Cellular Biology of Cardiac Sodium-Calcium Exchange

描述(申请人提供)：心肌细胞膜Na-Ca交换器(NCX)是从心肌细胞中排出钙的主要机制。因此，交换器是细胞内钙和心肌收缩能力的重要调节器。这一更新的持续长期目标是进一步了解Ncx在兴奋收缩(EC)偶联和起搏器活动中的意义。NCX水平上的遗传变化产生了意想不到的影响，使该领域了解到心肌细胞中EC偶联的新方面。提出的三个项目如下：1.心房特异性NCX基因敲除小鼠的窦房结活动。我们最近培育出了新的心房特异性NCX基因敲除(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。在完整的体外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内钙运动的变化。这些实验将解决心脏起搏器生成理论中的根本争议，并可能对该领域产生巨大影响。2.心房兴奋收缩偶联对基因改变的Ncx的适应。我们将验证这一假设，即当心房Ncx被敲除时，心房钙调节和EC偶联发生关键的适应。由于心房肌细胞缺乏与心室细胞横管相关的空间限制，我们可以检验NCX对EC偶联的调节依赖于空间梯度和细胞微域的假设。这些研究将为我们提供对心房收缩调节的重要见解，这可能具有重要的治疗意义。3.心肌兴奋收缩偶联对Ncx基因改变的适应性。我们已经培育出了能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通过L型钙通道的钙内流来耐受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。我们提出了增益的增加是因为缺乏NCX提高了二分裂隙钙，而升高的裂隙钙增加了EC的耦合保真度。这表明，NCX对细胞内钙离子的操纵可以作为一种肌力调节策略来替代-激动剂，从而避免细胞内钙超载及其有害后果。这一目标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偶联增益的机制的关键空白。这些知识是必不可少的，这样我们就可以学习如何在心力衰竭和缺血性心功能不全患者中操作NCX和裂解钙作为治疗靶点。

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.