Cellular Biology of Cardiac Sodium-Calcium Exchange

描述（由申请人提供）：心脏肌膜钠钙交换器（NCX）是从肌细胞中排出钙的主要机制。因此，交换器是细胞内Ca和心肌收缩力的重要调节剂。本次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水平的遗传改变具有意想不到的效果，其告知了该领域关于心脏细胞中EC偶联的新方面。这三个建议项目如下：1。心房特异性NCX敲除小鼠的窦房结活动。我们最近产生了新的心房特异性NCX敲除（KO）小鼠，这些小鼠活到成年，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，包括细胞电生理学和亚细胞Ca运动的变化。这些实验将解决心脏起搏器产生理论的根本争议，并可能对该领域产生巨大影响。2.心房兴奋-收缩耦合对遗传改变的NCX水平的适应。我们将检验以下假设：当心房NCX被敲除时，心房Ca调节和EC偶联发生关键性适应。由于心房肌细胞缺乏与心室细胞的横小管相关的空间限制，我们可以检验NCX调控EC偶联依赖于空间梯度和细胞微区的假设。 这些研究将为我们提供重要的见解，调节心房收缩力，可能有重要的治疗意义。3.心室兴奋-收缩偶联对遗传改变的NCX水平的适应。我们已经培育出了心室特异性NCX KO小鼠，这些小鼠可以存活到成年期。 来自这些小鼠的心室肌细胞通过减少通过L型Ca通道的Ca进入而耐受NCX消融，但通过增加EC偶联增益而不增加肌浆网Ca负荷来维持收缩性。我们已经提出，增益增加，因为缺乏NCX升高二进制分裂钙，和升高的分裂钙增加EC耦合保真度。这表明，裂缝钙操纵NCX可用作替代-激动剂作为一种变力性策略，避免细胞钙超载及其有害后果。在这一目标的实验将填补关键的空白，我们了解的机制，通过NCX消融提高EC耦合增益，而不会引起钙超载和细胞死亡。这些知识是必不可少的，这样我们就可以学习如何操纵NCX和裂缝钙作为治疗目标的患者与心力衰竭和缺血性心功能不全。

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.