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Mitochondrial Calcium Signaling in Heart

心脏中的线粒体钙信号传导

基本信息

批准号：
8967676
负责人：
George S. B. Williams
金额：
$ 13.24万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
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/8967676
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 Drug Formulations Environment Experimental Designs Failure Fluorometry Foundations Functional disorder Future Gene Expression Goals 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 Solutions 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 public health relevance research study sensor simulation temporal measurement therapeutic development tool

项目摘要

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.