The mitochondrial calcium uniporter in sinus node physiology and disease

描述(申请人提供)：窦房结(SAN)细胞是位于右心房外侧的心脏起搏细胞，需要适当的钙离子处理才能产生有节奏的心率(HR)。众所周知，窦房结细胞依赖慢去极化IF通道、肌浆钙释放和膜离子通道对细胞内钙的调制来调节心率。线粒体钙单转运体(MCU)是新近发现的一种40 kDa的蛋白，负责钙离子内流到线粒体。我发现，用拮抗剂RU360抑制MCU，可以阻止分离的SAN细胞对肾上腺素能受体激动剂异丙肾上腺素的反应增加搏动频率。当在移液管溶液中灌流ATP时，这一结果被逆转。基于这些数据，我假设线粒体Ca~(2+)是SAN细胞对异丙肾上腺素做出反应所需的信号，抑制MCU可以阻止线粒体将能量供应与需求相匹配。窦房结功能障碍(SND)是一种临床症状，主要表现为心脏起搏器调节失调，心率减慢，心律失常和猝死的风险增加。目前，SND的唯一治疗方法是手术植入永久起搏器，每年花费20亿美元，并与许多疾病有关。我们实验室最近报道了一种新的机制，即血管紧张素II(Ang II)输注的小鼠由于SAN细胞死亡而发生SND。线粒体Ca~(2+)是调节代谢过程所必需的信号，但过量的线粒体Ca~(2+)负荷可导致线粒体细胞死亡。在这里，我将测试一个新的概念，即MCU是线粒体钙离子进入的守门人，但具有允许线粒体钙超载、细胞死亡和SND的潜力。我培育出了具有遗传MCU抑制作用的小鼠，通过心肌定界转基因表达了一种显性阴性的MCU。我将使用这些新的小鼠来测试MCU在SAN生理学和Ang-II诱导的SND中的潜在作用。据我所知，这将是第一次在心脏组织中进行MCU的活体研究。本工作的目的是确定线粒体Ca~(2+)如何参与心脏起搏，并在SND血管紧张素转换酶II输注模型中探讨MCU抑制的作用。我的研究将使用两个特定的目标来测试MCU在SAN生理和疾病中的假设作用：目的1：确定通过MCU抑制钙内流的生理效应。目的2：在体内研究MCU抑制是否影响生理心率反应和/或保护窦房结细胞死亡和SND。

DESCRIPTION (provided by applicant): Sinoatrial node (SAN) cells are cardiac pacemaker cells located in the lateral right atrium that require proper Ca2+ handling to produce a rhythmic heart rate (HR). It is well accepted that SAN cells rely on slow depolarizing If channels, sarcoplasmic Ca2+ release and modulation of intracellular Ca2+ by membrane ion channels to modulate HR. The mitochondrial calcium uniporter (MCU) is a newly identified 40 kDa protein that is responsible for Ca2+ influx into mitochondria. I found that inhibiting MCU with the antagonist drug, RU360, blocks isolated SAN cells from increasing beating frequency in response to isoproterenol, a ¿-adrenergic receptor agonist. This outcome was reversed when ATP was perfused in the pipette solution. Based on these data, I hypothesize that mitochondrial Ca2+ is a required signal for SAN cells to respond to isoproterenol and that inhibiting MCU prevents mitochondria from matching energy supply with demand. Sinus node dysfunction (SND) is a clinical condition marked by dysregulation of the primary cardiac pacemaker, slowing of the HR and increased risk of arrhythmias and sudden death. At present the only treatment for SND is surgical implantation of a permanent pacemaker, which costs 2 billion dollars annually and is associated with numerous morbidities. Our lab recently reported a novel mechanism in which angiotensin II (Ang II) infused mice developed SND due to SAN cell death. Mitochondrial Ca2+ is a required signal for regulation of metabolic processes, but excessive mitochondrial Ca2+ load can lead to mitochondrial induced cell death. Here I will test the novel concept that the MCU is a gatekeeper for mitochondrial Ca2+ entry but with the potential to permit mitochondrial Ca2+ overload, cell death and SND. I developed mice with genetic MCU inhibition, by myocardial-delimited transgenic expression of a dominant-negative MCU. I will use these new mice to test the potential role of MCU in SAN physiology and in Ang-II induced SND. To my knowledge, this will be the first in vivo study of the MCU in cardiac tissue. The goal of thi work is to determine how mitochondrial Ca2+ contributes to cardiac pacing and explore the effect of MCU inhibition in an Ang II infusion model of SND. My studies will test the hypothesized role of the MCU in SAN physiology and disease using two Specific Aims: Aim 1: Determine the physiologic effects of inhibiting Ca2+ influx through the MCU. Aim 2: Determine whether MCU inhibition affects physiological HR responses and/or protects against SAN cell death and SND in vivo.