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良好的模式和功能的心脏。虽然许多早期心脏发生的关键基因已被以前的遗传学研究确定，建立和维持胚胎心脏功能所需的遗传网络仍有待探索。我们以前已经表明，钙稳态在维持斑马鱼胚胎心脏节律方面具有重要作用，并且NCX 1h（负责心脏中钙排出的主要分子之一）功能的丧失会消除同步心脏收缩并导致称为心脏颤动的混乱心脏运动。与NCX 1在钙稳态中的作用一致，我们观察到NCX 1h空斑马鱼胚胎心脏中异常的钙瞬变。这些观察结果表明，NCX 1h突变斑马鱼可以作为一种工具，用于研究钙调节网络的重要胚胎心脏功能。从斑马鱼tremblor/NCX 1h遗传模型的化学抑制筛选中，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-F7，一个新的小分子抑制心脏颤动的tremblor/NCX 1无效的遗传背景，我们的生化研究表明，线粒体蛋白VDAC 2是OK-F7的蛋白质的目标。此外，VDAC 2的过度表达恢复了缺乏NCX 1h活性的胚胎的节律性心脏收缩，这表明VDAC 2和线粒体在钙调节和胚胎心脏节律中起着关键作用。作为了解VDAC 2在胚胎心脏节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来评估VDAC 2在心脏发育中的需求（Aim 1）。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VDAC 2通道活性。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有助于深入了解OK-F7和VDAC 2抑制心脏颤动（Aim 2）的机制。最后，我们将研究OK-F7治疗是否可以恢复tremblor和其他具有钙处理缺陷的斑马鱼胚胎中的节律性钙波。我们还将确定其他VDAC蛋白的强制表达是否可以恢复缺乏NCX 1h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蛋白在分子水平上对胚胎心律的作用的理解（Aim 3）。本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重要的基因网络。从这项研究计划中获得的信息将揭示以前未被认识到的VDAC和线粒体在胚胎心脏功能中的作用。 公共卫生相关性：心脏纤维性颤动是一种心律失常，对健康构成严重威胁。我们假设，调节心脏节律的基本机制是保守的脊椎动物和斑马鱼tremblor突变体可以作为一个很好的动物模型，心脏颤动。该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.