Monday, April 29, 2024
4/29/2024
PROJECT SUMMARY The ability of the heart to contract continuously is vital to the organism and is dictated in part by the sarcomeres, the functional units of the contractile apparatus. Consequently, any errors in the formation, composition or homeostasis of the sarcomere structure lead to congenital heart defects (CHD) or various forms of cardiomyopathy. While the highly complex structure of the cardiac sarcomere and its function have been studied extensively for decades, comparatively little remains known about how the sarcomere structure is established in the first place, during de novo sarcomerogenesis. Current knowledge is largely qualitative as in depth mechanistic studies are challenging at such early stages of development. Here we will use human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to interrogate our hypothesis that de novo sarcomerogenesis occurs via a mode of assembly that involves formation of membrane-less organelles (MLOs) with distinct biophysical properties. In Aim 1 we will interrogate the biophysical properties of Z-bodies and Z-discs during early heart development and identify individual candidates and biological processes that dictate Z-body formation. We will use super-resolution and time-lapse microscopy, FRAP analysis and transmission electron microscopy. To identify drivers of Z-body formation we will use CRISPRi for candidates identified in the ACTN2 interactome and likely to be involved in formation of MLOs. Lastly, we will assess if Z-bodies possess distinct biochemical functions, including the presence of specific mRNA transcrips and/or local translational activity. In Aim 2 we will determine the mechanisms that underlie Z-body initiation at the onset of de novo sarcomerogenesis. We have described a role for WNT and RHO signaling in Z-body formation and we will identify additional mechanisms using small molecule screening. The successful completion of this comprehensive and detailed mechanistic study of Z-bodies holds the promise of delivering an unprecedented level of characterization of this critical stage of de novo sarcomerogenesis. It will encompass a thorough understanding of the formation and function of Z- bodies as a biomolecular condensates, as well as the identification of novel candidates involved in this process and their role in cardiac function.
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