Mitochondrial Calcium Signaling in Heart

心脏中的线粒体钙信号传导

• 批准号: 9102242
• 负责人: George S. B. Williams
• 金额: $ 16.92万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9102242
• 关键词: Back; Binding; Buffers; Calcium; Calcium Signaling; Cardiac; Cardiac Myocytes; Cell Death; Cell physiology; Cells; Cessation of life; Complement; Complex; Computer Simulation; Confocal Microscopy; Coupling; Development; Disease; Environment; Experimental Designs; Failure; Fluorometry; Formulation; Foundations; Functional disorder; Future; Gene Expression; Goals; Health; Heart; Heart Mitochondria; Human body; Image; Injury; Inner mitochondrial membrane; Investigation; Ischemia; Laboratories; Life; Link; Measurement; Measures; Membrane Potentials; Mentors; Methods; Mitochondria; Mitochondrial Matrix; Modeling; Molecular; Muscle Cells; Myocardial Ischemia; Oxygen; Partial Pressure; Physiological; Production; Rattus; Reperfusion Injury; Reperfusion Therapy; Research; Resources; Role; Series; Signal Transduction; Speed; System; Techniques; Technology; Testing; Theoretical model; Therapeutic; Time; Training; Ventricular; Work; blood pump; career; cell injury; heart cell; heart function; improved; in vivo; innovation; insight; mathematical model; millisecond; mitochondrial permeability transition pore; novel; novel therapeutics; prevent; protective effect; research study; sensor; simulation; temporal measurement; therapeutic development; tool

 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.

 描述（由申请人提供）：“心脏中的线粒体钙信号传导”PI：乔治S。B。威廉姆斯。 心脏依靠线粒体来提供大量的能量需求，这些能量需求与泵送血液到全身有关。线粒体基质中的钙（Ca 2+）影响几乎所有主要的线粒体功能（包括能量产生），并与心肌缺血-再灌注（IR）损伤期间的不可逆细胞损伤有关。尽管如此重要，线粒体Ca 2+（[Ca 2 +]）的水平和动态仍然知之甚少，仍然存在争议。PI和他的导师W. J. Lederer，沿着其他人最近对线粒体的重要发现（见背景），使线粒体Ca 2+信号的K25定量研究成为可能。初步实验表明，PI和他的导师可以测量[Ca 2 +]动态使用遗传编码，神经靶向Ca 2+传感器。该提案结合了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的进步：1）用[Ca 2 +]m指示剂体内转导心脏，使得能够同时测量新鲜分离的心肌细胞中的胞质[Ca 2 +]（[Ca 2 +]）i和[Ca 2 +]m; 2）停流荧光测定法，其提供准确的，高时间分辨率在分离的心肌线粒体中在生理[Ca 2 +]i水平下的线粒体Ca 2+通量的测量（毫秒）; 3）能够测量分离的线粒体中[Ca 2 +]m的实时缓冲的技术; 4）在活的分离的单个心肌细胞被成像时快速控制并同时测量其微环境中的氧分压的方法; 5）具有真实的[Ca 2 +]i和[Ca 2 +]m动力学和通量的计算模型，其使得能够更深入地研究心脏中线粒体和钙之间的复杂关系，并反过来为实验方法提供信息。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，这些实验和模拟将对心脏中的线粒体Ca 2+信号产生新的见解。该建议旨在研究[Ca 2 +]m在生理和病理生理条件下的动态。PI假设虽然线粒体Ca 2+通量在心脏中可能很小（威廉姆斯等人，PNAS 2013），线粒体仍然积累Ca 2+，并且在病理生理条件下升高的[Ca 2 +]m可能有助于IR损伤。为了研究这一假设，PI将寻求回答三个至关重要的问题：1）心肌线粒体内膜的Ca 2+通量有多大？2)健康的单个心室心肌细胞内[Ca 2 +]m的动态是什么？（3）[Ca ~（2+）]m水平升高是否与心肌IR损伤有关？通过回答前两个问题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。实时观察mPTP转换之前的[Ca 2 +]m水平对于获得IR损伤的新见解至关重要。PI具有挑衅性的新工具和技术，将首次允许比较IR损伤期间的[Ca 2 +]m动态与心脏正常条件下的[Ca 2 +] m动态。所提出的工作的一个额外的独特功能是并行实验和计算建模的结合。计算建模是重要的，在这里作为一种手段，以确认复杂的实验观察的解释。这是特别相关的，因为线粒体，特别是[Ca 2 +]m，是出了名的难以实验研究。此外，计算模型将为小的、可能在实验上不可见的[Ca 2 +]m瞬变提供定量测量，该瞬变必须与起搏期间线粒体的已知Ca 2+积累相关。通过使用一组集中的实验测试以及一个约束良好的计算模型来研究[Ca 2 +]m的动力学，这项工作将提供更多关于[Ca 2 +]m如何促进细胞生理学和病理生理学的见解，而不是单独使用任何一种方法。因此，拟议的工作应该为未来的调查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Ca 2+信号实验室之一，te Lederer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导师正在进行的研究（心脏Ca 2+信号传导），同时将其扩展到一个新的方向（IR损伤）。然而，最重要的是，拟议的调查支持PI的长期职业目标相结合的新的和定量的实验研究与理论建模，以扩大我们的心脏分子和细胞生理学的理解。