ADP: A Master Regulator for Bioenergetics and Ca2+/ROS Signaling in Heart

描述（由申请人提供）：心力衰竭是一个严重的医疗问题：在美国有超过500万人受到影响。慢性心力衰竭的原因是多方面的，包括生物能量缺乏，Ca2+超载和氧化应激。ADP是ATP形成的关键底物。此外，ADP是线粒体通透性过渡孔（mPTP）开放的有效抑制剂。尽管mPTP的分子身份仍未得到解决，但有两个不同的概念脱颖而出。一项研究表明mPTP不需要腺嘌呤核苷酸转位酶（ANT），而另一项研究则将mPTP描述为一个多蛋白复合物，ANT和线粒体肽酰脯氨酸顺式反式异构酶即亲环蛋白d （Cyp-D）是关键成分。有趣的是，ADP与ANT结合有助于心磷脂稳定呼吸链超复合体，从而提高ATP生成的效率。心磷脂的氧化破坏了这些超复合物的稳定性，并与衰老、糖尿病性心肌病、缺血再灌注损伤和心力衰竭相关的线粒体功能障碍有关。此外，ADP抑制无机磷酸盐Pi的外排，无机磷酸盐Pi是形成Ca2+-Pi复合物的关键线粒体Ca2+缓冲液。最后，线粒体分裂蛋白DLP1的激活导致线粒体分裂、ROS生成和mPTP打开。综上所述，这些结果使我们假设ADP与ANT的结合在线粒体功能中起着两个基本作用：通过稳定心磷脂- etc复合物来提高ATP的生成效率，通过减少ROS的生成和DLP1的激活来抑制mPTP。生理上，ADP介导的mPTP抑制最大限度地减少线粒体ROS的过量产生和线粒体Ca2+的释放，以优化兴奋-收缩-代谢（ECM）耦合的有效性。病理上，这种ADP调节机制的缺陷导致能量衰竭、氧化应激和Ca2+失调，从而增加心脏对损伤的易感性”。我们提出了两个具体目的：具体目的1：确定ADP抑制mPTP的机制。假设：ADP稳定心磷脂完整性，抑制线粒体内DLP1活性，从而防止cypd非依赖性mPTP打开。此外，ADP通过抑制mPTP减少ROS的产生，从而使ROS对mPTP的反馈激活最小化。专项目的2：评价ADP在心脏保护中的作用。假设：患病模型心脏的mPTP对Ca2+诱导的开放表现出更高的敏感性，这是由于其对首次撞击应激的易感性，包括氧化应激、高DLP1活性和/或Ca2+缓冲能力降低。维持最佳矩阵ADP水平可以减轻这种增加的mPTP脆弱性。代谢信号和Ca2+/氧化还原/细胞死亡信号之间相互作用的干扰是包括心脏病在内的疾病发病机制的基础。这一应用的探索性和高风险想法，如果得到验证，将在广泛的疾病机制和治疗方面开辟新天地。

DESCRIPTION (provided by applicant): Heart failure is a serious medical problem: over 5 million people in the US affected. The cause for chronic heart failure is multifaceted and includes bioenergetic deficiency, Ca2+ overload, and oxidative stress. ADP is the key substrate for ATP formation. Moreover, ADP is a potent inhibitor for the opening of mitochondrial permeability transition pore (mPTP). Although the molecular identity of mPTP is still unsolved, 2 different concepts stand out. One indicates that adenine nucleotide translocase (ANT) is not required for mPTP, whereas the other depicts mPTP as a multi-protein complex, the ANT and the mitochondrial peptidyl-prolyl cis-trans isomerase known as cyclophilin-D (Cyp-D), are the key components. Interestingly, binding of ADP to ANT facilitates cardiolipin to stabilize respiratory chain supercomplexes so that the efficiency of ATP generation is enhanced. Oxidation of cardiolipin destabilizes these supercomplexes and has been linked to mitochondrial dysfunction associated with aging, diabetic cardiomyopathy, ischemia-reperfusion injury, and heart failure. Furthermore, ADP inhibits efflux of inorganic phosphate Pi, the key mitochondrial Ca2+ buffer that forms Ca2+-Pi complex. Finally, activation of the mitochondrial fission protein, DLP1 causes mitochondrial fission, ROS generation, and mPTP opening. Taken together, these results lead us to hypothesize that "binding of ADP to ANT serves two fundamental roles in mitochondrial function: enhancing ATP generation efficiency by stabilizing cardiolipin-ETC complexes and inhibiting mPTP by decreasing ROS generation and DLP1 activation. Physiologically, ADP- mediated mPTP inhibition minimizes excessive mitochondrial ROS generation and Ca2+ release from mitochondria in order to optimize the effectiveness of excitation-contraction-metabolism (ECM) coupling. Pathologically, defects of this ADP regulatory mechanism lead to energetic failure, oxidative stress, and Ca2+ dysregulation that enhance cardiac vulnerability to injury". We propose 2 Specific Aims: Specific Aim 1: To determine the mechanisms for ADP inhibition of mPTP. Hypothesis: ADP stabilizes cardiolipin integrity and inhibits DLP1 activity in the mitochondria and thus protects against Cyp-D-independent mPTP opening. Moreover, ADP decreases ROS generation via mPTP inhibition, and thus minimizes feedback activation of mPTP by ROS. Specific Aim 2: To assess the role of ADP in cardiac protection. Hypothesis: The mPTP in hearts of diseased models exhibits increased sensitivity to Ca2+-induced opening due to its predisposition to the first hit stresses including oxidative stress, high DLP1 activity, and/or diminished Ca2+ buffering capacity. Maintenance of optimal matrix ADP levels alleviates this increased mPTP vulnerability. Disturbances in the interaction between metabolic signaling and Ca2+/redox/cell death signaling are fundamental in disease pathogenesis including cardiac diseases. The exploratory and high-risk ideas in this application, if validated, will break new ground in a wide spectrum of disease mechanisms and treatments.