Project Summary The thin filament is a multi-subunit regulatory machine. Proper regulation of cardiac contraction requires communication among, and controlled movement of, individual thin filament proteins. The goal of this application is to understand how post-translational modifications (PTMs) and human cardiomyopathy mutations, located at conserved interfaces between thin filament subunits, affect protein-protein associations, modulate muscle function, and/or lead to disease. Drosophila melanogaster benefits from robust experimental tools that permit efficient, yet comprehensive, scrutiny of the most proximal consequences of thin filament perturbations. This animal model will continue to help us discern the mechanistic basis of contractile regulation and, importantly, of myopathic responses to molecular insults. Mice, however, are more genetically and physiologically similar to humans. Using a unique combination of techniques including high-speed video and cryo-electron microscopy, in silico modeling, and mechanical assays we will define, for the first time, the structural and functional effects of specific PTMs and cardiomyopathy mutations, located at interfacial seams between thin filament subunits, from the molecular to the tissue level. Therefore, a highly integrative approach will be employed that relies, in part, on a pioneering strategy to express human actin variants in Drosophila for purification and biophysical analysis, and upon several new fly models of actin and troponin T (TnT)-based cardiomyopathies. The latter will be complemented by murine models. Aim 1 will focus on determining the effects of actin acetylation on tropomyosin (Tm) positioning and cardiac performance using recombinant human proteins, flies, and mice. We will test the hypothesis that acetylation of K326 and K328 on actin, residues we previously showed bind to and help orient Tm such that it prevents actomyosin cycling, discourages inhibitory Tm positioning and promotes cardiac contraction. For Aim 2 we will delineate how certain actin and TnT cardiomyopathy mutations uniquely affect myocardial relaxation. We will test the hypothesis that particular actin and TnT lesions disturb distinct, critical interfacial contacts with Tm, which differentially alters Tm-based inhibition of contraction and force production to initiate discrete cardiac pathologies. For Aim 3, we will ascertain if the same actin PTMs investigated in Aim 1, improve or worsen myocardial dysfunction in murine and fly cardiomyopathy models. We will test the hypothesis that enhanced cardiac contractility, conferred by actin pseudo-acetylation, will improve and aggravate the pathological phenotypes in models of dilated and hypertrophic cardiomyopathy, respectively. Overall, this work is significant since it will provide critical structural and functional information necessary to understand how the thin filament machine operates normally and during disease. Additionally, our efforts will yield genotype...