TRPV4 and Calcium Dependent Ventricular Arrhythmia Following Ischemia-Reperfusion

项目概要/摘要： 室性心律失常是心肌梗死（MI）后死亡的主要原因，发病率高 在老龄化人口中。我们实验室的最新数据表明， 受体电位香草酸4（TRPV 4）阳离子通道增加心室心肌细胞中的表达， 年龄增长。缺血再灌注（I/R）与细胞内钙超载和低渗应激有关。 心肌Ca 2+超负荷通过增加心肌细胞内Ca 2+浓度，干扰兴奋-收缩偶联，促进心律失常 舒张期Ca 2+释放和膜去极化。因此，该提议检验了低渗的假设， I/R期间的应激激活TRPV 4介导的Ca 2+内流，这有助于Ca 2+过载， 胚胎发生具体目的1是研究TRPV 4在离体心肌细胞Ca 2+内流中的作用， 膜电位和低渗胁迫后的Ca 2+稳态。膜片钳记录 将在来自老龄小鼠（24-26个月， 高TRPV 4表达）、年轻小鼠（3-6个月，具有低TRPV 4表达）和具有可诱导的 转基因心脏特异性TRPV 4过表达（年轻TRPV 4过表达者）。分离的心肌细胞 来自相同组，但具有额外的GCaMP 6 f Ca 2+传感器转基因的小鼠也将经受 低渗应激和细胞内Ca 2+稳态将使用激光扫描共聚焦显微镜进行评估。 在存在和不存在TRPV 4拮抗作用（HC 067047，1 M）的情况下，在显微镜下观察。具体目标二是 研究TRPV 4在Langendorff灌注的离体心脏I/R中的促凋亡作用。 左前降支（LAD）的可逆性结扎将诱导老年、年轻和年轻TRPV的I/R 4 心脏过度表达。来自LAD供应心肌的心肌细胞的锐利微电极记录将 在存在和不存在TRPV 4拮抗作用的情况下，在I/R之前、期间和之后服用。相同的 方案将在Langendorff灌注的Young和Young TRPV 4过表达的离体心脏上进行 (with GCaMP 6 f Ca 2+传感器），同时通过激光扫描共聚焦成像左心室游离壁 显微镜这种原位成像将允许测量心脏中致心律失常的舒张期Ca 2+释放事件。 完整的心脏该项目研究了一种新的治疗靶点，用于预防钙依赖性 老年人群心肌梗死后的室性心律失常此外，该提案还规定， 对申请人进行心血管研究的宝贵培训。

Project Summary/Abstract: Ventricular Arrhythmia is a leading cause of death following Myocardial Infarction (MI), with high incidence among aging populations. Recent data from our laboratory show that the osmotically activated Transient Receptor Potential Vanilloid 4 (TRPV4) cation channel increases expression in ventricular cardiomyocytes with advancing age. Ischemia-Reperfusion (I/R) is associated with both Ca2+ overload and hypoosmotic stress on the myocardium. Ca2+ overload disturbs excitation-contraction coupling and promotes arrhythmia by increasing diastolic Ca2+ release and membrane depolarization. Thus, this proposal tests the hypothesis that hypoosmotic stress during I/R activates TRPV4-mediated Ca2+ influx, which contributes to Ca2+ overload and arrhythmogenesis. Specific Aim 1 is to investigate the role of TRPV4 in isolated cardiomyocyte Ca2+ influx, membrane potential, and Ca2+ homeostasis following hypoosmotic stress. Patch clamp recording of membrane potential and membrane Ca2+ currents will be measured in cardiomyocytes from Aged mice (24-26 month, with high TRPV4 expression), Young mice (3-6 month, with low TRPV4 expression), and Young mice with inducible transgenic cardiac-specific TRPV4 overexpression (Young TRPV4 Overexpressors). Isolated cardiomyocytes from the same groups, but with an additional GCaMP6f Ca2+ sensor transgene𝜇𝜇will also be subjected to hypoosmotic stress and intracellular Ca2+ homeostasis will be assessed using laser scanning confocal microscopy in the presence and absence of TRPV4 antagonism (HC067047, 1 M). Specific Aim 2 is to investigate the arrhythmogenic role of TRPV4 in Langendorff-perfused isolated hearts subjected to I/R. Reversible ligation of the left anterior descending artery (LAD) will induce I/R in Aged, Young, and Young TRPV4 Overexpressor hearts. Sharp microelectrode recordings of cardiomyocytes from LAD-supplied myocardium will be taken prior to, during, and following I/R in the presence and absence of TRPV4 antagonism. The same protocol will be conducted on Langendorff perfused Young and Young TRPV4 Overexpressor isolated hearts (with the GCaMP6f Ca2+ sensor) while imaging of the left-ventricular free wall by laser scanning confocal microscopy. This in situ imaging will allow for measurement of arrhythmogenic diastolic Ca2+ release events in the intact heart during I/R. This project investigates a novel therapeutic target for preventing Ca2+-dependent ventricular arrhythmias following myocardial infarction in aging populations. Additionally, the proposal provides valuable training to the applicant in conducting cardiovascular research.