MITOCHONDRIAL MANIPULATION AND ANALYSIS IN DROSOPHILA HEARTS

描述（由申请人提供）：线粒体融合在心肌细胞中的作用存在争议;一些人认为融合在这些细胞中没有作用，其中线粒体接触受到物理限制，一些人认为它是严格病理性的，因为它促进SR-线粒体钙转运，打开线粒体渗透性转换孔，一些人认为线粒体融合对线粒体健康至关重要。目前没有发表的体内数据解决这个问题，使用小鼠基因敲除的未发表结果不一致。在这个修订后的R21，我们提出的研究，将使用在体内的线粒体外膜和内膜融合蛋白的遗传操作，以确定线粒体融合在果蝇心管的作用。我们将使用心脏基因操作，心脏表型和活心肌细胞研究的组合，使用新的果蝇（果蝇）心肌病模型和荧光蛋白指示剂模型，我们已经产生。我们将使用基因互补，取代人的线粒体融合蛋白抑制果蝇线粒体融合蛋白，询问哺乳动物线粒体融合蛋白的作用。果蝇心管（在细胞、转录和功能水平）与哺乳动物的心脏非常相似，可以使用类似的表型分析技术对心脏结构和功能进行分析，从而可以将这种互补/拯救数据外推到高等生物。由于果蝇心脏管可以在室温和正常大气中存活数小时，因此它们是活心肌细胞研究的最佳选择。这种机械细胞生物学和完整心脏生理学的结合在哺乳动物系统中是不可能的。 拟议的研究将不会解决一个重要的未回答的科学问题，但将为科学界产生新的试剂和技术，这将广泛推进使用果蝇平台的心脏研究。事实上，目前的研究是对果蝇心脏系统假设检验研究能力的一次雄心勃勃的测试。我们的目标是：SA#1。生成具有心脏特异性线粒体融合缺陷的新型果蝇遗传模型，类似于我们和其他人正在开发的心脏特异性线粒体融合蛋白缺失小鼠模型。 SA #2。开发和部署创新技术，通过使用表达基因编码的荧光探针的新型飞线，以小鼠模型不可能的方式分析所得心脏表型，用于心肌细胞钙信号传导、线粒体和肌浆网形态测定和相互作用、ATP/ADP比率、ROS（过氧化氢）产生和线粒体氧化还原电位。 公共卫生相关性：这些研究将开始回答重要的基本问题，线粒体融合的作用，在体内心脏功能的神经-肌浆拴系。本文所述的模型、技术和方法将适用于黑腹果蝇中任何未来的遗传心肌细胞特异性操作，并且将建立用于需要心脏特异性基因操作和表型分析的研究的有用的新的高通量分析平台。

DESCRIPTION (provided by applicant): The role of mitochondrial fusion is disputed in cardiac myocytes; some believe fusion has no role in these cells where mitochondrial contact is enforced by physical constraints, some believe it is strictly pathological because it promotes SR-mitochondrial calcium transport that opens mitochondrial permeability transition pores, and some contest that mitochondrial fusion is essential to mitochondrial health. There are currently no published in vivo data addressing this issue and unpublished results using mouse knockouts are inconsistent. In this revised R21 we propose studies that will use in vivo genetic manipulation of mitochondrial outer and inner membrane fusion proteins to define the role of mitochondrial fusion in Drosophila heart tubes. We will use a combination of cardiac gene manipulation, cardiac phenotyping, and live cardiomyocyte research using new Drosophila melanogaster (fruit fly) cardiomyopathy models and fluorescent protein indicator models that we have generated. We will use genetic complementation, substituting human mitofusins for suppressed Drosophila mitochondrial fusion protein, to interrogate the roles of mammalian mitochondrial fusion proteins. The Drosophila heart tube is (at the cellular, transcriptional, and functional level) sufficiently like the mammalian heart to permit analysis of cardiac structure and function using analogous phenotyping techniques, permitting extrapolation of such complementation/rescue data to higher organisms. Because viable Drosophila heart tubes can be maintained for many hours at room temperature and normal atmosphere, they are optimal for live cardiomyocyte studies. This combination of mechanistic cell biology and intact cardiac physiology is not possible in mammalian systems. The proposed studies will not address an important unanswered scientific question, but will generate new reagents and techniques for the scientific community that will broadly advance cardiac studies using the Drosophila platform. Indeed, the current studies are an ambitious test of the capabilities of hypothesis testing research in the Drosophila cardiac system. Our Specific Aims are: SA#1. Generate novel Drosophila genetic models with cardiac-specific mitochondrial fusion defects analogous to the cardiac-specific mitofusin null mouse models we and others are developing. SA#2. Develop and deploy innovative techniques for analysis of resulting cardiac phenotypes in ways not possible with mouse models through the use of novel fly lines expressing genetically-encoded fluorescent probes for cardiomyocyte calcium signaling, mitochondrial and sarcoplasmic reticular morphometry and interactions, ATP/ADP ratio, ROS (hydrogen peroxide) production, and mitochondrial redox potential. PUBLIC HEALTH RELEVANCE: These studies will begin to answer important fundamental questions about the role of mitochondrial fusion and mitochondrial-sarcoplasmic tethering on in vivo cardiac function. The models, techniques and approaches described herein will be applicable to any future genetic cardiomyocyte-specific manipulations in Drosophila melanogaster, and will establish a useful new high-throughput analytical platform for studies requiring cardiac- specific gene manipulation and phenotyping.