Abstract P1059: Establishing The Mechanistic Basis Of Dilated Cardiomyopathy Associated With The Skeletal Muscle Actin Mutation R256H

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Actin, which is expressed in all eukaryotic cells, is an essential component of the cardiac and skeletal muscle sarcomere thin filament. Mutations in actin have been associated with a wide range of diseases, including skeletal myopathies and cardiomyopathies. Humans express six actin isoforms with two that are thought to be highly enriched in sarcomeres and differ by only four amino acids: skeletal muscle actin (ACTA1) and cardiac actin (ACTC1). While ACTC1 is exclusively expressed in the adult heart, ACTA1 is predominantly expressed in skeletal muscle with lower levels detected in cardiac muscle. As such, ACTA1 mutations are well known to cause skeletal myopathy with minimal effects on cardiac function. However, I previously reported a family carrying a novel heterozygous mutation in ACTA1 (ACTA1 R256H) with dilated cardiomyopathy (DCM) and no clinical evidence of skeletal myopathy. The objective of this study is to elucidate the molecular mechanism(s) by which ACTA1 R256H may cause heart failure. I generated ACTA1 R256H/+ human pluripotent stem cells (hPSCs) using Cas9/CRISPR gene editing. Using traction force microscopy, I observed that ACTA1 R256H/+ hPSC-derived cardiomyocytes display reduced contractility. To define relevant mechanisms, I devised a novel approach to purify milligram quantities of recombinant human ACTA1, which has served as a remarkable challenge for the field. Importantly, this purification technique circumvents contamination from endogenous actins and produces functional protein capable of polymerization. Moreover, in vitro motility assays demonstrated that reconstituted human thin filaments containing recombinant ACTA1 are functional and activated in a calcium-regulated manner. Intriguingly, purified ACTA1 R256H appears to have a polymerization defect, suggesting a possible mechanism by which this mutation leads to a defect in contractility. Together, these studies suggest for the first time that a mutation in skeletal muscle actin, ACTA1 R256H, causes cardiomyocyte hypocontractility while also suggesting a potential molecular mechanism. Finally, this study also introduces a new purification method essential for biochemical and biophysical analysis of human ACTA1 mutations.