The Actc A295S Hypertrophic Cardiomyopathy Mutation Promotes Thin Filament Disinhibition and Enhances Contractile Activity

Abstract

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease of the heart. HCM mutations in sarcomeric genes often engender hypercontractile myocardium that displays increased Ca2+-sensitivity of force, enhanced cross-bridge cycling, and incomplete relaxation, which precede abnormal wall thickening. The alpha-cardiac actin (ACTC) Ala295Ser substitution causes a highly penetrant disease with diverse phenotypes. To help resolve the mechanistic basis of the disorder, we generated several Drosophila models that limit genetic diversity and pathological complexity. We created lines with inducible wildtype UAS-Act57BWTand mutant UAS-Act57BA295Scardiac actin transgenes to examine the effects of A295S actin on the fly heart. High-speed video microscopy and motion analysis of beating hearts revealed A295S actin expression significantly reduced diastolic volumes and prolonged systolic intervals, consistent with elevated contractile properties at rest and during contraction. In situ treatment of Act57BA295S hearts with blebbistatin, under low intracellular Ca2+ conditions, caused significantly larger increases in myocyte length vs. Act57BWTcontrols. These data suggest A295S actin induces excessive thin filament disinhibition, myosin binding, and resting tension. We also expressed indirect flight muscle (IFM)-targeted Act88FWT or Act88FA295S transgenes in an IFM actin-null background. Confocal microscopy resolved hypercontracted Act88FA295S fibers. Reconstituted Act88FA295S thin filaments, using bovine cardiac troponin-tropomyosin, displayed significantly enhanced Ca2+-sensitivity of activation vs. control filaments in regulated in vitro motility experiments. Finally, in silico modelling revealed the A295S mutation influences the orientation of nearby actin residues that mediate critical electrostatic interactions with tropomyosin. Such mutation-induced propagated effects likely lower the energy required to relieve tropomyosin-mediated inhibition and promote force production. Overall, enhanced contractile activity from the molecular through the cellular level is consistent with the early hyperdynamic contractile properties frequently associated with HCM and could trigger ACTC A295S-associated pathology. This work was supported by R37HL36153 and 1R01HL124091.