Delineate the Pathophysiological Effect of Cardiomyocyte-specific Mitochondrial Stress

摘要/摘要 在美国，心力衰竭(HF)仍然是一个主要的健康问题和显著的死亡率。而缺陷存在于 线粒体功能与心衰的病理生理密切相关，没有线粒体靶向治疗 到目前为止在临床上都是成功的，这表明我们对 线粒体功能障碍与心力衰竭发生的机制。正常的线粒体功能依赖于 维持内膜电位(m)。在心肌细胞(CM)中，m的扰动不仅直接 影响ATP的产生，但也影响多种信号通路，调节氧化还原平衡，钙离子 动态平衡和线粒体质量控制。然而，CM的损失在多大程度上以及如何单独 损害心脏的能量、收缩能力和电活动(即，心衰的特征)仍然不清楚。这 部分原因是缺乏有效和选择性地在体内操纵CM线粒体的方法 布景。现有的去极化的药理学方法既缺乏细胞(CM与非CM)，也缺乏器官 (心脏与非心脏的)特异性。为了克服这一技术障碍，我们开发了一种创新的 线粒体靶向光遗传技术(称为mLumOpto)，并证明它可以诱导 用荧光素酶-荧光素对发射的体内外动态、选择性CMm去极化 细胞内生物发光照明。该提案的主要目标是利用mLumOpto技术 直接在完整心脏诱导CM特异性m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CM特异的m单独去极化 足以引起不利的心脏重构和心力衰竭的发展，这将用以下方法进行测试 明确的目标。特异性目标1将充分描述mLumOpto介导的m去极化的特征 CMS(ACMS)体外培养，并建立荧光素剂量依赖关系。MLumOpto介导的m如何去极化 急性和慢性影响ACM的功能和健康，将分别被解剖。特定目标2将 测定CM特异性m去极化对完整心脏的急性效应。第一，mLumOpto在体内的介导 将确定特定于CM的m去极化。然后，CMm去极化对心脏的急性影响 将在体外和体内检测完整心脏的新陈代谢、泵功能和电生理。 具体目标3将描绘适度的CMm去极化对心脏重构的慢性影响(在 重量、组织学、生化、电生理和分子水平)导致收缩 功能障碍和心力衰竭的发展。还将检验CMm去极化引起的HF的可逆性。 这项研究的成功完成不仅将为CM的动态控制带来一项创新技术 自由移动的动物的线粒体，但也有新的发现，将把我们带到新的途径 未来线粒体靶向心力衰竭疗法的开发和翻译。

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.