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) ).