Missense mutations in alpha-tropomyosin (encoded by TPM1) can result in cardiomyopathic phenotypes, including dilated and hypertrophic forms. When considering tropomyosin mutations that are likely to cause significant disruption, the Y261 residue is of particular interest due to extensive interactions with the N-terminus of troponin T (TnT1) in a computational model of myofilament activation. We hypothesized that missense mutations at this residue, such as Y261C, Y261E, Y261F, and Y261Q, would lead to reductions in TnT1 affinity for actin-tropomyosin and cause corresponding alterations in myofilament function. We tested this hypothesis using co-sedimentation assays, in vitro motility measurements, and in human engineered heart tissues (EHTs) expressing mutant tropomyosin. Binding data show that Y261E and Y261Q mutations in tropomyosin decrease the affinity of TnT1 for actin-tropomyosin filaments, which was strongly correlated with increased Ca 2+ sensitivity (higher pCa 50 values) in regulated in vitro motility assays. Mutation-based shifts in pCa 50 could be reproduced in a myofilament activation model by reducing nearest-neighbor interactions, reinforcing the role of TnT1 in coupling adjacent tropomyosins. EHTs composed of human iPSC-derived cardiomyocytes seeded on decellularized porcine myocardium were fabricated to assess the effects of the Y261C mutation on force production and kinetics. EHTs were transduced via adenoviral delivery of either wild-type or Y261C mutant TPM1. Y261C-expressing EHTs demonstrated a 20% increase in relaxation times compared to wild-type, consistent with increased myofilament Ca 2+ sensitivity. We conclude that missense mutations to the Y261 residue of tropomyosin can destabilize its binding with TnT1, ultimately reducing the ability of tropomyosin to inhibit actin-myosin activity at low Ca 2+ and leading to poor twitch relaxation.
Nistala et al. (Sun,) studied this question.