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.