Molecular basis of Ca2+ leak in heart

描述（由申请人提供）：心脏细胞中钙（Ca2+）的细胞和亚细胞运动是细胞收缩和影响电行为的基础。PI和他的共同导师最近发现了心脏细胞中Ca2+运动的新特征，这对理解心脏功能具有深远的意义。在这里，PI建议通过定量数学研究与实验测试相结合来研究“看不见的Ca2+泄漏”的新发现。Ca2+泄漏是细胞内储存细胞器Ca2+的损失，并通过平衡sarco/内质网Ca2+ atp酶（SERCA）泵的摄取，在维持健康的细胞Ca2+含量方面起着至关重要的作用。了解Ca2+泄漏及其分子基础对于细胞生理学、病理生理学（包括心力衰竭和心律失常）的实验和理论研究以及开发新的治疗方法至关重要。SR Ca2+泄漏的计划调查将利用PI和他的共同导师最近发明的新颖和非常有效的数学工具，这将使单个心室肌细胞Ca2+信号的完全随机数学调查成为可能。PI和他的共同导师的工作提供了Ca2+泄漏的两个组成部分的初步数学和生物学特征：Ca2+火花（见介绍）和“看不见的”非火花Ca2+泄漏。这两种成分似乎在正常和心律失常Ca2+信号传导行为中都起作用，但尚未在分子水平上表征。提出的工作将通过将数学建模研究与单小鼠心室肌细胞实验相结合来测试这种关键的Ca2+信号行为。共聚焦Ca2+成像与同时膜片钳实验将在酶解细胞中进行，以告知建模和测试结果。Ca2+火花，[Ca2+]i瞬态和膜电流将在对照小鼠（C57BL/6）和突变小鼠的肌细胞中进行研究，这些小鼠的心脏ryanodine受体（RyR2） （C57BL/6- R2474S）产生Ca2+依赖性心律失常（见[1]）。PI和他的共同导师的初步工作表明，在控制和突变的心脏细胞中，Ca2+泄漏特征存在深刻的差异。对心脏中SR Ca2+泄漏的拟议研究旨在解决心脏Ca2+信号传导的两个至关重要的问题：1)健康肌细胞中SR Ca2+泄漏的分子机制是什么？2)致心律失常的RyR2突变如何影响SR Ca2+泄漏？所提出的工作的独特之处在于在丰富的互动环境中结合建模和实验，并在此类工作中取得了良好的成功记录。对于PI来说，这项研究很好地支持了他的长期计划，即将理论研究与实际和翔实的测试结合起来，以期扩大我们对心脏细胞功能的理解。

DESCRIPTION (provided by applicant): The cellular and subcellular movement of calcium (Ca2+) in heart cells underlies cellular contraction and influences electrical behavior. The PI and his co-mentors have recently discovered new features of Ca2+ movement in heart cells that have profound implications for understanding heart function. Here, the PI proposes to investigate the novel discovery of "invisible Ca2+ leak" by combining quantitative mathematical investigations with experimental tests. Ca2+ leak is the loss of Ca2+ from intracellular storage organelles and plays a vital role in maintaining healthy cellular Ca2+ content by balancing uptake from the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) pump. Understanding Ca2+ leak and its molecular basis is essential for experimental and theoretical examination of cellular physiology, pathophysiology (including heart failure and arrhythmias), and developing new therapeutics. The planned investigation of SR Ca2+ leak will exploit novel and very efficient mathematical tools recently invented by the PI and his co-mentors that will enable a fully stochastic mathematical investigation of the Ca2+ signaling in single cardiac ventricular myocytes. Work by the PI and his co-mentors has provided preliminary mathematical and biological characterization of two components of Ca2+ leak: Ca2+ sparks (see introduction) and "invisible," non-spark Ca2+ leak. These two components appear to play a role in both normal and arrhythmogenic Ca2+ signaling behavior but have yet to be characterized at the molecular level. The proposed work will test this critical Ca2+ signaling behavior by combining mathematical modeling investigations with single mouse ventricular myocyte experiments. Confocal Ca2+ imaging with simultaneous patch clamp experiments will be carried out in enzymatically dissociated cells to inform the modeling and test the findings. Ca2+ sparks, [Ca2+]i transients, and membrane currents will be investigated in myocytes from control mice (C57BL/6) and from mutant mice with specific alterations in the cardiac ryanodine receptor (RyR2) (C57BL/6- R2474S) that produce Ca2+-dependent arrhythmias (see [1]). Preliminary work by the PI and his co-mentors suggest that there are profound differences in the Ca2+ leak characteristics in the control and mutant heart cells. The proposed investigation into SR Ca2+ leak in heart seeks to address two critically important questions on cardiac Ca2+ signaling: 1) What is the molecular mechanism of SR Ca2+ leak in healthy myocytes? and 2) how do arrhythmogenic RyR2 mutations affect SR Ca2+ leak? The unique feature of the proposed work is the combination of modeling and experiments in a richly interactive environment with a strong record of success in such work. For the PI, the investigation nicely supports his long-term plan to combine theoretical investigations with practical and informative tests with the prospect of broadening our understanding of cardiac cellular function.