We recently demonstrated that the sensory nerve neuropeptide substance P (SP) is required for the development of cardiac fibrosis in response to elevations in myocardial stress, specifically pressure overload (PO). Due to its localization to sensory nerves projecting to coronary arteries, SP is likely one of the first mediators released in response to changes in coronary pressure/flow. We believe that this places SP up- stream of multiple pro-fibrotic cascades, and we have identified several cell-specific effects initiated by SP in response to PO. Therefore, targeting SP holds real therapeutic potential, however, dissection of these cell- specific pathways is firstly required. What makes SP an even more attractive therapeutic target is the existence of two isoforms of its receptor, the neurokinin-1 receptor (NK-1R). We believe that the full length NK-1R mediates the physiological actions of SP, while the truncated isoform mediates the pro-fibrotic effects. Thus, it may be possible to selectively target the adverse effects of SP, while leaving the physiological actions intact. This proposal will examine the role of these NK-1R isoforms in mediating the cell-specific actions of SP on multiple pathways involved in cardiac fibrosis: 1) focal cardiomyocyte necrosis and the subsequent macrophage response; 2) SP/endothelin-1 interactions at the level of the cardiac fibroblast; and 3) mast cell- specific proteases. Our overall hypothesis is that SP acts via the truncated NK-1R to modulate cell-specific molecular pathways to promote cardiac fibrosis and diastolic dysfunction. Specific aim 1 will examine the extent to which SP activation of the NK-1R on sympathetic nerves initiates focal cardiomyocyte necrosis, a known stimulus for fibrosis. Further, the contribution to fibrosis of SP activated macrophages in response to focal necrosis will also be examined. Specific aim 2 will examine the synergistic actions of SP and endothelin-1 on cardiac fibroblast phenotype and function. This includes investigating truncated NK-1R activation of the membrane type 1 matrix metalloproteinase/TGF-β1 pathway in vitro. Myofibroblast-specific NK-1R-deficient mice will be used to examine the role of myofibroblast-specific NK-1Rs in vivo under conditions of PO. Specific aim 3 will identify the importance of mast cell-specific NK-1Rs in mediating the release of mast cell-derived pro-fibrotic molecules including tryptase and chymase. This proposal is significant and innovative because it will establish a neuropeptide as a mediator of cardiac fibrosis and diastolic dysfunction, as well as identifying cell-specific mechanisms by which SP promotes fibrosis. Of high significance is the existence of the two NK-1R isoforms, of which we believe the truncated isoform mediates the pro-fibrotic actions of SP. Thus, this proposal will identify a unique, actionable therapeutic target for diastolic dysfunction since antagonists, biologics, or gene therapy approaches specific to the truncate...