Delineate the Pathophysiological Effect of Cardiomyocyte-specific Mitochondrial Stress

总结/摘要 心力衰竭（HF）在美国仍然是主要的健康问题和显著的死亡率。虽然缺陷， 线粒体功能与HF的病理生理学密切相关，没有靶向治疗 到目前为止，在临床上已经取得了成功，这表明我们仍然没有足够的了解， 线粒体功能障碍和HF发展的相关机制。正常的线粒体功能依赖于 维持内膜电位（Inner membrane potential，简称Em）。在心肌细胞（CM）中，心肌细胞的扰动不仅直接 影响ATP的产生，但也影响各种信号通路，调节氧化还原平衡，Ca 2 + 稳态和线粒体质量控制。然而，在何种程度上，以及如何损失CM的净利润 损害心脏能量学、收缩性和电活动（即，HF的特征）仍然不清楚。这 部分是由于缺乏在体内有效和选择性地操纵CM线粒体的方法， 设置.现有的去甲肾上腺素药理学方法缺乏细胞（CM与非CM）和器官 （心脏与非心脏）特异性。为了克服这一技术障碍，我们开发了一种创新的 肿瘤靶向发光遗传学（称为mLumOpto）技术，并证明它可以诱导 在体外和体内，动态的，选择性的CM去极化，用双发射的 细胞内生物发光照明。该提案的主要目标是利用mLumOpto技术 在完整心脏中直接诱导CM特异性心室肌去极化，并描绘急性（即<24小时）和 慢性（即2周）对心脏功能的影响。我们的假设是，仅CM特异性去极化即可 足以诱导不良心脏重塑和HF发展，将采用以下方法进行检测 具体目标。特异性目的1将充分表征孤立成人中mLumOpto介导的视网膜去极化 CMs（ACMs）体外，并建立剂量依赖性。mLumOpto介导的去极化 急性和慢性影响ACM功能和健康，分别将被解剖。具体目标2将 确定完整心脏中CM特异性去极化的急性效应。首先，mLumOpto介导的体内 将测定CM特异性去极化率。然后，观察了CM-100对心肌去极化的急性效应， 将在体外和体内检查完整心脏的代谢、泵功能和电生理学。 具体目标3将描述中度CM去极化对心脏重塑的慢性影响（在 重量分析、组织学、生物化学、电生理学和分子水平），导致收缩性 功能障碍和HF发展。还将研究CM去极化诱导HF的可逆性。 这项研究的成功完成不仅将导致一个创新的技术，为动态控制的CM 线粒体在自由移动的动物，但也有新的发现，将导致我们的新途径， 开发和转化未来的脑血管靶向HF治疗。

SUMMARY/ABSTRACT Heart failure (HF) remains a major health problem and significant mortality in the United States. While defects in mitochondrial function are strongly implicated in the pathophysiology of HF, no mitochondrial-targeted therapy has been successful in the clinic to date, indicating that we still do not possess sufficient understanding of the mechanisms connecting mitochondrial dysfunction and HF development. Normal mitochondrial functions rely on maintaining the inner membrane potential (m). In the cardiomyocyte (CM), m perturbations not only directly affect ATP production, but also influence a variety of signaling pathways that modulate redox balance, Ca2+ homeostasis, and mitochondrial quality control. However, to what extent and how the loss of CM m alone impairs cardiac energetics, contractility, and electrical activity (i.e., hallmarks of HF) remains poorly defined. This is due, in part, to a lack of methods for effectively and selectively manipulating CM mitochondria in the in vivo setting. Existing pharmacological approaches to depolarize m lack both cell (CM versus non-CM) and organ (cardiac versus non-cardiac) specificity. To overcome this technical barrier, we developed an innovative mitochondrial-targeted luminoptogenetic (termed mLumOpto) technology and demonstrated that it can induce dynamic, selective CM m depolarization both in vitro and in vivo, with luciferase-luciferin pair-emitted intracellular bioluminescent illumination. The primary goal of this proposal is to utilize the mLumOpto technology to directly induce CM-specific m depolarization in intact hearts, and delineate the acute (i.e. <24 hours) and chronic (i.e. 2 weeks) impacts on cardiac functions. Our hypothesis is that CM-specific m depolarization alone is sufficient to induce adverse cardiac remodeling and HF development, which will be tested with the following Specific Aims. Specific Aim 1 will fully characterize mLumOpto-mediated m depolarization in isolated adult CMs (ACMs) in vitro, and establish luciferin dose dependency. How the mLumOpto-mediated m depolarization acutely and chronically impacts ACM function and health, respectively, will be dissected. Specific Aim 2 will determine the acute effect of CM-specific m depolarization in intact hearts. First, mLumOpto-mediated in vivo CM-specific m depolarization will be determined. Then, the acute effects of CM m depolarization on cardiac metabolism, pump function, and electrophysiology in intact hearts will be examined both ex vivo and in vivo. Specific Aim 3 will delineate the chronic effects of moderate CM m depolarization on cardiac remodeling (at gravimetric, histologic, biochemical, electrophysiological, and molecular levels) that lead to contractile dysfunction and HF development. The reversibility of CM m depolarization-induced HF will also be examined. Successful completion of this research will lead to not only an innovative technology for dynamic control of CM mitochondria in freely-moving animals, but also new findings that will lead us to novel avenues for the development and translation of future mitochondrial-targeted HF therapies.