Novel Tools to Probe Trafficking and Function of Calcium Channel Signaling Complexes in Heart

SUMMARY Heart failure (HF) affects >6 million people in the United States and accounts for 400,000 deaths each year. 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) is the dominant pathway for Ca2+ entry into CMs and initiates excitation-contraction coupling, regulates action potential duration, and controls other essential Ca2+-dependent. 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 cardiac pathophysiology; and that posttranslationally regulating CaV1.2 functional expression is a prospective 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. We have developed several novel tools to bridge this gap including; a transgenic mouse expressing a YFP-fused pore-forming α1C subunit with an extracellular epitope tag in the heart, and engineered nanobodies that permit bi-directional regulation of CaV1.2 functional expression. We combine these tools with two unique resources - a catalogue of proteins located in CaV1.2 nanodomains in heart defined using a proximity-labeling proteomics assay, and the Pakistan Genome Resource, which contains gene sequence and extensive phenotype data from >80,000 individuals with high rates of consanguinity that result in a prevalence of genetic mutations including heterozygous null and missense mutations in α1C. 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 nanobodies that increase functional expression of CaV1.2 and evaluate their efficacy in preventing progression to HF after myocardial infarction. 3) Assess functional impact of α1C loss-of-function mutations in humans.