Regulation of Coronary Blood Flow

项目总结 这个项目的目标是更好地理解新陈代谢偶联的机制。 通过改变流向心脏的冠脉血流量，心肌氧需求和氧供应之间的关系。 冠状动脉血管平滑肌氧化还原敏感型电压门控性钾通道(即Kv1.x) 已知对于增加心肌血流量(MBF)是必不可少的 心脏负荷。CVSM中KV1通道活性如何响应来自 活跃的心肌是未知的。在这里，我们提出了KV1通道代谢调节的重要作用 胞质辅助Kvβ亚基的四聚体组装的活性，这些亚基是醛酮类的成员 还原酶(AKR)超家族和结合氧化和还原的吡啶核苷酸(如NAD(H))与高 亲和力。我们的初步数据与全球假设一致，即NAD(H)和NAD(H)之间的相互作用 Kvβ1和Kvβ2可精确控制局部血流量与心脏需氧量之间的耦合。 在目标1中，我们将描述冠状动脉kvβ亚单位在调节代谢充血中的作用。 回应。为此，我们将使用无创性心肌声学造影(MCE)来测量MBF 麻醉WT和Kvβ缺失动物给药后心脏负荷的变化 去甲肾上腺素(NE)。CVSM中Kvβ1和Kvβ2在代谢性充血中的细胞特异性贡献将是 在双转基因动物中可诱导Kvβ在Null中的特异性表达 背景动物。体外电生理学和肌图学将测试Kvβ在改变 KV1的激活、失活和代谢性血管扩张分别具有电压依赖性。在目标2中，我们 将阐明Kvβ介导的代谢偶联的机制。我们将利用电生理学来测量， Kvβ亚基在特定操作中对KV1活性调节的相对功能贡献 改变CVSM中NADH/NAD氧化还原比的细胞代谢。我们将量化这些变化 使用基因编码的荧光生物传感器和MALDI-MS成像在CVSM中进行NADH/NAD氧化还原，以及 Kvβ催化NAD(H)周转在冠脉血管扩张调节中的作用 增强MBF。在目标3中，我们将阐明Kvβ在运动心血管适应中的作用 条件反射。为了做到这一点，我们将让WT和转基因动物接受强制跑步机跑步 测量MBF适应性、冠脉血管扩张能力、Kv激活和 停用属性。这一目标还将解决生理性心肌梗死的总体依赖性 运动能力对冠脉Kvβ依赖性MBF变化的适应和增强。

PROJECT SUMMARY The goal of this project is to gain a better understanding of mechanisms that underlie metabolic coupling between myocardial oxygen demand and oxygen supply via changes in coronary blood flow to the heart. Redox-sensitive voltage-gated potassium channels (i.e., Kv1.x) in coronary vascular smooth muscle (CVSM) are known to be essential to the enhancement of myocardial blood flow (MBF) in response to increased cardiac workload. How Kv1 channel activity is enhanced in CVSM in response to metabolic signals from an active myocardium is unknown. Here, we propose an essential role for metabolic regulation of Kv1 channel activity by the tetrameric assembly of cytosolic auxiliary Kvβ subunits, which are members of the aldo-keto reductase (AKR) superfamily and bind oxidized and reduced pyridine nucleotides (e.g., NAD(H)) with high affinity. Our preliminary data are consistent with the global hypothesis that interactions between NAD(H) and Kvβ1 and Kvβ2 impart precise control over the coupling between regional MBF with cardiac oxygen demand. In Aim 1, we will delineate the role of coronary Kvβ subunits in the regulation of the metabolic hyperemia response. To do this, we will use non-invasive myocardial contrast echocardiography (MCE) to measure MBF as a function of cardiac workload in anesthetized WT and Kvβ-null animals upon administration of norepinephrine (NE). The cell-specific contribution of Kvβ1 and Kvβ2 in CVSM to metabolic hyperemia will be examined in double transgenic animals with inducible smooth muscle-specific expression of Kvβ in null background animals. In vitro electrophysiology and myography will test the relative roles for Kvβ in altering the voltage-dependence of Kv1 activation and inactivation and metabolic vasodilation, respectively. In Aim 2, we will elucidate the mechanism of Kvβ-mediated metabolic coupling. We will measure, using electrophysiology, the relative functional contribution of Kvβ subunits to regulation of Kv1 activity upon specific manipulations in cellular metabolism that alter the redox ratio of NADH:NAD+ in CVSM. We will quantify changes in NADH/NAD+ redox in CVSM using genetically-encoded fluorescent biosensors and MALDI-MS imaging, and determine the role of NAD(H) turnover by Kvβ catalysis in regulation of coronary vasodilation and enhancement of MBF. In Aim 3, we will clarify the role of Kvβ in cardiovascular adaptation to exercise conditioning. To do this, we will subject WT and genetically-modified animals to a forced treadmill running exercise protocol before measuring adaptations in MBF, coronary vasodilatory capacity, and Kv activation and inactivation properties. This aim will also address the overall dependence of physiological myocardial adaptations and enhancement of exercise capacity on coronary Kvβ-dependent changes in MBF.