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.
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