Figure 6. Danicamtiv increases motility speed in the presence of regulatory proteins and these effects on muscle contraction can be recapitulated computationally.
Black = DMSO control. Pink = 10 μM danicamtiv. A and B) Unregulated in vitro motility speed as a function of the myosin concentration on the flowcell surface. The speed decreases if there is not at least one active myosin head bound to actin at any given time. Thus, if the duty ratio increases with drug, less myosin will be required to reach saturation. 10 μM danicamtiv decreases motility speed at higher myosin concentrations but increases speed at low myosin concentrations, indicative of a higher duty ratio, despite having a smaller working stroke. A) shows the measured speed and B) shows normalized data. ~40 filaments were tracked across four fields of view from two different experimental preparations. C and D) Regulated in vitro motility speed using thin filaments decorated with troponin and tropomyosin as a function of calcium. The data were fitted with the Hill equation and the fitted values ± standard error are: Vmax values are 386 ± 8 vs. 135 ± 7 nm/s (P < 0.001), pCa50 values are 5.76 ± 0.02 vs. 6.1 ± 0.09 (P = 0.01), and the Hill coefficients are 3.4 ± 0.4 vs 3.1 ± 1.5 (P = 0.85) for the control vs. 10 μM danicamtiv, respectively C) Shows the measured speed and D) shows the data normalized to the fitted Vmax and Vmin. Each point represents average speed with error bars showing the standard deviation of ~40–60 filaments imaged from 4–6 fields of view from 2–3 experimental replicates. E) Simulated force-calcium relationship from FiberSim. To simulate danicamtiv, we incorporated increased actin attachment, reduced myosin working stroke, and an increase in the population of active myosin heads. The simulations recapitulate the shift seen in the motility experiments. 5 replicates were conducted, the shaded region shows the range of values, and the solid line shows the mean. F) Simulated twitch in response to a calcium transient using the same simulation parameters. Danicamtiv increases the maximal force, slows kinetics, and G) increases the force-time integral.μ