Elucidating the molecular mechanisms regulating embryonic cardiac rhythmicity

描述(由申请人提供)：胚胎的生长和存活需要一个有良好模式和功能的心脏。虽然许多对早期心脏发生至关重要的基因已被先前的遗传学研究确定，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律失常中起着重要作用，NCX1h是负责心脏钙排出的主要分子之一，其功能的丧失会取消心脏同步收缩，并导致称为心脏纤颤的心脏混乱运动。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。这些观察表明，NCX1h突变斑马鱼可以作为一种工具来研究对胚胎心脏功能至关重要的钙调节网络。从斑马鱼颤抖/NCX1h遗传模型的化学抑制屏幕上，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。我们在颤抖/NCX1缺失的遗传背景中发现了一个新的抑制心脏颤动的小分子OK-F7，我们的生化研究表明线粒体蛋白VDAC2是OK-F7的蛋白靶点。此外，在缺乏NCX1h活性的胚胎中，VDAC2的过表达可以恢复节律性心脏收缩，这表明VDAC2和线粒体在钙调节和胚胎心律失常中起着关键作用。作为了解VDAC2在胚胎心律失常中作用的第一步，我们建议通过功能获得和功能丧失两种方法评估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治疗是否改变了VDAC2通道的活性。我们还将研究OK-F7对线粒体钙内流的影响。从这一系列研究中获得的信息将为深入了解OK-F7和VDAC2抑制心脏颤动(AIM2)的机制提供依据。最后，我们将研究OK-F7治疗是否能恢复颤抖和其他有钙处理缺陷的斑马鱼胚胎的节律性钙波。我们还将确定强制表达其他VDAC蛋白是否可以恢复缺乏NCX1h活性的胚胎的节律性心脏收缩。这一系列研究的成功将在分子水平上加深我们对VDAC蛋白在胚胎心律失常中作用的理解(Aim3)。我们这项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失常至关重要的基因网络。从这项研究计划中获得的信息将揭示VDAC和线粒体在胚胎心脏功能中以前未被认识到的作用。 公共卫生相关性：心脏颤动是一种严重威胁健康的心律失常。我们假设调控心律节律性的基本机制在脊椎动物中是保守的，斑马鱼颤抖突变体可以作为心脏纤颤的良好动物模型。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。

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.