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Henry M. Colecraft

Heart disease remains the leading cause of death in the United States, a trend likely to worsen with the inexorable increase in aging of the population. Deepened fundamental insights into molecular mechanisms that underlie normal cardiac physiology, and how their dysregulation contributes to heart disease is essential for identifying new drug targets and developing new therapeutics to combat heart disease. Ca2+ cycling is indispensable for heart function, and derangement of cardiomyocyte (CM) Ca2+ signaling is a prominent hallmark of heart disease. The L-type Ca2+ channel (CaV1.2) as the dominant pathway for Ca2+ entry initiates excitation-contraction coupling, regulates action potential duration, and controls other essential Ca2+-dependent processes in CMs. The overall hypotheses motivating this proposal are: that there is spatial and functional heterogeneity of CaV1.2 in heart cells that is critical for normal cardiac physiology; that derangement of CaV1.2 molecular and functional organization caused by chronic stress or genetic mutations contributes prominently to human cardiac pathophysiology; and that regulating CaV1.2 functional expression is a promising approach for treating heart diseases. There is foundational, but preliminary, evidence in the literature to support these hypotheses. However, the capacity to build on these initial observations to forge a more complete understanding of the molecular and functional heterogeneity of CaV1.2 signaling complexes in heart cells in physiology and disease is hampered by a dearth of methods to visualize and selectively manipulate such distinctive Ca2+ signaling complexes. Our preliminary data show we have successfully developed novel tools to break this impasse: a transgenic mouse inducibly expressing a yellow fluorescent protein (YFP)-fused dihydropyridine (DHP)-resistant CaV1.2 pore-forming α1C subunit with an extracellular epitope tag in the heart; a knock-in mouse expressing DHP-resistant α1C without a YFP tag; nanobodies (Nbs) to CaV1.2 auxiliary β subunits; and engineered Nbs that permit bi-directional regulation of CaV1.2 functional expression. We combine these tools with two unique resources central to this program project grant (PPG) application - a catalogue of proteins located in CaV1.2 nanodomains in heart defined using proximity proteomics (Project 2); and the Pakistan Genome Resource (PGR) (Core A), which contains gene sequencing and extensive phenotype data from >80,000 individuals with high rates of consanguinity enabling identification of genetic mutations, including heterozygous null (loss of function) and missense mutations in numerous proteins including α1C. Preliminary bioinformatics analyses of the PGR database suggests individuals with α1C haploinsufficiency have a higher propensity for heart failure (HF). Together, these provide a unique opportunity to probe molecular and functional heterogeneity of CaV1.2 macromolecular complexes in heart, contribution of their derangement to cardiac pathophysiology in humans, and their utility as druggable targets for heart disease. We propose three Aims:

  1. Develop novel tools to probe dynamic trafficking, organization, and regulation of CaV1.2 signaling complexes in live CMs in physiology and disease

  2. Develop engineered Nbs that increase functional expression of CaV1.2 and evaluate their efficacy in preventing progression to HF

  3. Assess functional impact of α1C loss-of-function mutations in humans

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