Roles of Rad and other CaV1.2 neighboring proteins in regulating cardiac function in health and disease

Rad 和其他 CaV1.2 邻近蛋白在健康和疾病中调节心脏功能中的作用

• 批准号: 10628915
• 负责人: Steven O Marx
• 金额: $ 42.77万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-15 至 2028-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10628915
• 关键词: Ablation; Acute; Adrenergic Agents; Adrenergic Agonists; Adrenergic beta-Agonists; Adrenergic beta-Antagonists; Affinity; Alanine; Animals; Arrhythmia; Attenuated; Binding; Biochemical; Biological Assay; Biological Markers; C-terminal; Calcium; Calcium Channel; Cardiac; Cardiac Myocytes; Collaborations; Complex; Congestive Heart Failure; Coronary artery; Cyclic AMP-Dependent Protein Kinases; Deletion Mutation; Deterioration; Disease; Disinhibition; Echocardiography; Electrocardiogram; Electrophysiology (science); Enzymes; Exercise stress test; Exons; Exposure to; Fluorescence Resonance Energy Transfer; Forskolin; Genetic; Goals; Health; Heart; Heart Atrium; Heart Contractilities; Heart Rate; Heart failure; Heterozygote; Human; Human Inbreeding; Hypertrophy; In Vitro; Incidence; Individual; Inherited; Isoproterenol; Knock-in Mouse; Label; Lead; Left; Ligation; Mass Spectrum Analysis; Mechanics; Medical; Methods; Missense Mutation; Modeling; Molecular; Monitor; Monomeric GTP-Binding Proteins; Mus; Mutate; Mutation; Myocardial; Myocardial dysfunction; N-terminal; Nervous System; Outcome; Pakistan; Participant; Pathologic; Peroxidases; Phenotype; Phosphorylation; Phosphorylation Site; Physiological; Physiology; Probability; Property; Protein Dephosphorylation; Proteins; Proteomics; Regulation; Regulatory Pathway; Role; Sarcomeres; Serine; Serum; Signal Pathway; Signal Transduction; Site; Structure; Sympathetic Nervous System; Syndrome; Testing; Transgenic Organisms; Ventricular; aorta constriction; ascorbate; beta-adrenergic receptor; cellular targeting; cohort; consanguineous family; desensitization; gain of function; genome resource; heart function; inducible gene expression; inhibitor; insight; loss of function; mortality; mutant; novel; overexpression; premature; pressure; prevent; proband; rare variant; symptom treatment; voltage

The overarching goal of this PPG is to define the molecular mechanisms that regulate local Ca2+ signaling in normal and failing hearts with unprecedented precision. There are three subaims shared by the four projects: 1) explore the precise role of adrenergic signaling in modulating calcium in normal and failing hearts; 2) define novel mechanisms of interactions between t-tubule and SR calcium channels; 3) develop new understandings of genetic based mechanics of inherited forms of CV disease involving calcium. Our long-term goals are to elucidate mechanisms underlying the progressive decompensation towards advanced HF, and to identify novel cellular targets and signaling pathways to prevent and treat the symptoms of HF, and to prevent arrhythmias. Project 2 focuses on the regulation of the Ca2+ channel by the adrenergic nervous system, both under physiological and pathological conditions. Recently, we identified the mechanism by which -adrenergic agonists stimulate voltage-gated Ca2+ channels. We expressed 1C or 2B subunits conjugated to ascorbate- peroxidase in mouse hearts and used multiplexed, quantitative proteomics to track hundreds of proteins in close proximity to CaV1.2. We observed that the Ca2+ channel inhibitor Rad, a monomeric G-protein, is enriched in the CaV1.2 micro-environment but is depleted during -adrenergic stimulation. PKA-catalyzed phosphorylation of specific serine residues on Rad decreases its affinity for auxiliary -subunits and relieves constitutive inhibition of CaV1.2 observed as an increase in channel open probability. We created knock-in mice with alanine substitutions of the four PKA phosphorylation sites. The stimulatory effect of isoproterenol or forskolin on Ca2+ current is completely eliminated when Rad can no longer be phosphorylated in atrial and ventricular cardiomyocytes. Furthermore, loss of PKA phosphorylation of Rad markedly diminished the adrenergic stimulation of the Ca2+ transient and sarcomere shortening. One of our goals is to investigate the relevance of RRAD in humans through analyses of common and rare variants in 20,000 cases with HF, 45,000 cases with MI and equivalent number of controls for each outcome, and to understand the consequences of RRAD deletion and missense mutations through deep phenotyping of carriers and non-carriers of these mutations in consanguineous families in the Pakistan Genome Resource (PGR). Three specific aims are proposed: (1) To determine the impact of -adrenergic stimulation of CaV1.2 in HF. We will use knock-in mice with PKA phosphorylation sites of Rad mutated to alanine, and mice lacking the Rad-subunit interaction in heart. (2) To determine upstream and downstream regulatory pathways modulating adrenergic stimulation of CaV1.2 in physiological and HF states. (3) To explore the role of Rad in regulating cardiac function in humans. We anticipate that this multi-pronged approach will yield novel mechanistic insights into adrenergic signaling in HF and more specifically the adrenergic regulation of Ca2+ channels in HF.

