Abstract Body: Skeletal muscle actin (ACTA1) is robustly expressed in skeletal muscle but has trace expression in the heart. While ACTA1 variants are known causes of skeletal myopathies, their role in cardiomyopathy is controversial. We previously identified the ACTA1 R256H variant in individuals with dilated cardiomyopathy (DCM). Here, we define R256H ACTA1 as a pathogenic DCM variant through integrated in vivo, biochemical, and structural analyses.

Acta1^R256H/+ knock-in mice have skeletal muscle weakness but normal baseline cardiac function consistent with the tissue expression pattern of Acta1. Because cardiac Acta1 expression increases under stress, we subjected mice to transaortic constriction, which raised cardiac Acta1 from <1% to ~5% of total sarcomeric actin. This modest increase was sufficient to produce marked hypocontractility and fibrosis. Human ACTA1^R256H/+ iPSC-derived cardiomyocytes similarly exhibited impaired contractility, supporting pathogenicity in a human model.

To dissect how R256H ACTA1 causes potent contractile defects, we purified recombinant R256H ACTA1. Molecular dynamics predicted reduced nucleotide binding which was consistent with experimental findings of impaired R256H thermal stability, nucleotide exchange, and polymerization kinetics. Next, we directly evaluated the effect of R256H ACTA1 on biochemical contractility. R256H ACTA1 filaments moved normally with cardiac myosin in the absence of regulatory troponin/tropomyosin (Tn-Tpm) but showed severe motility arrest upon addition of Tn-Tpm. Incorporation of small amounts of R256H ACTA1 into wild type filaments also disrupted motility. To understand the structural changes responsible for this potent effect, we resolved R256H ACTA1 filament by cryo-EM. We found disruption of the ACTA1 K240 and Tpm E117 interaction, a contact that stabilizes the open, myosin-accessible Tpm state, providing a structural mechanism for the R256H contractile defect.

Collectively, we show that an ACTA1 variant, R256H, causes DCM through potent defects in multiple actin biochemical functions related to structural changes altering actin-Tpm interactions. Our work reveals that other cardiomyopathy-associated ACTA1 variants are likely pathogenic with a similar dominant effect on cardiac contractility.