Solutions
Muscle mechanics solution concentrations were formulated by solving equations describing ionic equilibria (Godt and Lindley, 1982) and all concentrations are listed in mM unless otherwise noted. Skinning solution: 50 BES, 30.83 K propionate, 10 Na azide, 20 EGTA, 6.29 MgCl2, 6.09 ATP, 1 DTT, 20 BDM, 50 Leupeptin, 275 Pefabloc, and 1 E-64; with 1% Triton-X100 (wt/vol) and 50% glycerol (wt/vol). Storage solution: same as skinning solution without Triton-X100. Relaxing solution: pCa 8.0 (pCa = -log10[Ca2+]), 5 EGTA, 5 MgATP, 1 Mg2+, 0.3 Pi, 35 phosphocreatine, 300 U/mL creatine kinase, pH 7.0, at 200 ionic strength adjusted with Na methanesulfonate. Activating solution: same as relaxing solution, but with pCa 4.5.
Mavacamten (MYK-461) was purchased from Axon Medichem (Reston, VA, USA) and dissolved in dimethylsulfoxide (DMSO) to give a 1 mM stock solution. This was then mixed with relaxing and activating solutions to yield experimental solutions containing 0.5 µM mavacamten and 0.05% DMSO (vol/vol). The 0.5 μM mavacamten concentration was chosen as an intermediate between the IC50 value of 0.3 μM for inhibiting myosin ATPase activity in biochemical assays using murine and bovine myosin (though isometric force appeared less sensitive to mavacmaten than ATPase (Green et al., 2016) ) and plasma levels that effectively relieved left ventricular outflow tract obstruction in clinical trial patients (350-695 ng/ml = 1.28-2.54 μM; (Heitner et al., 2019) ).