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

摘要 在美国，心力衰竭(HF)影响着600万人，每年导致40万人死亡。 加深了对正常心脏生理基础的分子机制的基本见解，以及 它们的失调导致心脏病是识别新药靶点和开发新药物的关键 对抗心脏病的治疗学。钙循环对心脏功能是不可或缺的，而心脏功能紊乱 心肌细胞(CM)钙信号是心脏病的一个显著标志。L型钙通道(CaV1.2) 是钙离子进入细胞质雄性不育系的主要途径，启动兴奋-收缩偶联，调节作用 潜在的持续时间，并控制其他必要的钙依赖。推动这一提议的总体假设 是：在心脏细胞中存在CaV1.2的空间和功能异质性，这对正常心脏至关重要 生理学；慢性应激或压力引起的CaV1.2分子和功能组织的紊乱 基因突变对心脏病理生理学有重要作用；翻译后调节 CaV1.2功能表达是治疗心脏病的一种有前景的方法。有基础的，但是 初步的，文献中支持这些假设的证据。然而，建立在这些初始基础上能力 观察以形成对CaV1.2分子和功能异质性的更完整的理解 生理学和疾病中心脏细胞中的信号复合体由于缺乏可视化的方法而受到阻碍。 并选择性地操纵这种独特的钙信号复合体。我们已经开发了几个新的工具来 弥合这一差距包括；表达融合了YFP-形成孔的α1C亚单位的转基因小鼠 心脏中的细胞外表位标记，以及允许双向调控CaV1.2的工程纳米体 函数表达式。我们将这些工具与两个独特的资源相结合--位于 用邻近标记蛋白质组学方法确定的心脏中的CaV1.2纳米结构域，以及巴基斯坦基因组 资源，其中包含来自&gt；80,000个高危个体的基因序列和广泛的表型数据。 导致包括杂合缺失和错义在内的基因突变盛行的血缘关系比率 α1C基因突变。我们提出了三个目标：1)开发新的工具来调查动态的人口贩运、组织和 CaV1.2信号复合体在活体细胞质雄性不育中的生理和疾病调控；2)开发工程化 增加CaV1.2功能表达的纳米抗体及其在预防肿瘤进展中的作用 心肌梗死后转为心力衰竭。3)评估α1C功能缺失突变对人类的功能影响。

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.