Cardiomyocyte TRPV4 and cardiac dysfunction following ischemia-reperfusion in the aged heart.

项目摘要/摘要 高龄是冠心病和心肌梗死的主要危险因素。一个主要原因 心肌梗死后心律失常和组织损伤的主要原因是再灌注损伤(缺血-再灌注或I-R)。 当心肌细胞内钙浓度过高时，这一过程称为钙超载。我们的实验室 新近发现的瞬时受体电位香草素成员4(TRPV4)阳离子通道高度 在老年(但不年轻)心脏的心肌细胞中表达，并与细胞内钙有关 低渗应激后的超负荷。心肌组织在I-R期间发生明显的渗透性变化， 再灌流与心肌细胞的大量低渗应激有关。因此，这项提议考验着 中心假说认为，TRPV4参与了I-R后钙超载和心功能不全。为了测试 这一假设使用了年轻人(3-6个月，TRPV4低表达)和老年人(24-26个月，高表达) TRPV4表达)C576BL/6小鼠、年轻心肌细胞特异性TRPV4转基因小鼠(“过度表达”) TRPV4高表达)和老年TRPV4基因敲除小鼠(无TRPV4表达)。来补充 这种遗传方法我们还利用了TRPV4的特定拮抗剂的药理学方法。特定的 目的1验证TRPV4导致心肌细胞钙超载、过度收缩和 I-R后心律失常模拟I-R(联合代谢和渗透)对分离的心肌细胞的影响 应激)将被用来研究TRPV4对钙信号通路的影响。 共聚焦荧光显微镜。同样，编码GCaMP6f钙的转基因小鼠心脏 传感器将受到体外I-R，并在完整的器官内监测促心律失常的钙信号。 我们将利用兰登多夫灌流的心脏来测试TRPV4对I-R后心脏收缩功能和心律失常的影响。 R.特殊目的2验证了TRPV4导致心肌细胞死亡、组织损伤和 I-R后不良心脏重塑线粒体膜的荧光成像方法 电位和质膜完整性将阐明TRPV4在心肌细胞功能障碍和 模拟I-R后或体外灌流心脏I-R后实时死亡翻译研究使用 口服生物可利用的TRPV4抑制剂将用于检测TRPV4在不良心脏重塑中的作用 以及体内I-R后的组织损伤。该项目的目标是严格检查TRPV4的作用 离子通道在I-R后心功能障碍和组织损伤中的作用，其长期目标是将我们的 研究发现心肌梗塞后老年人的新治疗方法。

Project Summary/Abstract Advancing age is the primary risk factor for coronary artery disease and myocardial infarction (MI). A major cause of arrhythmia and tissue damage following MI is reperfusion injury (ischemia-reperfusion or I-R) which associates with excessive calcium concentration within cardiomyocytes, a process termed calcium overload. Our laboratory recently showed that the transient receptor potential vanilloid, member 4 (TRPV4) cation channel is highly expressed within cardiomyocytes of the aged (but not young) heart and contributes to intracellular calcium overload following hypoosmotic stress. Osmotic changes in cardiac tissue are pronounced during I-R, and reperfusion associates with substantial hypoosmotic stress on cardiomyocytes. Therefore, this proposal tests the central hypothesis that TRPV4 contributes to calcium overload and cardiac dysfunction following I-R. To test this hypothesis we utilize young (3-6 months, with low TRPV4 expression) and aged (24-26 months, with high TRPV4 expression) C576BL/6 mice, young cardiomyocyte-specific TRPV4 transgenic mice (“overexpressors” with high TRPV4 expression), and aged TRPV4 knock-out mice (with no TRPV4 expression). To complement this genetic approach we also utilize a pharmacological approach with specific antagonists of TRPV4. Specific Aim 1 tests the hypothesis that TRPV4 produces cardiomyocyte calcium overload, excessive contractility, and arrhythmia following I-R. Isolated cardiomyocytes subjected to simulated I-R (combined metabolic and osmotic stress) will be used to examine the effect of TRPV4 on calcium signaling modalities using high-resolution confocal fluorescence microscopy. Similarly, transgenic mouse hearts which encode the GCaMP6f calcium sensor will be subjected to ex vivo I-R with pro-arrhythmic calcium signaling monitored within the intact organ. Langendorff-perfused hearts will be utilized to test the role of TRPV4 on contractility and arrhythmia following I- R. Specific Aim 2 tests the hypothesis that TRPV4 contributes to cardiomyocyte death, tissue damage, and adverse cardiac remodeling following I-R. Fluorescence imaging approaches of mitochondrial membrane potential and plasma membrane integrity will elucidate the role of TRPV4 on cardiomyocyte dysfunction and death in real time following simulated I-R or in ex vivo perfused hearts following I-R. Translational studies using orally bioavailable TRPV4 inhibitors will be used to examine the role of TRPV4 in adverse cardiac remodeling and tissue damage following I-R in vivo. The goal of this project is to rigorously examine the role of the TRPV4 ion channel in cardiac dysfunction and tissue damage following I-R, with the long-term goal of translating our research findings into new treatments for aged individuals following MI.