Figure 1. Steady state properties of β-cardiac myosin treated with danicamtiv.

A) The steady-state myosin ATPase rate was measured using the NADH coupled assay. The steady state myosin ATPase rate is plotted verses a function of actin concentration. Data were fitted by a hyperbolic function to calculate the maximal cycling rate (V_max) and actin affinity (K_m) with the Michaelis Menten equation. Treatment with 10 μM danicamtiv increased the maximal rate by ~1.2 fold from 5.9 to 7.0 s⁻¹ (P = 0.028) and decreased the K_m from 14.0 to 8.1 μM (P = 0.01). Each point represents the average rate from four independent trials with error bars showing the standard deviation. Statistical testing done using a 2-tailed T-test. Black = DMSO control. Pink = 10 μM danicamtiv. B) Speed of actin translocation in the unregulated in vitro motility assay. The addition of 10 μM danicamtiv decreased motility speed ~55% (P = 4×10⁻⁶). Thick horizontal lines show the average speed with standard deviation shown by the thin horizontal lines. Points represent the average speed of all filaments in a field of view for a single technical replicate measured across N = 3 independent experiments. Statistical testing done using a 2-tailed T-test. C) Scheme of myosin’s mechanochemical cross-bridge cycle. Myosin’s rate limiting step is actin attachment, so the predominant population of motors reside in the pre-working stroke M.ADP.Pi state during steady state cycling. The steady-state ATPase is thus limited by actin attachment (k_att) which is rapidly followed by the mechanical working stroke and phosphate release. In vitro motility speed is limited by the ADP release rate (k₊₅’).