Integrative Investigation of Mitochondrial Dysfunction

描述（由申请人提供）：线粒体膜电位（DYm）是线粒体功能的关键调节器，驱动ATP和活性氧（ROS）的产生。在分离的心肌细胞中DYm的动态振荡可导致电生理振荡，显著影响心肌细胞功能并导致细胞水平上的不可兴奋性。然而，在完整心脏内测量代谢功能时空梯度的技术挑战阻碍了对线粒体不稳定特性在介导全局氧化应激和相关机电功能障碍中的功能后果的直接研究。自最初提交该提案以来，我们已经开发了一种半定量成像技术，用于在器官水平上以亚细胞分辨率测量DYm的时空动态。在本提案中，我们将测量扩展到进行ROS成像，以研究ROS诱导的ROS释放是否是完整正常和肥厚心脏中DYm不稳定的机制。先前的工作确定了线粒体苯二氮卓受体（mBZR）作为预防心律失常的潜在有吸引力的候选者。然而，针对mBzR的药物干预通过直接抑制l型钙电流显著影响收缩和钙处理特性。这可能限制了这些药物的临床应用，并提出了DYm稳定性本身与改变机械-电气特性的直接相关性的重要问题。我们将通过使用靶向mBZR表达的基因转移方法直接解决这一重要问题。这将揭示IMAC通过mBZR过表达改变线粒体、机械和电学特性的作用。从实际的角度来看，这些研究将帮助我们确定mBZR表达的调节是否是改变机械-电气特性的可行策略。如果是这样，未来的基因沉默模拟IMAC阻断可能是合理的。

DESCRIPTION (provided by applicant): The mitochondrial membrane potential (DYm) is a key regulator of mitochondrial function that drives the production of ATP and reactive oxygen species (ROS). Dynamic oscillations of DYm in isolated cardiac myocytes can result in electrophysiological oscillations that significantly impact myocyte function and lead to inexcitability at the cellular level. However, technical challenges in measuring spatio-temporal gradients in metabolic function within the intact heart have precluded a direct investigation of the functional consequences of unstable mitochondrial properties in mediating global oxidative stress and associated mechano-electrical dysfunction. Since the original submission of this proposal, we have developed a semi-quantitative imaging technique for measuring the spatio-temporal dynamics of DYm with subcellular resolution at the organ level. In this proposal, we extend our measurements to perform ROS imaging in order to investigate if ROS-induced ROS-release is a mechanism of DYm instability within the intact normal and hypertrophied heart. Previous work identified the mitochondrial benzodiazepine receptor (mBZR) as a potentially attractive candidate for preventing arrhythmias. However, pharmacological interventions that target mBzR significantly impact contractile and calcium handling properties by directly suppressing the L-type calcium current. This may limit the clinical utility of these agents and raises important questions regarding the direct relevance of DYm stability per se in altering mechano-electrical properties. We will directly address this important issue by using a gene transfer approach that targets mBZR expression. This will uncover the role of IMAC through mBZR overexpression in altering mitochondrial, mechanical, and electrical properties. From a practical perspective, these studies will help us determine if modulation of mBZR expression is a viable strategy for altering mechano-electrical properties. If so, future gene silencing to mimic IMAC blockade may be warranted.