本PPG的首要目标是确定调节局部Ca 2+信号传导的分子机制， 以前所未有的精确度测量正常和衰竭的心脏这四个项目共有三个次级目标： 1)探索肾上腺素能信号在正常和衰竭心脏中调节钙的确切作用; 2）定义 t-小管和SR钙通道相互作用的新机制; 3）发展新的认识 涉及钙的遗传性CV疾病的遗传机制。我们的长期目标是 阐明晚期HF进行性失代偿的潜在机制，并确定新的 细胞靶点和信号传导途径，以预防和治疗HF的症状，并预防心律失常。 项目2关注肾上腺素能神经系统对钙通道的调节， 生理和病理条件。最近，我们确定了β-肾上腺素能神经元 激动剂刺激电压门控Ca 2+通道。我们表达了与抗坏血酸结合的β 1C或β 2B亚基， 过氧化物酶在小鼠心脏和使用多重，定量蛋白质组学跟踪数百种蛋白质， 接近CaV1.2。我们观察到钙通道抑制剂Rad，一种单体G蛋白， 在CaV1.2微环境中富集，但在β-肾上腺素能刺激期间耗尽。PKA催化 Rad上特定丝氨酸残基的磷酸化降低了其对辅助β-亚基的亲和力， CaV1.2的组成性抑制被观察为通道开放概率的增加。我们创造了敲门 四个PKA磷酸化位点被丙氨酸取代的小鼠。异丙肾上腺素的刺激作用 当心房肌中Rad不再被磷酸化时， 心室肌细胞此外，Rad的PKA磷酸化的丧失显著降低了Rad的表达。 肾上腺素能刺激Ca 2+瞬变和肌节缩短。我们的目标之一是调查 通过分析20，000例HF病例中的常见和罕见变异， 每种结局的MI病例和同等数量的对照，并了解 RRAD缺失和错义突变通过这些基因的携带者和非携带者的深度表型分析， 巴基斯坦基因组资源（PGR）中的近亲家庭突变。三个具体目标是 目的：（1）研究β-肾上腺素能刺激对心衰大鼠CaV_（1.2）的影响。我们将使用基因敲入小鼠 Rad的PKA磷酸化位点突变为丙氨酸，而缺乏Rad-PKA亚基相互作用的小鼠， 心(2)为了确定调节肾上腺素能刺激的上游和下游调节途径， CaV1.2在生理和HF状态下。(3)探讨Rad在人类心脏功能调节中的作用。 我们预计，这种多管齐下的方法将产生新的机制的见解肾上腺素能信号， HF，更具体地说，HF中的肾上腺素能调节Ca 2+通道。