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因子，可驱动ATP和活性氧（ROS）的产生。分离的心肌细胞中DYm的动态振荡可导致电生理振荡，其显著影响肌细胞功能并导致细胞水平的不兴奋性。然而，测量完整心脏内代谢功能的时空梯度的技术挑战已经排除了介导全局氧化应激和相关机械电功能障碍的不稳定线粒体特性的功能后果的直接调查。由于最初提交的这一建议，我们已经开发了一种半定量成像技术，用于测量DYm的时空动态与亚细胞分辨率在器官水平。在这个建议中，我们扩展我们的测量进行ROS成像，以调查如果ROS诱导的ROS释放是完整的正常和肥大的心脏内DYm不稳定的机制。以前的工作确定了线粒体苯二氮卓受体（mBZR）作为一个潜在的有吸引力的候选人，用于预防心律失常。然而，靶向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。这可能会限制这些药物的临床应用，并提出了关于DYm稳定性本身在改变机械-电气特性方面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个重要问题。这将揭示IMAC通过mBZR过表达在改变线粒体、机械和电特性中的作用。从实践的角度来看，这些研究将帮助我们确定是否mBZR表达的调制是一个可行的策略，改变机电性能。如果是这样，未来的基因沉默模仿IMAC封锁可能是必要的。 公共卫生相关性：该提案致力于：1）开发综合成像技术，以揭示心脏肥大中改变的代谢特性易导致电和收缩功能障碍的机制;和2）使用基因转移方法，通过关键受体的表达水平调节线粒体功能来调节电和收缩特性。

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. PUBLIC HEALTH RELEVANCE: This proposal is dedicated to: 1) developing integrative imaging techniques to uncover mechanisms by which altered metabolic properties in cardiac hypertrophy predispose to electrical and contractile dysfunction; and 2) using gene transfer approaches to modulate electrical and contractile properties by regulating mitochondrial function through the expression levels of a key receptor.