MITOCHONDRIAL BIOGENESIS DURING CARDIAC DEVELOPMENT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要： 利用我们在心脏开发方面的专业知识和罗切斯特大学医学中心线粒体研究和创新小组的资源，我们最近开始测试这一假说，即发育中的心脏细胞内钙信号的变化调节线粒体的生物发生、结构和功能。为了验证这一假说，我们正在研究以下具体目标：1.确定线粒体在心脏发育过程中的空间分布及其功能意义；2.研究钙瞬变在心脏发育过程中细胞外基质偶联中的作用；3.确定钙瞬变在心脏发育过程中线粒体生物发生中的作用和机制；以及4.研究钙依赖的线粒体生物发生或分布异常在心肌病发生中的作用。在进行这些实验时，我们希望定义在心肌细胞分化和心脏发育过程中发生的正常事件，并确定这些事件在人类疾病中是如何被破坏的，例如非致密性心肌病。 初步数据 在过去的一年里，我们已经开始使用完整的胚胎心脏和培养的胚胎心肌细胞来研究发育中的心脏线粒体的生物发生，以确定线粒体结构和功能是如何随着心脏的发育而演变的。这些实验使用了荧光和多光子显微镜来检查活的和固定的样品。此外，我们最近已经开始检查URMC电子显微镜核心中的小鼠胚胎第9.5天、11.5天和13.5天的心脏，以确定随着心脏成熟亚细胞结构的变化。我们主要研究了心肌的结构和线粒体数量、结构的变化以及与其他细胞结构的联系。 在这些实验过程中，我们注意到，与经典的描述相比，这些心肌细胞中的线粒体可能具有异常的内部结构。特别是在较年轻的标本中，线粒体往往具有较少的有组织的脊。事实上，这些线粒体中的许多似乎是大的、双膜的液泡，尽管每个细胞内都有很大的差异。在许多情况下，我们观察到相同的线粒体区域，在一个区域出现正常的隆起，而在另一个毗邻区域出现“空泡化”。 这种线粒体的出现以前就被观察到过，被认为是一种正常的发育现象。Shepard(1998，Anat Rec，252：383)观察到，在胚胎心脏的早期分化过程中存在空泡化的线粒体，当时细胞被认为是糖酵解的，但当细胞被氧化时，后期心脏的线粒体看起来更“正常”。根据Shepard的说法，这两种形式的线粒体之间的过渡涉及到内膜的管状凹陷；这些结构最终演变为脊。然而，这还没有得到确凿的证明。 这些研究中需要解决的问题 首先，我们希望利用生物医学研究技术中心的技术进行电子断层扫描，从E9.5、11.5和13.5的心脏创建线粒体的三维重建，以确定这些出现“异常”的线粒体的确切结构，以及这种结构是如何随着心脏的成熟而演变的。 在未来，我们还将研究转基因动物(钙通道和钠/钙交换基因敲除小鼠)中钙信号的中断如何影响发育中心脏的线粒体生物发生。最后，我们将使用这些技术来研究随着心脏发育而发生的线粒体与收缩装置的联系的演变。 方法 野生型(C57BL/6)、CaV1.2基因缺失和Ncx1基因缺失的小鼠将被处死，用于这些实验，使用标准的人道程序。胚胎将从E9.5、11.5和13.5的定时妊娠中获得。此外，野生型心脏可能来自E16.5胎儿、出生后1-2天、出生后10天和成年小鼠。请注意，空小鼠将不会在以后的年龄进行研究，因为它们已经死于心力衰竭。 标本将在林格氏溶液或2.5%戊二醛中解剖，用1.0%四氧化二臭氧固定后，在分级的乙醇系列中脱水，渗透并嵌入EPON/Araldite树脂和在70oC聚合的树脂块中。一旦确定了方向，块将被修剪并连续薄切，用于断层和非断层成像，并放置在铜网上，铜网用醋酸铀酰和柠檬酸铅染色。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Using our expertise in cardiac development and the resources of the Mitochondrial Research and Innovation Group at the University of Rochester Medical Center, we have recently begun to test the hypothesis that alterations in intracellular Ca2+ signaling in the developing heart regulate mitochondrial biogenesis, structure, and function. To test this hypothesis, we are investigating the following specific aims: 1. to determine the spatial distribution of mitochondria and its functional significance during cardiac development, 2. to examine the role of calcium transients in ECM coupling during cardiac development, 3. to define the role and mechanism of calcium transients in mitochondrial biogenesis during cardiac development, and 4. to examine the role of abnormal calcium-dependent mitochondrial biogenesis or distribution in the development of cardiomyopathy. In performing these experiments, we hope to define the normal events that occur during cardiac myocyte differentiation and cardiac development as well as to determine how these events are disrupted in human disease, for example, non-compaction cardiomyopathy. Preliminary Data In the last year, we have begun to study mitochondrial biogenesis in the developing heart using whole embryonic hearts and cultured embryonic myocytes to determine how mitochondrial structure and function evolved as the heart develops. These experiments have been performed using epifluorescence and multiphoton microscopy to examine live and fixed samples. In addition, we have recently begun to examine hearts from mouse embryonic day (E) 9.5, 11.5, and 13.5 embryos in the electron microscopy core at URMC to determine changes in subcellular structure as the heart matures. We have concentrated on ventricular myocardial architecture and on changes in mitochondrial numbers, structure, and association with other cellular structures. In the course of these experiments, we have noticed that mitochondria in these myocytes can have abnormal internal structure compared to their classic description. Particularly in younger specimens, mitochondria tend to have fewer organized cristae. In fact, many of these mitochondria appear to be large, double-membraned vacuoles, although there is large variation within each cell. In many cases, we have observed areas of the same mitochondrion with normal appearing cristae in one region and "vacuolization" in another, adjoining region. This mitochondrial appearance has been observed before and is thought to be a normal developmental phenomenon. Shepard (1998, Anat Rec, 252:383) observed that the vacuolated mitochondria are present during early differentiation in the embryonic heart, when cells are thought to be glycolytic, but that mitochondria in the later heart, when the cells are oxidative, appear more "normal." According to Shepard, the transition between these two forms of mitochondria involves the presence of tubular invaginations of the inner membrane; these structures eventually evolve into cristae. However, this has not been conclusively demonstrated. Question to be addressed in these studies Initially, we wish to use the skills of the Biomedical Research Technology Center to perform electron tomography to create three dimensional reconstructions of mitochondria from hearts at E9.5, 11.5, and 13.5 to define the exact structure of these "abnormal" appearing mitochondria and how this structure evolved as the heart matures. In the future, we will also examine how disruption of calcium signaling in transgenic animals (calcium channel and sodium/calcium exchanger knock-out mice) affects mitochondrial biogenesis in the developing heart. Finally, we will use these techniques to study the evolution of mitochondrial association with the contractile apparatus that occurs as the heart develops. Methods Wild-type (C57BL/6), CaV1.2 null, and Ncx1 null mice will be sacrificed for these experiments using standard humane procedures. Embryos will be harvested from timed pregnancies at E9.5, 11.5, and 13.5. In addition, wild-type hearts may be harvested from E16.5 fetuses, postnatal day 1-2, postnatal day 10, and adult mice. Please note that null mice will not be studied at the later ages, as they have died of heart failure. Specimens will be dissected in either Ringer's solution or 2.5% glutaraldehyde, post fixed with 1.0% Osmium tetroxide, dehydrated in a graded series of ethanol, infiltrated and embedded into EPON/Araldite resin and resin blocks polymerized at 70oC. Once the orientation is confirmed, the block will be trimmed and serially thin sectioned for tomographic and non-tomographic imaging and placed onto copper grids, which are stained with uranyl acetate and lead citrate.