The role of VSNL1 in human heart rate regulation

VSNL1在人体心率调节中的作用

• 批准号: 10750747
• 负责人: ZANIAR GHAZIZADEH
• 金额: $ 8.08万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-09-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750747
• 关键词: Action Potentials; Adrenergic Agents; Adrenergic Agonists; Adrenergic Receptor; Adrenergic beta-Antagonists; Affect; Agonist; Arrhythmia; Atrial Function; Attenuated; Autonomic nervous system; Biological; Bradycardia; CRISPR/Cas technology; Calcium; Cardiac conduction system; Cardiovascular Physiology; Cardiovascular system; Cell Line; Cell model; Cell physiology; Cells; Cellular biology; Central Nervous System; Characteristics; Cholinergic Agonists; Clinical; Data; Derivation procedure; Development; Electrocardiogram; Electrophysiology (science); Endocrine; Etiology; Exercise; Gene Expression Profiling; Generations; Genetic Databases; Genetic Engineering; Genomics; Goals; Health; Heart; Heart Atrium; Heart Rate; Human; Human Activities; In Vitro; Individual; Knock-in; Knock-out; Lead; Longevity; Magnetic Resonance Imaging; Maps; Mendelian randomization; Methodology; Methods; Modeling; Molecular; Molecular Profiling; Morbidity - disease rate; Muscle Cells; Nervous System; Neurotransmitter Receptor; Neurotransmitters; Nodal; Outcome; Pacemakers; Participant; Pattern; Periodicity; Phenotype; Physiological; Physiology; Population Genetics; Precision therapeutics; Principal Investigator; Proteins; Proteomics; Protocols documentation; Regulation; Reporter; Rest; Role; Secondary to; Signal Pathway; Signal Transduction; Sinoatrial Node; Stimulus; Stress Tests; System; Tachycardia; Technology; Testing; Time; VSNL1 gene; Validation; Variant; Ventricular; Ventricular Function; biobank; cardiac magnetic resonance imaging; cardiovascular effects; cell type; cholinergic; clinical database; clinical phenotype; cohort; design; differentiation protocol; embryonic stem cell; epidemiology study; gain of function; genetic variant; human data; human embryonic stem cell; human embryonic stem cell line; in vitro Model; loss of function; mortality; new therapeutic target; nodal myocyte; null mutation; paracrine; patch clamp; response; sensor; single-cell RNA sequencing; stem cells; tool; trafficking; transcriptomics

PROJECT SUMMARY In the heart, pacemakers or sinoatrial node cells (SAN) initiate and maintain a rhythmic beating pattern that can respond to external stimuli, including neurotransmitters, paracrine, and endocrine signals. In addition to its clinical importance, resting heart rate is associated with lifespan across species, and has strong correlation with longevity within individuals in several large epidemiologic studies. Despite their key role in human health and physiology, the exact intracellular mechanism to maintain precise rhythmic oscillation is unknown, given the limited access to these cells in the heart. The principal investigator hypothesizes that gene expression analysis of the human sinoatrial node cell will identify cellular and physiologic features of human pacemaking function. Recent technologies have enabled generating functional human SAN from embryonic stem cells (hPSC-SAN) and performing molecular characterization at the single cell level. VSNL1, a calcium sensing protein, was identified as a marker specific to SAN cells. I hypothesize that VSNL1 is uniquely involved in heart rate regulation. Analyzing the genetic variants in VSNL1 gene and their effect on heart rate in large biobanks will enable validation of the functional role of this protein in heart rate physiology. The applicant will use large biobank cohorts and genetically engineered human embryonic stem cells to pursue the following aims: First, determine the association between genetic variants in VSNL1 with cardiovascular physiology in several large biobanks. Preliminary data in UK biobank (UKBB) cohort suggests significant association between genetic variants in VSNL1 gene and baseline heart rate. The applicant will further study the effect of VSNL1 variants on heart rate with exercise, atrial and ventricular function (on cardiac MRI), and cardiovascular morbidity and mortality outcomes in UKBB and other more diverse cohorts. Second, understand the effect of genetic variants associated with heart rate in UKBB on molecular and electrophysiological characteristics of hPSC-SAN cells in vitro. In preliminary data, the applicant has successfully confirmed the specific expression of VSNL1 in hPSC-SAN cells and their absence in the human embryonic stem cells (hESC) derived ventricular myocytes. Using CRISPR-Cas9 technology, the applicant has generated knock-out models of VSNL1 gene in hESC. The applicant will use hESC lines carrying VSNL1 null mutation to generate hPSC-SAN lacking functional VSNL1. Analyzing beating rate, calcium activity, and action potential using patch clamp will elucidate the cell type-specific role of VSNL1 in human SAN biology. Third, perform functional studies to test the role of VSNL1 in hPSC-SAN response to adrenergic and cholinergic signals. Recent studies support that VSNL1 is involved in neurotransmitter receptor trafficking. The applicant will generate loss of function (LoF) and gain of function (GoF) VSNL1 variants in hESC using the previously established protocols. The applicant will generate SAN cells from hESCs carrying knock-out, GoF, or LoF VSNL1 variants to study the effect of various adrenergic and cholinergic neurotransmitters on SAN cells’ beating rate, calcium, activity, and action potential.

