MITOCHONDRIAL BIOGENESIS DURING CARDIAC DEVELOPMENT

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 摘要： 利用我们在心脏发育方面的专业知识和罗切斯特大学医学中心线粒体研究与创新小组的资源，我们最近开始测试这一假设，即发育中心脏细胞内Ca 2+信号的改变调节线粒体的生物发生、结构和功能。 为了验证这一假设，我们正在研究以下具体目标：1。研究心肌发育过程中线粒体的空间分布及其功能意义。研究钙瞬变在心脏发育过程中ECM偶联中的作用，3.明确钙瞬变在心脏发育过程中线粒体生物合成中的作用和机制。研究钙依赖性线粒体生物发生或分布异常在心肌病发展中的作用。 在进行这些实验时，我们希望定义心肌细胞分化和心脏发育过程中发生的正常事件，以及确定这些事件在人类疾病中如何被破坏，例如致密化不全性心肌病。 初步数据 在过去的一年里，我们已经开始研究线粒体生物发生在发育中的心脏使用整个胚胎心脏和培养的胚胎肌细胞，以确定如何线粒体结构和功能演变的心脏发展。 这些实验已经进行了使用落射荧光和多光子显微镜检查活的和固定的样品。 此外，我们最近开始在URMC的电子显微镜核心中检查小鼠胚胎（E）9.5，11.5和13.5天胚胎的心脏，以确定心脏成熟时亚细胞结构的变化。 我们集中在心室心肌结构和线粒体数量的变化，结构，并与其他细胞结构的关联。 在这些实验的过程中，我们注意到这些肌细胞中的线粒体与其经典描述相比可能具有异常的内部结构。 特别是在年轻的标本中，线粒体往往有较少的组织嵴。 事实上，这些线粒体中的许多似乎是大的，双膜空泡，尽管每个细胞内有很大的变化。 在许多情况下，我们观察到同一个区域，在一个区域出现正常的嵴，而在另一个相邻区域出现空泡化。 这种线粒体外观以前曾被观察到，并被认为是正常的发育现象。 谢泼德（1998，Anat Rec，252：383）观察到，空泡化的线粒体存在于胚胎心脏的早期分化期间，此时细胞被认为是糖酵解的，但是在后期心脏中的线粒体，此时细胞是氧化的，看起来更“正常”。根据谢泼德的说法，这两种形式的线粒体之间的过渡涉及内膜的管状内陷的存在;这些结构最终演变成嵴。 然而，这一点尚未得到最终证明。 这些研究要解决的问题 最初，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，以创建E9.5，11.5和13.5心脏线粒体的三维重建，以定义这些“异常”出现的线粒体的确切结构以及这种结构如何随着心脏成熟而演变。 将来，我们还将研究转基因动物（钙通道和钠/钙交换器敲除小鼠）中钙信号的破坏如何影响发育中心脏的线粒体生物合成。 最后，我们将使用这些技术来研究线粒体与心脏发育过程中发生的收缩装置的关系。 方法 使用标准人道程序处死野生型（C57 BL/6）、CaV1.2缺失和Ncx 1缺失小鼠用于这些实验。 将在E9.5、11.5和13.5时从定时妊娠中收获胚胎。 此外，野生型心脏可从E16.5胎仔、出生后第1-2天、出生后第10天和成年小鼠收获。 请注意，无效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。 将在林格氏溶液或2.5%戊二醛中解剖样本，用1.0%四氧化锇后固定，在梯度系列乙醇中脱水，渗透并包埋到EPON/Araldite树脂和在70 ℃下聚合的树脂块中。 一旦确认了方向，将对块进行修剪并连续薄切片，用于断层扫描和非断层扫描成像，并将其放置在铜网格上，用醋酸双氧铀和柠檬酸铅染色。

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.