Cardiac myosin inhibitor MYK-581 prevents development of hypertrophic cardiomyopathy phenotype in human iPSC models

Abstract Background Mutations in genes encoding for sarcomeric proteins such as myosin (MYH7) or troponin T (TNNT2) are linked to hypertrophic cardiomyopathy (HCM). Human induced pluripotent stem (hiPSC) models recapitulate key cellular features of HCM (e.g., cellular hypertrophy, myofibrillar disarray, mitochondrial dysfunction). Cardiac myosin inhibitors (CMIs) such as mavacamten are used clinically with symptomatic obstructive HCM patients, but whether they can prevent the development of HCM is not known. Here, we test the hypothesis that CMI treatment targeting the molecular defects common to many HCM-linked mutations can prevent the development of HCM in vitro. Objective To determine whether a mavacamten analogue (MYK-581) can prevent the development of the cellular HCM phenotype in hiPSC-models carrying pathogenic HCM gene variants within thin (TNNT2-I79N, TNNT2-R92Q) and thick filaments (MYH7-R403Q). Method After differentiation to cardiomyocytes (CMs), hiPSC-CMs were cultured from Day 16 to 30 in a well-established maturation media (1,2) that promotes adult-like electrophysiology, Ca2+ handling and mitochondrial metabolism. To test the effect of CMI treatment, MYK-581 (0.1 µM, 14 days) or vehicle was included in the maturation media. Afterwards, cellular phenotypes (cell volume, sarcomere organization morphology, intracellular Ca2+ handling, action potential AP, mitochondrial function) were assessed and compared to isogenic control hiPSC-CM using published protocols (3). Result MYK-581 treatment prevented the development of cellular hypertrophy and restored normal sarcomere organization in hiPSC-CM HCM models (Fig. 1A). Metabolically, all three HCM hiPSC-CM models exhibit significantly impaired mitochondrial function (Fig. 1B, left panel), which is also commonly observed in HCM patients. Treatment with MYK-581 prevented the mitochondrial dysfunction, as evidenced by normalized mitochondrial oxygen consumption and ATP production rates both in thin filament and thick filament hiPSC-CM HCM models (Fig. 1B, right panel). MYK-581 treatment restored intracellular Ca2+ handling by normalizing cytosolic Ca2+ buffering parameters (Table 1), which normalized end-diastolic Ca2+ concentrations and Ca2+ transient decay kinetics (Tau, s: Isogenic control:0.38±0.12, TnT-I79N+vehicle: 0.45±0.12, TnT-I79N+MYK: 0.38±0.11, p0.01 vs I79N+vehicle, n=34-45 cells per group). MYK-581 treatment also prevented the pro-arrhythmic AP triangulation (Fig. 1C) and AP alternans that is typically observed in HCM hiPSC-CMs. Conclusion These findings in human cardiomyocytes carrying pathogenic HCM mutations suggest that CMI treatment with MYK-581 can prevent the development of cellular features of HCM. Our results suggest that further studies beyond in vitro models are warranted.Figure 1.Cellular HCM phenotype Table 1.**p0.001 vs vehicle, N=18-25