Danicamtiv reduces myosin’s working stroke but enhances contraction by activating the thin filament

Figures

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  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 (Vmax) and actin affinity (Km) 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 Km 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…

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  Figure 2.. Stopped-flow kinetics measured with and without 10 μM danicamtiv. Black = DMSO control. Pink = 10 μM danicamtiv. A) The rates of ATP-induced actomyosin dissociation were measured in the stopped flow. Transients were well fitted by the sum of two exponential functions, where the observed rate of the fast phase (kfast) is plotted as a function of ATP concentration. Data were fitted with a hyperbolic function to obtain K1’ and k+2’. There are no differences in either K1’ or k+2’ with or without danicamtiv (P = 0.64 and P = 0.99, respectively). Each point represents an independent measurement over 3 experimental days. B) The rate of ADP release from actomyosin was measured using stopped flow techniques, and the fluorescence transients were fitted with single exponential functions. Note, both 0 and 10 μM danicamtiv are plotted and overlay. There is no statistically significant difference in the rate of ADP release the absence or presence of danicamtiv (P = 0.96). C) The overall ADP binding affinity to actomyosin was measured by mixing an actomyosin solution containing increasing concentrations of ADP with 50 μM ATP (concentrations after mixing) measured using a competition experiment.…

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  Figure 3.. Single molecule optical trapping reveals that danicamtiv reduces the size of myosin’s working stroke without altering detachment kinetics. Black = DMSO control. Pink = 10 μM danicamtiv. A) Cartoon schematic of the optical trapping assay. An actin filament is strung between two optically-trapped beads and lowered onto a pedestal bead sparsely bound with myosin. B) Optical trapping data traces showing the stochastic binding of myosin to actin. Binding interactions are shown in grey or pink and detached states are shown in black. C) Time forward ensemble averages of myosin’s working stroke reveal a ~50% reduction in the size of myosin’s total working stroke in the presence of danicamtiv. D) The cumulative distribution of the total working stroke displacements at 10 μM ATP is well fit by a single cumulative Gaussian function (dotted lines) with average values of 4.9 ± 9.7 nm versus 3.0 ± 9.0 nm for DMSO control and 10 μM danicamtiv, respectively (P < 0.001 using a two-tailed T-test). N = 2076 binding interactions for control and 4776 binding events for 10 μM danicamtiv. E) The cumulative distributions of attachment durations at 10 μM ATP. Single exponential functions were fit to the…

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  Figure 4.. Danicamtiv does not alter myosin’s load-dependent detachment kinetics at 1 mM ATP. Black = DMSO control. Pink = 10 μM danicamtiv. A) An isometric force clamp was used to maintain actin at an isometric position during myosin binding interactions. To do this, the motor bead (M) was moved to hold the transducer bead (T) at an isometric position. Data traces are shown. B) Plots of actomyosin attachment duration versus the average resistive force applied during the binding event. Data are exponentially distributed at each force. Each point represents an independent actomyosin binding interaction. The data were fitted with the Bell equation using maximum likelihood estimation and 95% confidence intervals were calculated for each parameter by bootstrapping. The detachment rate in the absence of load, k0, was not different between control and 10 μM danicamtiv, 61 (−21/+40) vs 89 (−18/+24) s−1 (P = 0.17). These values are consistent with our measurements of the rates of ADP release from stopped-flow experiments. The distance to the transition state, d, which measures the load-sensitivity of the detachment rate, was not different between control and 10 μM danicamtiv, 1.40(−0.37/+0.46) vs…

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  Figure 5.. Danicamtiv increases myosin’s attachment rate in a single turnover stopped flow assay. Black = DMSO control. Pink = 10 μM danicamtiv. A) Schematic conceptually describing the single turnover assay used to measure myosin’s attachment and detachment rates. In this assay, myosin and pyrene labeled actin are pre-incubated and then mixed with a sub-saturating concentration of ATP. The pyrene fluorescence increases as myosins detach from actin, and the increase in fluorescence reports the rate of detachment of myosin from actin (kdet). Myosin then reattaches to actin, quenching the fluorescence and reporting the attachment rate (katt). B) Fluorescence transients from the single turnover assay. Data were fitted as described in the Supplemental Methods. C) The average second-order rate of detachment (kdet) was similar with and without 10 μM danicamtiv (3.8 ± 0.9 vs. 4.2 ± 0.8 s−1; P = 0.23). D) The second-order rate of attachment (katt) increased with the addition of 10 μM danicamtiv (0.0040 ± 0.0002 vs 0.0063 ± 0.0007 μM−1·s−1; P = < 0.001). For C and D, the thick lines show the average values, and the error bars show the standard deviation. The individual points are the fitted…

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  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…