The myosin mutation E525K was identified in a patient with dilated cardiomyopathy. Recent work on isolated myosin has led to the hypothesis that this mutation has two main effects: (1) stabilization the interacting heads motif and (2) increasing myosin ATPase activity of isolated heads. This ultimately leads to hypocontractility due to impaired myosin recruitment. Here, we present the first force and contractility measurements from human induced pluripotent stem cell (hiPSC) derived cardiomyocytes carrying the heterozygous E525K mutation. These cells were generated on the WTC11 background with a green fluorescent protein (GFP) tag on α-actinin. Our data revealed a 65% reduction in sarcomere contraction in single E525K/WT cells and a 39% decrease in maximal twitch force in engineered heart tissues (EHTs). Paradoxically, isolated myofibrils showed a 45% increase in peak force under high calcium stimulation (pCa 4.0) and no difference at sub-maximal calcium. To validate these findings, we introduced the E525K mutation into a different hiPSC genetic background (UC2), generating wild-type, heterozygous, and homozygous lines. These cells confirmed reduced force at the tissue level in a stepwise manner (WT/WT > E525K/WT > E525K/E525K). Myofibrils from these new lines also showed decreased force with the mutation, mirroring the EHTs. Forces measured with the UC2 lines were significantly less that for WTC11. Interestingly, we observed no change in contractile kinetics for either cell background, suggesting no alteration in myosin cycling rate under loaded condition, in contrast with previous findings. This difference may be due to changes in myosin function under loaded conditions. Our results highlight the need for comprehensive biochemical and biophysical investigations of myosin mutations and emphasizes the importance of understanding the interplay between the myosin chemomechanical cycle and genetic background.
Robeson et al. (Sun,) studied this question.