased expression of GRP-78 (sensor of ER stress) and Gadd153 (a transcription factor induced by ER stress), and activation of caspase-12( a protease which plays a central role in ER stress-induced apoptosis). Increased expression of GRP-78 and Gadd153 and activation of caspase-12 was also observed in the heart following myocardial infarction (MI). Inhibition of GSK-32 inhibited 2-AR-stimulated increases in Gadd153 levels. Well-known pharmacological ER stressors (brefeldin A, thapsigargin and tunicamycin) activated GSK-32 and increased cardiac myocyte apoptosis. Salubrinal, known to protect cells from ER stress, inhibited 2-AR-stimulated increases in cardiac myocyte apoptosis in vitro. Infusion/treatment of mice with salubrinal reduced the extent of 2-AR-stimulated and MI-induced left ventricular dysfunction and cardiac myocyte apoptosis in the heart. These observations have led to our central hypothesis that 2-AR-stimulated induction of ER stress activates GSK-32, and activation of GSK-32 plays a pro-apoptotic role in 2-AR-stimulated apoptosis by augmenting ER stress and involving mitochondrial death pathway. Studies of Specific Aim 1 are focused on understanding the proximal signaling pathway leading to ER stress and cardiac myocyte apoptosis. Specific Aim 2 will use Gadd153 knockout mice to investigate the in vivo role of ER stress in apoptosis and myocardial remodeling following 2-AR stimulation and MI. Specific aim 3 will use pharmacologic, adenoviral and siRNA strategies to define the role of ER stress in the activation of GSK-32, and get an insight into the mechanism by which active GSK-32 augments ER stress and activates mitochondrial death pathway. Specific aim 4 will use transgenic mice with cardiac-specific overexpression of dominant negative GSK-32 to investigate the role of GSK-32 in cardiac myocyte apoptosis and myocardial remodeling following 2-AR stimulation and MI. The proposed studies investigating the role of ER stress in cardiac myocyte apoptosis and myocardial remodeling may uncover novel therapeutic strategies for the treatment of heart failure. PUBLIC HEALTH RELEVANCE: Heart failure represents a major cause of morbidity and mortality among veterans. Increased sympathetic activity is known to cause adverse effects in the heart. Recent studies have shown that 2-adrenergic receptor (2-AR) blocker therapy improves survival and reduces hospitalizations in patients with chronic heart failure. This has added 2-AR blockers to the therapy of heart failure. Despite the improved survival, the disease process is still pre-eminent in affecting the morbidity and mortality of patients with chronic heart disease. The objectives of this project are directed towards understanding the intracellular signals initiated by increased sympathetic activity during heart failure. Thus, the proposed studies investigating the role of endoplasmic reticulum stress in cardiac myocyte apoptosis and myocardial remodeling following 2-AR stimulation and myocardial infarction may uncover novel therapeutic strategies for the treatment of heart failure.