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的基础，并影响电行为。PI和他的同事最近发现了心脏细胞中Ca2+运动的新特征，这些特征对理解心脏功能具有深远的意义。在这里，PI建议通过将定量数学研究与实验测试相结合来研究“不可见的Ca2+泄漏”的新发现。Ca2+渗漏是细胞内储存细胞器中Ca2+的丢失，通过平衡肌浆网/内质网Ca2 + ATP酶（SERCA）泵的摄取，在维持健康细胞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）的实验和理论检查以及开发新的治疗方法至关重要。 SR Ca2+泄漏的计划研究将利用PI及其共同导师最近发明的新颖且非常有效的数学工具，这将使单个心室肌细胞中Ca2+信号传导的完全随机数学研究成为可能。PI及其合作导师的工作提供了Ca2+泄漏的两种成分的初步数学和生物学表征：Ca2+火花（见引言）和"不可见"的非火花Ca2+泄漏。这两种成分似乎在正常和致炎性Ca2+信号传导行为中发挥作用，但尚未在分子水平上表征。拟议的工作将测试这一关键的Ca2+信号行为相结合的数学建模研究与单个小鼠心室肌细胞实验。将在酶促解离的细胞中进行具有同步膜片钳实验的共焦Ca2+成像，以告知建模并测试发现。将在对照小鼠（C57 BL/6）和心脏ryanodine受体（RyR2）发生特异性改变的突变小鼠（C57 BL/6-R2474 S）（可产生Ca2+依赖性心律失常）的肌细胞中研究Ca2+火花、[Ca2 +] i瞬变和膜电流（见[1]）。PI和他的合作导师的初步工作表明，对照和突变心脏细胞中的Ca2+泄漏特征存在深刻的差异。 对心脏SR Ca 2+渗漏的研究旨在解决心脏Ca 2+信号传导中的两个关键问题：1）健康心肌细胞SR Ca 2+渗漏的分子机制是什么？以及2）促细胞凋亡RyR2突变如何影响SR Ca 2+渗漏？所提出的工作的独特之处是在一个丰富的交互式环境中结合建模和实验，在这种工作中取得了很大的成功。对于PI来说，这项研究很好地支持了他的长期计划，即将联合收割机理论研究与实际和信息丰富的测试相结合，并扩大我们对心脏细胞功能的理解的前景。

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.