PATHOGENESIS AND IN VIVO SUPPRESSION OF THIN FILAMENT-BASED CARDIOMYOPATHIES

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-
phenotype information in a less complex model system (Drosophila) that limits genetic modifiers and
environmental factors to help establish paradigms for disease processes involved in cardiac remodeling.

Grant Number: 5R01HL124091-09
NIH Institute/Center: NIH
Principal Investigator: Anthony Cammarato