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

总结 心力衰竭（HF）影响美国> 600万人，并且每年造成40万人死亡。 加深了对正常心脏生理学基础的分子机制的基本见解，以及如何 它们的失调会导致心脏病，这对于确定新的药物靶点和开发新的 治疗心脏病的药物Ca 2+循环对于心脏功能是不可或缺的， 心肌细胞（CM）Ca 2+信号传导是心脏病的显著标志。L型钙通道（CaV1.2） 是Ca 2+进入CM的主要途径，并启动兴奋-收缩偶联，调节作用 电位持续时间，并控制其他必需的Ca 2+依赖性。激发这一提议的总体假设 CaV1.2在心脏细胞中存在空间和功能异质性，这对正常心脏至关重要。 生理学;慢性应激或应激引起CaV1.2分子和功能组织紊乱 基因突变对心脏病理生理学有显著影响; CaV1.2的功能性表达是治疗心脏疾病的一种有前景的方法。有基础，但 初步，文献中的证据来支持这些假设。然而，在这些初步基础上发展的能力 观察，以更全面地了解CaV1.2的分子和功能异质性 心脏细胞中的信号复合物在生理学和疾病中的作用受到缺乏可视化方法的阻碍 并选择性地操纵这种独特的Ca 2+信号复合物。我们开发了几种新的工具， 弥合这一差距，包括：一个转基因小鼠表达YFP融合的孔形成α1C亚基， 心脏中的细胞外表位标签，以及允许双向调节CaV1.2的工程纳米抗体 函数表达式我们联合收割机将这些工具与两种独特的资源相结合--位于 使用邻近标记蛋白质组学测定和巴基斯坦基因组定义心脏中的CaV1.2纳米结构域 资源，其中包含基因序列和广泛的表型数据，来自> 80，000个高 导致遗传突变（包括杂合无效和错义）流行的近亲比率 α1C突变。我们提出了三个目标：1）开发新的工具来探测动态贩运，组织， CaV1.2信号复合物在生理和疾病中的调节; 2）开发工程化的 增加CaV1.2的功能表达并评估其在预防进展中的功效的纳米抗体 心肌梗死后的HF。3)评估人类α1C功能丧失突变的功能影响。

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.