Mitochondrial Calcium Signaling in Heart

 描述(申请人提供)：“线粒体钙信号在心脏”PI：乔治S.B.威廉姆斯。摘要：心脏依靠线粒体来满足与将血液输送到全身相关的巨大能量需求。线粒体基质中的钙离子影响线粒体的几乎所有主要功能(包括能量产生)，并与心肌缺血再灌注损伤中不可逆转的细胞损伤有关。尽管有如此重要的意义，但线粒体钙离子的水平和动态仍然知之甚少，并且仍然存在争议。第一次，PI和他的导师W.J.莱德尔博士开发的创新方法，以及其他人最近的关键线粒体发现(见背景)，使拟议的K25线粒体钙信号定量研究成为可能。初步实验表明，PI和他的导师可以使用遗传编码的线粒体目标钙传感器动态测量[钙]+。这一建议结合了PI开发或增强的五种关键工具来研究[Ca~(2+)]_m。这些包括技术和方法上的进步：1)用[Ca~(2+)]_m指示剂在体内转导心脏，使得能够同时测量新鲜分离的心肌细胞的胞浆[Ca~(2+)]_i([Ca~(2+)])_i和[Ca~(2+)]_m；2)停流荧光法提供准确的、高时间分辨率(毫秒)的线粒体在生理[Ca~(2+)]_i水平下的线粒体Ca~(2+)通量的测量；3)能够实时测量分离的线粒体中[Ca~(2+)]_m的缓冲；4)快速控制和同时测量单个活的单个心肌细胞成像时微环境中氧分压的方法；5)具有真实的[Ca~(2+)]i和[Ca~(2+)]m动力学和通量的计算模型，使更深入地研究心肌线粒体和钙之间的复杂关系，进而为实验方法提供指导。正是这些工具的独特组合使PI能够进行一系列具有挑战性的实验和计算模拟，这些实验和计算模拟将对心脏中的线粒体钙信号产生新的见解。本研究旨在研究生理和病理生理条件下细胞内钙离子浓度的动态变化。PI假设，虽然心肌中线粒体的钙流量可能很小(Williams等人，PNAS 2013)，但线粒体仍然积累钙，在病理生理条件下，升高的[钙]m可能有助于IR损伤。为了研究这一假说，PI将试图回答三个至关重要的问题：1)跨心肌线粒体内膜的钙流量有多大？2)健康单个心室肌细胞内的[钙]m动力学是什么？以及3)[钙]m水平的升高是否有助于心肌IR损伤？回答前两个问题所获得的理解对于解释与第三个问题有关的结果至关重要。线粒体死亡，通过不可逆的线粒体通透性转换孔(MPTP)开口，与IR损伤相关的大量细胞死亡有关。实时观察MPTP转变前的[Ca~(2+)]_m水平对于获得对IR损伤的新认识至关重要。PI具有挑衅性的新工具，这项技术将首次允许将IR损伤期间的[Ca~(2+)]m动力学与心脏正常条件下的[Ca~(2+)]m动力学进行比较。拟议工作的另一个独特之处是并行实验和计算建模的结合。在这里，作为确认对复杂实验观测的解释的一种手段，计算建模具有重要意义。这一点尤其相关，因为众所周知，线粒体，特别是[Ca~(2+)]_m，很难通过实验进行研究。此外，计算模型将提供对微小的、实验上可能看不见的[Ca~(2+)]_m瞬变的定量测量，这些瞬变肯定与已知的线粒体在起搏期间积累Ca~(2+)有关。通过使用一组有针对性的实验测试和一个受良好约束的计算模型来研究[Ca~(2+)]m的动力学，这项工作将提供比这两种方法单独实现的更多关于[Ca~(2+)]m如何对细胞生理学和病理生理学做出贡献的见解。因此，拟议的工作应为今后的研究和治疗方法的发展奠定坚实的基础。对于PI来说，这项研究在世界上最具创新性的钙信号实验室之一--TE Leder实验室提供了令人兴奋的最先进的培训。事实上，PI提出的工作很好地补充了Mentor正在进行的研究(心脏钙信号)，同时将其扩展到一个新的方向(IR损伤)。然而，最重要的是，拟议的研究支持了PI的长期职业目标，即将新颖和定量的实验研究与理论建模相结合，以拓宽我们对心脏分子和细胞生理学的理解。

 DESCRIPTION (provided by applicant): "Mitochondrial Calcium Signaling in Heart" PI: George S. B. Williams. Summary: The heart relies on mitochondria to fuel the massive energy demand associated with pumping blood throughout the body. Calcium (Ca2+) in the mitochondrial matrix influences nearly every major mitochondrial function (including energy production) and is linked to irreversible cell damage during myocardial ischemia-reperfusion (IR) injury. Despite such significance, the level and dynamics of mitochondrial Ca2+ ([Ca2+]) are still poorly understood and remain controversial. For the first time, innovative methods developed by the PI and his mentor Dr. W. J. Lederer, along with key recent mitochondrial discoveries by others (see Background), enable the proposed K25 quantitative investigation of mitochondrial Ca2+ signaling. Preliminary experiments show that the PI and his mentor can measure [Ca2+] dynamically using genetically encoded, mitochondrially targeted Ca2+ sensors. This proposal combines five critical tools developed or enhanced by the PI to investigate [Ca2+]m. These include both technical and methodological advancements: 1) In vivo transduction of the heart with a [Ca2+]m indicator, enabling the simultaneous measurement cytosolic [Ca2+] ([Ca2+])i and [Ca2+]m in freshly isolated cardiomyocytes; 2) Stopped-flow fluorometry that provides accurate, high temporal resolution (millisecond) measurement of mitochondrial Ca2+ fluxes under physiological [Ca2+]i levels in isolated cardiac mitochondria; 3) Technique capable of measuring real-time buffering of [Ca2+]m in isolated mitochondria; 4) Method to rapidly control and simultaneously measure the partial pressure of oxygen in the microenvironment of a living isolated single cardiomyocyte while it is