Cellular Biology of Cardiac Sodium-Calcium Exchange

描述（由申请人提供）：心脏肌层钠钙交换器（NCX）是从肌细胞中挤出钙的主要机制。因此，交换剂是细胞内钙离子和心脏收缩性的重要调节剂。这项更新的持续长期目标是进一步了解NCX在兴奋收缩（EC）耦合和起搏器活动中的意义。NCX水平的遗传改变产生了意想不到的影响，这为心脏细胞中EC偶联的新方面提供了信息。拟建的三个项目如下：心房特异性NCX敲除小鼠窦房结活性。我们最近生产了一种新的心房特异性NCX敲除（KO）小鼠，它们可以活到成年，但表现出严重的心动过缓和窦房结（SAN）功能障碍。在完整的离体SAN制剂和分离的SAN细胞中，我们将研究这些适应的潜在机制，包括细胞电生理和亚细胞钙运动的变化。这些实验将解决心脏起搏器生成理论中的基本争议，并可能对该领域产生巨大影响。2. 心房兴奋-收缩偶联对基因改变的NCX水平的适应。我们将验证心房NCX被敲除时心房Ca调节和EC偶联发生关键适应的假设。由于心房肌细胞缺乏与心室细胞横向小管相关的空间约束，我们可以验证NCX对EC偶联的调节取决于空间梯度和细胞微域的假设。这些研究将为我们提供关于心房收缩性调节的重要见解，这可能具有重要的治疗意义。3. 心室兴奋-收缩耦合对基因改变的NCX水平的适应。我们已经培育出能够存活到成年的脑室特异性NCX KO小鼠。这些小鼠的心室肌细胞通过减少l型钙通道的钙进入来耐受NCX的消融，但通过增加EC偶联增益而不增加肌浆网钙负荷来维持收缩性。我们提出，增益增加是因为NCX的缺乏提高了双向间隙Ca，而升高的间隙Ca增加了EC耦合的保真度。这表明NCX操纵裂隙钙可以作为一种替代-激动剂的肌力增强策略，避免细胞钙超载及其有害后果。本目的实验将填补我们对NCX消融在不引起钙超载和细胞死亡的情况下提高EC偶联增益的机制的理解中的关键空白。这一知识是必不可少的，因此我们可以学习如何操纵NCX和裂Ca作为心力衰竭和缺血性心功能障碍患者的治疗靶点。

DESCRIPTION (provided by applicant): The cardiac sarcolemmal Na-Ca exchanger (NCX) is the primary mechanism for the extrusion of Ca from myocytes. As such, the exchanger is an important regulator of intracellular Ca and cardiac contractility. The continuing long-term objective of this renewal is to further understand the significance of NCX in excitation contraction (EC) coupling and pacemaker activity. Genetic alterations in the level of NCX have had unexpected effects that have informed the field about novel aspects of EC coupling in cardiac cells. The three proposed projects are as follows: 1. Sinoatrial Node Activity in Atrial-Specific NCX Knockout Mice. We have recently produced novel atrial-specific NCX knockout (KO) mice that live into adulthood, but exhibit profound bradycardia and sinaotrial node (SAN) dysfunction. In both intact ex vivo SAN preparations and in isolated SAN cells, we will study the underlying mechanism for these adaptations, including changes in cellular electrophysiology and subcellular Ca movements. These experiments will resolve fundamental disputes in cardiac pacemaker generation theory and may have an enormous impact on the field. 2. Adaptations of Atrial Excitation-Contraction Coupling to Genetically Altered Levels of NCX. We will test the hypothesis that critical adaptations in atrial Ca regulation and EC coupling occur when atrial NCX is knocked out. Since atrial myocytes lack the spatial constraints associated with transverse-tubules of ventricular cells, we can test the hypothesis that regulation of EC coupling by NCX depends on spatial gradients and cellular microdomains. These studies will provide us with important insights into regulation of contractility in the atrium that may have important therapeutic implications. 3. Adaptations of Ventricular Excitation-Contraction Coupling to Genetically Altered Levels of NCX. We have produced ventricular-specific NCX KO mice that survive into adulthood. Ventricular myocytes from these mice tolerate ablation of NCX by reducing Ca entry through L-type Ca channels, yet maintain contractility by increasing EC coupling gain without increasing sarcoplasmic reticulum Ca load. We have proposed that gain increases because lack of NCX elevates diadic cleft Ca, and that elevated cleft Ca increases EC coupling fidelity. This suggests that cleft Ca manipulation by NCX could be used as an alternative to -agonists as an inotropic strategy that avoids cellular Ca overload and its deleterious consequences. The experiments in this aim will fill critical gaps in our understanding of the mechanism through which NCX ablation raises EC coupling gain without provoking Ca overload and cell death. This knowledge is essential so that we can learn how to manipulate NCX and cleft Ca as therapeutic targets in patients with heart failure and ischemic cardiac dysfunction.