Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle characterized by ventricular wall thickening, hyperdynamic contractile properties, and impaired relaxation. It is commonly caused by mutations in sarcomeric proteins. The alpha-cardiac actin ACTC M305L HCM mutation is located near the nucleotide-binding site and residues predicted to help confine tropomyosin to an inhibitory position along thin filaments. We generated several Drosophila models of the disorder to evaluate the effects of the lesion from the organ through molecular level. In all muscles tested, phenotypic disturbances correlated with mutant protein load. For example, relative to wildtype actin, when highly overexpressed in the fly heart, the variant significantly reduced cardiac output, prolonged systole, and restricted diastolic volumes. Preliminary force measurements from jump muscle fibers corroborated a dose-dependent, M305L actin-induced pathological effect. Moreover, we observed greater incremental decreases in flight ability with increasing amounts of mutant versus wildtype actin expression in indirect flight muscles (IFM). Flight was abolished when the mutant was highly overexpressed and destructive myosin-induced hypercontraction was evident, suggesting disrupted contractile regulation and poorly inhibited actomyosin associations. However, regulated in vitro motility parameters, including Vmax, nH, and pCa50, of thin filaments reconstituted from M305L or wildtype IFM actin and bovine cardiac troponin-tropomyosin, were indistinguishable. Likewise, no overt differences in computationally-derived electrostatic interaction energy landscapes between M305L and wildtype actin-tropomyosin filaments were observed, suggesting unperturbed tropomyosin-positioning in the absence of troponin. Nonetheless, over sparsely populated beds of myosin, a significantly higher percentage of troponin-free M305L versus wildtype actin-tropomyosin filaments were motile, implying tropomyosin is less effective at blocking actomyosin associations on mutant filaments. Mispositioning of tropomyosin may contribute to the supranormal contraction and compromised relaxation observed in our in vivo models and, potentially, in human patients.