Elucidating the molecular mechanisms regulating embryonic cardiac rhythmicity

阐明调节胚胎心律的分子机制

• 批准号: 7889281
• 负责人: JAU-NIAN CHEN
• 金额: $ 36.83万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-15 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7889281
• 关键词: Adult; Animal Model; Arrhythmia; Biochemical; Biological Process; Blood Circulation; Calcium; Calcium Oscillations; Cardiac; Cardiac Myocytes; Characteristics; Chemicals; Data; Defect; Development; Disease; Embryo; Embryonic Heart; Ensure; Gene Components; Genes; Genetic; Genetic Models; Goals; Growth; Health; Heart; Heart Diseases; Homeostasis; In Situ Hybridization; Maintenance; Membrane; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Molecular Genetics; Movement; Mus; Nutrient; Pattern; Periodicity; Preventive; Process; Proteins; Regulation; Regulatory Pathway; Research; Role; Staging; Therapeutic; VDAC2 gene; VDAC3 gene; Vertebrates; Voltage-Dependent Anion Channel; Zebrafish; base; cardiogenesis; gain of function; heart function; in vitro activity; insight; loss of function; mutant; novel; overexpression; programs; public health relevance; small molecule; success; tool; voltage gated channel

DESCRIPTION (provided by applicant): A well-patterned and functioning heart is required for the growth and survival of embryos. While many genes critical for early cardiogenesis have been identified by previous genetic studies, genetic networks required for establishing and maintaining embryonic cardiac function remain to be explored. We have previously shown that calcium homeostasis has an important role in maintaining embryonic cardiac rhythmicity in zebrafish and that loss of function of NCX1h, one of the primary molecules responsible for calcium extrusion in the heart, abolishes synchronized cardiac contraction and leads to chaotic cardiac movements known as cardiac fibrillation. Consistent with the role of NCX1 in calcium homeostasis, we observed abnormal calcium transients in NCX1h null zebrafish embryonic hearts. These observations suggest that the NCX1h mutant zebrafish can serve as a tool for studying the calcium- regulatory networks important for embryonic cardiac function. From a chemical-based suppression screen on the zebrafish tremblor/NCX1h genetic model, we identified a critical component of the gene network governing embryonic cardiac function. We discovered that OK-F7, a novel small molecule suppresses cardiac fibrillation in the tremblor/NCX1 null genetic background, and our biochemical study indicated that the mitochondrial protein VDAC2 is the protein target of OK-F7. Furthermore, over expression of VDAC2 restores rhythmic cardiac contractions in embryos lacking NCX1h activity, suggesting a critical role for VDAC2 and mitochondria in calcium regulation and embryonic cardiac rhythmicity. As the first step toward understanding the role for VDAC2 in embryonic cardiac rhythmicity, we propose to evaluate the requirement of VDAC2 in cardiac development by both gain-of-function and loss-of-function approaches (Aim1). Second, to understand how the interaction of OK-F7 and VDAC modulates calcium homeostasis, we propose to evaluate whether OK-F7 treatment changes VDAC2 channel activity. We will also investigate the impact of OK-F7 on mitochondrial calcium influx. Information obtained from this line of study will provide insight into the mechanism by which OK-F7 and VDAC2 suppress cardiac fibrillation (Aim2). Finally, we will investigate whether OK-F7 treatment can restore rhythmic calcium waves in tremblor and other zebrafish embryos that have calcium-handling defects. We will also determine whether forced expression of other VDAC proteins can restore rhythmic cardiac contractions in embryos lacking NCX1h activity. The success of this line of study will further our understanding of the role for VDAC proteins in embryonic cardiac rhythmicity at the molecular level (Aim3). Our overall goal of this research program is to gain insight into gene networks important for calcium homeostasis and embryonic cardiac rhythmicity through multi-disciplinary studies. Information obtained from this research program will reveal previously unrecognized roles for VDAC and mitochondria in embryonic cardiac function. PUBLIC HEALTH RELEVANCE: Cardiac fibrillation is a form of cardiac arrhythmia that poses serious health threats. We hypothesize that the fundamental mechanisms regulating cardiac rhythmicity are conserved among vertebrates and that the zebrafish tremblor mutant can serve as a good animal model for cardiac fibrillation. The studies in this proposal will help elucidate cellular and molecular mechanisms underlying cardiac fibrillation and ultimately contribute to the development of new preventive and therapeutic approaches.

描述（由申请人提供）：胚胎的生长和存活需要结构良好且功能良好的心脏。虽然之前的遗传学研究已经鉴定了许多对早期心脏发生至关重要的基因，但建立和维持胚胎心脏功能所需的遗传网络仍有待探索。我们之前已经证明，钙稳态在维持斑马鱼胚胎心脏节律方面具有重要作用，NCX1h（负责心脏钙排出的主要分子之一）功能丧失，会消除同步心脏收缩并导致混乱的心脏运动（称为心脏颤动）。与 NCX1 在钙稳态中的作用一致，我们在 NCX1h 缺失的斑马鱼胚胎心脏中观察到异常的钙瞬变。这些观察结果表明，NCX1h 突变斑马鱼可以作为研究对胚胎心脏功能重要的钙调节网络的工具。通过对斑马鱼颤抖/NCX1h 遗传模型进行基于化学的抑制筛选，我们确定了控制胚胎心脏功能的基因网络的关键组成部分。我们发现OK-F7这种新型小分子可以在颤抖/NCX1无效遗传背景下抑制心脏颤动，并且我们的生化研究表明线粒体蛋白VDAC2是OK-F7的蛋白靶标。此外，VDAC2 的过度表达可以恢复缺乏 NCX1h 活性的胚胎的节律性心脏收缩，这表明 VDAC2 和线粒体在钙调节和胚胎心脏节律性中发挥着关键作用。作为了解 VDAC2 在胚胎心律性中作用的第一步，我们建议通过功能获得和功能丧失方法评估 VDAC2 在心脏发育中的需求 (Aim1)。其次，为了了解 OK-F7 和 VDAC 的相互作用如何调节钙稳态，我们建议评估 OK-F7 治疗是否会改变 VDAC2 通道活性。我们还将研究 OK-F7 对线粒体钙流入的影响。从这一系列研究中获得的信息将有助于深入了解 OK-F7 和 VDAC2 抑制心脏颤动的机制 (Aim2)。最后，我们将研究 OK-F7 治疗是否可以恢复震颤和其他具有钙处理缺陷的斑马鱼胚胎的节律性钙波。我们还将确定其他 VDAC 蛋白的强制表达是否可以恢复缺乏 NCX1h 活性的胚胎的节律性心脏收缩。这一系列研究的成功将进一步加深我们对 VDAC 蛋白在分子水平上胚胎心律性中的作用的理解 (Aim3)。我们该研究计划的总体目标是通过多学科研究深入了解对钙稳态和胚胎心律性重要的基因网络。从该研究项目获得的信息将揭示 VDAC 和线粒体在胚胎心脏功能中先前未被认识的作用。 公众健康相关性：心颤是心律失常的一种形式，对健康构成严重威胁。我们假设调节心脏节律的基本机制在脊椎动物中是保守的，并且斑马鱼震颤突变体可以作为心脏颤动的良好动物模型。该提案中的研究将有助于阐明心脏颤动的细胞和分子机制，并最终有助于开发新的预防和治疗方法。