Thursday, December 26, 2024
12/26/2024
Project Summary Experiments outlined in this application, suggest a novel paradigm, in which we add a new layer of complexity to the current understanding of β-adrenergic receptor (βAR)-mediated regulation of CaV1.2 channels and EC- coupling. We propose that stimulation of βARs initiates dynamic augmention of CaV1.2 channel abundance, enhanced cooperative gating of CaV1.2 channels, and de novo couplon formation in the sarcolemma of young ventricular myocytes, to amplify Ca2+ influx into these cells and tune EC-coupling in times of high demand. Our preliminary data suggest that a pre-synthesized pool of sub-sarcolemmal, CaV1.2 channels-containing vesicles/endosomes, resides in cardiomyocytes and can be mobilized to the sarcolemma in times of high metabolic or hemodynamic demand. We hypothesize that insertion of these ‘new’ channels into the sarcolemma occurs in a PKA- and CaMKII-mediated phosphyorylation dependent fashion, while subsequent internalization of the mobile pool when demand decreases, occurs in a CaN-mediated dephosphorylation dependent manner. Further preliminary data suggests that the BAR-domain containing protein BIN1, choreographs this response, with roles in microtubule and actin mediated CaV1.2 targeting to the sarcolemma and in trafficking channels out of early endosomes and recycling them back to the cell surface. Strikingly, this dynamic regulatory process is absent in ventricular myocytes isolated from aged mice where we find cardiac BIN1 protein levels are almost doubled. Increased expression of BIN1 in the brain with aging is associated with Alzheimer’s Disease and defects in intracellular trafficking termed ‘endosomal traffic jams’. We hypothesize that increased levels of BIN1 with aging, could cause analogous endosomal traffic jams in ventricular myocytes, leading to enhanced basal CaV1.2 expression at the sarcolemma and depletion of the readily insertable pool of channels. We propose a model in which BIN1 acts as a hub for CaV1.2 channel delivery to the sarcolemma in ventricular myocytes, and suggest that altered distribution and activity of CaV1.2 channels, and reduced responsivity to βAR stimulation with aging is mediated by changes in BIN1 expression. Specific Aim 1 tests the hypothesis that βAR activation stimulates age-dependent dynamic augmentation of sarcolemmal CaV1.2 channel abundance and clustering. Specific Aim 2 tests the hypothesis that changes in BIN1 expression underlie the altered dynamics and retention of CaV1.2 channels with aging. Finally, specific Aim 3 tests the hypothesis that age-related differences in CaV1.2 channel dynamics and trafficking in ventricular myocytes leads to impaired EC-coupling during the ‘fight or flight’ response. To achieve these aims, we employ a multi-faceted approach using state-of-the-art methods and analyses including super-resolution imaging, patch clamp electrophysiology, TIRF and confocal imaging
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