项目摘要 在心脏中，起搏器或窦房结细胞（SAN）启动并维持有节奏的跳动模式， 对外界刺激作出反应，包括神经递质、旁分泌和内分泌信号。除了其 临床重要性，静息心率与跨物种的寿命相关，并且与 在几个大型流行病学研究中，尽管它们在人类健康中发挥着关键作用， 在生理学上，维持精确节律振荡的确切细胞内机制是未知的， 心脏中这些细胞的获取受到限制。首席研究员假设基因表达 人窦房结细胞的分析将鉴定人窦房结细胞的细胞和生理特征。 起搏功能最近的技术已经能够从胚胎中产生功能性的人类SAN 干细胞（hPSC-SAN），并在单细胞水平上进行分子表征。VSNL 1，钙 传感蛋白，被鉴定为SAN细胞特异性的标志物。我假设VSNL 1是唯一 参与心率调节。VSNL 1基因变异及其对心率的影响 在大型生物库中的研究将能够验证这种蛋白质在心率生理学中的功能作用。申请人 将使用大型生物库队列和基因工程人类胚胎干细胞来进行以下研究 目的：首先，确定VSNL 1基因变异与心血管生理学之间的关联， 几个大型生物银行英国生物库（UKBB）队列的初步数据表明， VSNL 1基因的遗传变异和基线心率。申请人将进一步研究VSNL 1的作用 运动心率、心房和心室功能（心脏MRI）以及心血管发病率的变化 以及UKBB和其他更多样化队列的死亡率结局。第二，了解基因的影响 UKBB中与心率相关的变异体对hPSC-SAN分子和电生理特性的影响 体外细胞在初步数据中，申请人已经成功证实了VSNL 1在大肠杆菌中的特异性表达。 hPSC-SAN细胞及其在人胚胎干细胞（hESC）衍生的心室肌细胞中的缺失。 使用CRISPR-Cas9技术，申请人已经在hESC中产生了VSNL 1基因的敲除模型。的 申请人将使用携带VSNL 1无效突变的hESC系来产生缺乏功能性VSNL 1的hPSC-SAN。 用膜片钳技术分析搏动频率、钙离子活性和动作电位，将阐明细胞类型特异性 VSNL 1在人类SAN生物学中的作用。第三，进行功能研究以测试VSNL 1在hPSC-SAN中的作用。 对肾上腺素能和胆碱能信号的反应。最近的研究支持VSNL 1参与了 神经递质受体运输申请人将产生功能丧失（LoF）和功能获得（GoF） 使用先前建立的方案在hESC中检测VSNL 1变体。申请人将生成SAN单元， 携带敲除、GoF或LoF VSNL 1变体的hESC研究各种肾上腺素能和胆碱能神经递质的作用 神经递质对SAN细胞的搏动率、钙、活性和动作电位的影响。