being imaged; 5) A computational model with realistic [Ca2+]i and [Ca2+]m dynamics and fluxes that enables a deeper investigation of the complex relationship between mitochondria and calcium in heart, and in turn informs the experimental approaches. It is the unique combination of these tools that enables the PI to carry out the proposed set of challenging experiments and computational simulations that will yield new insights into mitochondrial Ca2+ signaling in heart. This proposal seeks to investigate [Ca2+]m dynamics during physiological and pathophysiological conditions. The PI hypothesizes that while mitochondrial Ca2+ fluxes are likely small in heart (Williams et al., PNAS 2013), mitochondria still accumulate Ca2+ and under pathophysiological conditions elevated [Ca2+]m may contribute to IR injury. To investigate this hypothesis, the PI will seek to answer three critically important questions: 1) How large are Ca2+ fluxes across the inner mitochondrial membrane of a cardiac mitochondrion?; 2) What are [Ca2+]m dynamics within a healthy single ventricular cardiomyocyte?; and 3) Do elevations in [Ca2+]m levels contribute to cardiac IR injury? The understanding gained by answering the first two questions will be critical to interpreting the results related to the third question. Mitochondrial death, via irreversible mitochondrial permeability transition pore (mPTP) openings, is linked to the vast cell death associated with IR injury. The real-time observation of [Ca2+]m levels that precede the mPTP transitions is critical to gaining new insights into IR injury. The PI has provocative new tools an techniques that will for the first time allow the comparison of [Ca2+]m dynamics during IR injury with [Ca2+]m dynamics under normal conditions in heart. An additional unique feature of the proposed work is the combination of parallel experiments and computational modeling. Computational modeling is significant here as a means to confirm the interpretation of complex experimental observations. This is particularly relevant as mitochondria, especially [Ca2+]m, are notoriously difficult to investigate experimentally. Furthermore, the computational model will provide quantitative measures for the small, likely experimentally invisible, [Ca2+]m transients that must be associated with the known accumulation of Ca2+ by mitochondria during pacing. By investigating the dynamics of [Ca2+]m using a set of focused experimental tests alongside a well-constrained computational model, this work will provide more insights into how [Ca2+]m contributes to cellular physiology and pathophysiology than either approach could achieve alone. The proposed work should thus establish a robust foundation for future investigations and the development of therapeutic approaches. For the PI, this investigation provides exciting state-of-the-art training in one of the most innovative Ca2+ signaling laboratories in the world, te Lederer laboratory. In fact, the proposed work by the PI complements nicely with the ongoing research by the mentor (cardiac Ca2+ signaling) while extending it in a new direction (IR injury). Most importantly, however, the proposed investigation supports the PI's long-term career goal of combining novel and quantitative experimental investigations with theoretical modeling to broaden our understanding of cardiac molecular and cellular physiology.