Functional role of IP3 receptors in the regulation of cardiac myofibroblasts

心血管疾病仍然是美国死亡的首要原因，心肌梗死(MI)和随后的 心力衰竭是这种致命的主要后遗症。最近的研究强化了这样一个概念： 心脏成纤维细胞(FBS)比心肌细胞研究得更少，远不止是简单的 细胞外基质周转的调节者；事实上，这些细胞(及其激活的表型，myoFBs)似乎 成为心力衰竭进展过程中的关键人物。尽管对心肌纤维化的研究相对较少，但它仍然是 缺血性心脏病的主要病理生理特征。三磷酸肌醇受体(IP3Rs) 细胞内钙释放通道是否位于主要的钙库--内质网(ER) 在细胞内。虽然IP3Rs在心肌细胞中的作用已经被研究过，特别是在 肥厚和心律失常的背景下，它们在心脏FBS激活中的确切功能 到目前为止，缺血性侮辱还没有被研究过。人和小鼠FBS表达IP3Rs的三种亚型 它们的活动和水平受到几种刺激的调节。我们假设IP3R在 在梗死心脏中心脏FBS的激活，在本方案中，我们将在体内验证这一假说，例如 活体和体外。我们有初步数据显示，IP3R在心脏中的表达(所有异构体)都增加了 心肌梗死后空腹血糖。此外，全基因组关联研究显示，IP3- 介导的信号转导通路与缺血性心脏病。然而，权威性的功能研究 在活体内研究IP3Rs在心肌纤维化中的机制作用尚不清楚。使用Cre/Lox重组 技术，我们建立了一种新的小鼠模型(IP3RKO)，在该模型中，IP3R在激活的心肌肌纤维中被消融。 这一模型为评估IP3R在心脏纤维化中的功能贡献提供了一个精巧的工具，并允许 我们需要克服在单个IP3R基因被敲除或KO后遇到的困难。我们的预赛 研究表明，在心肌梗死后，IP3RKO小鼠表现出显著的减少纤维化和减轻心肌梗死 功能障碍与对照IP3RCre和IP3RFLOX窝产仔比较。我们将进一步评估功能 IP3Rs与不同时间点和不同时间段心脏FBS心肌纤维化的关系 对既定刺激的反应，以评估它们的增殖、迁移、分泌和收缩 容量。此外，我们还将研究IP3R在小鼠胚胎FBS和人类心脏中的反应。 FBS。为了描述观察到的表型背后的分子机制，我们建议探索 钙介导的途径有：线粒体功能障碍、自噬、内质网应激和炎症。这个 拟议的研究具有非常重要的意义和创新性，因为它们将：(A)对 使用特定的CRE/LOX KO模型，IP3R在心肌梗死后心脏空腹血糖中的功能作用；(B)先前描述 在心肌纤维化的病理生理学中，IP3R和自噬之间的未知联系；(C)确定 创新的治疗策略，特别针对过度的缺血后心肌纤维化。

Cardiovascular disorders remain the first cause of mortality in US, with myocardial infarction (MI) and subsequent heart failure as the major sequela underlying such lethality. Recent studies have reinforced the concept that cardiac fibroblasts (FBs), which are less investigated than cardiomyocytes, are much more than simple regulators of extracellular matrix turnover; indeed, these cells (and their activated phenotype, myoFBs) seem to be key players in heart failure progression. Although relatively understudied, myocardial fibrosis remains one of the major pathophysiologic features of ischemic cardiac disorders. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular calcium release channels located on the endoplasmic reticulum (ER), the main calcium reservoir within the cell. While the role of IP3Rs in cardiomyocytes has been previously investigated, especially in the context of hypertrophy and arrhythmias, their exact function in the activation of cardiac FBs following an ischemic insult has not been explored hitherto. Human and murine FBs express all three isoforms of IP3Rs and their activity and levels are modulated by several stimuli. We hypothesize that IP3Rs play a key role in the activation of cardiac FBs in the infarcted heart and in the present proposal we will test this hypothesis in vivo, ex vivo, and in vitro. We have preliminary data showing that IP3R expression (all isoforms) is increased in cardiac FBs following MI. Moreover, genome-wide association studies revealed a significant association between IP3- mediated signal transduction pathways and ischemic heart disease. However, definitive functional studies examining the mechanistic role of IP3Rs in cardiac fibrosis in vivo are missing. Using a Cre/lox recombination technique, we generated a novel mouse model (IP3RKO) in which IP3Rs are ablated in activated cardiac myoFBs. This model provides an exquisite tool to evaluate the functional contribution of IP3R to cardiac fibrosis and allows us to overcome the difficulties encountered following the knockdown or KO of a single IP3R gene. Our preliminary studies show that following MI, IP3RKO mice display a significantly reduced fibrosis and attenuated myocardial dysfunction compared with control IP3RCre and IP3Rflox littermates. We will further assess the functional relationships between IP3Rs and cardiac fibrosis in primary isolated cardiac FBs at different time points and in response to established stimuli, in order to assess their proliferative, migratory, secretory, and contractile capacity. Additionally, we will examine IP3R-mediated responses in murine embryonic FBs and human cardiac FBs. To delineate the molecular mechanisms underlying the observed phenotype, we propose to explore the following calcium-mediated pathways: mitochondrial dysfunction, autophagy, ER stress, and inflammation. The proposed studies are highly significant and innovative as they will: (a) provide the first assessment of the functional role of IP3R in post-MI cardiac FBs using a specific Cre/lox KO model; (b) delineate previously unrecognized connections between IP3R and autophagy in the pathophysiology of cardiac fibrosis; (c) identify innovative therapeutic strategies that specifically target excessive post-ischemic cardiac fibrosis.