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模式良好且功能正常的心脏。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。我们之前的研究表明，钙稳态在维持斑马鱼胚胎心脏节律中起着重要作用，而ncx1（心脏中负责钙挤压的主要分子之一）功能的丧失，会破坏心脏同步收缩，导致被称为心脏颤动的心脏混乱运动。与NCX1在钙稳态中的作用一致，我们在NCX1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，ncx1突变斑马鱼可以作为研究胚胎心脏功能重要的钙调节网络的工具。通过对斑马鱼震颤/ ncx1遗传模型的化学抑制筛选，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。我们发现了一种新的小分子OK-F7在tremblor/NCX1零遗传背景下抑制心脏颤动，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的过表达可以恢复有节奏的心脏收缩，这表明VDAC2和线粒体在钙调节和胚胎心律节律中起着关键作用。作为了解VDAC2在胚胎心脏节律中的作用的第一步，我们建议通过功能获得和功能丧失两种方法来评估VDAC2在心脏发育中的需求（ai1）。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处理是否会改变VDAC2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（Aim2）的机制。最后，我们将研究OK-F7处理是否可以恢复震颤和其他有钙处理缺陷的斑马鱼胚胎的节律性钙波。我们还将确定在缺乏ncx1活性的胚胎中，强迫表达其他VDAC蛋白是否可以恢复有节奏的心脏收缩。这一研究的成功将进一步加深我们在分子水平上对VDAC蛋白在胚胎心律节律中的作用的理解（Aim3）。我们的研究项目的总体目标是通过多学科研究来深入了解钙稳态和胚胎心律失常的重要基因网络。从这个研究项目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。

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.