Dilated cardiomyopathy mutation in the converter domain of human cardiac myosin alters motor activity and response to omecamtiv mecarbil

Abstract

We investigated a dilated cardiomyopathy (DCM) mutation (F764L) in human β-cardiac myosin by determining its motor properties in the presence and absence of the heart failure drug omecamtive mecarbil (OM). The mutation is located in the converter domain, a key region of communication between the catalytic motor and lever arm in myosins, and is nearby but not directly in the OM-binding site. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing the F764L mutation, and compared it to WT with in vitro motility as well as steady-state and transient kinetics measurements. In the absence of OM we demonstrate that the F764L mutation does not significantly change maximum actin-activated ATPase activity but slows actin sliding velocity (15%) and the actomyosin ADP release rate constant (25%). The transient kinetic analysis without OM demonstrates that F764L has a similar duty ratio as WT in unloaded conditions. OM is known to enhance force generation in cardiac muscle while it inhibits the myosin power stroke and enhances actin-attachment duration. We found that OM has a reduced impact on F764L ATPase and sliding velocity compared with WT. Specifically, the EC₅₀ for OM induced inhibition of in vitro motility was 3-fold weaker in F764L. Also, OM reduces maximum actin-activated ATPase 2-fold in F764L, compared with 4-fold with WT. Overall, our results suggest that F764L attenuates the impact of OM on actin-attachment duration and/or the power stroke. Our work highlights the importance of mutation-specific considerations when pursuing small molecule therapies for cardiomyopathies.

Keywords: actin; cardiomyopathy; enzyme kinetics; heart failure; myosin; omecamtiv mecarbil.

Scheme 1.

Scheme of myosin ATPase cycle.

Figure 1.

Steady-state ATPase and in vitro motility. A, the actin-activated ATPase activity of purified M2β-S1 WT and F764L in MOPS 20 buffer at 25 °C was determined as a function of actin concentration. The data at each actin concentration represents the average ± S.E. from 5 to 6 separate preparations. B, the sliding velocity of M2β-S1 WT and F764L in MOPS 20 buffer was analyzed manually via ImageJ. 120 filaments from 4 different protein preparations (30 filaments from each, 0.24–0.4 μm M2β-S1 loading concentration) were pooled together and fit to the Gaussian function. The average velocity is 1547 ± 19 nm/s for WT, and 1300 ± 17 nm/s for F764L.

Figure 2.

Impact of OM on steady-state motor properties. A, the actin-activated ATPase of purified M2β-S1 F764L in the presence of 0.1% DMSO or 10 μm OM. The data at each actin concentration represents the average ± S.D. from three separate preparations. B, the ATPase activity of purified M2β-S1 WT and F764L was determined as a function of OM concentration at 20 μm actin. Traces were fit to a dose-response curve to determine the EC₅₀. The data at each OM concentration represents the average ± S.E. of three separate preparations. C, in vitro motility was performed in the presence of varying concentrations of OM (0–10 μm in 0.1% DMSO) to determine the EC₅₀ with 0.4 μm M2β-S1 WT or F764L. The relative velocities of M2β-S1 WT and F764L are normalized to the velocities at 0 μm OM from three separate preparations (90 filaments). Error bars represent mean ± S.E. D, in vitro motility was performed in the presence of varying concentrations of OM (0–200 μm in 1% DMSO). Relative velocities of M2β-S1 WT or F764 were normalized to the velocities at 0 μm OM from two different protein preparations. Inset highlights the difference in velocity at high OM concentrations (velocity at 200 μm OM: WT, 4.4 ± 0.2 nm/s; F764L, 12.8 ± 0.6 nm/s). Error bars represent mean ± S.E. In the OM titration experiments shown in C and D, additional KCl was added to the activation buffer to bring it to 100 mm KCl.

Figure 3.

Single turnover measurements. Single turnover sequential mix stopped-flow experiments were performed by mixing 0.25 μm M2β-S1 with 0.2 μm mant-ATP, aged for 10 s, and then mixed with varying concentrations of actin (2.5–50 μm) in the presence of 0.1% DMSO or 10 μm OM. Data were collected from 3 to 4 different protein preparations. The fast phase of the fluorescence transients was plotted as a function of actin concentration and fit to a hyperbolic function for WT (A) and F764L (B). C, representative fluorescence transients in the presence of 40 μm actin (average of 2–3 transients) fit to a two-exponential function (values for the fast phase rate constant and relative amplitude for WT DMSO, k = 6.24 ± 0.03 s⁻¹; A = 0.90; WT OM, k = 4.23 ± 0.03 s⁻¹; A = 0.90; F764L DMSO, k = 5.68 ± 0.02 s⁻¹, A = 0.89; F764L OM, k = 6.91 ± 0.03 s⁻¹, A = 0.91).

Figure 4.

ATP binding and hydrolysis. Tryptophan fluorescence enhancement was used to monitor ATP binding/hydrolysis by mixing 1 μm M2β-S1 WT or F764L with varying concentrations of ATP. The fluorescence transients were fit to a two-exponential function. The observed fast phase was plotted as a function of ATP concentration and fit to a hyperbolic function to determine maximum rate of ATP hydrolysis and apparent affinity for ATP in the presence of 0.1% DMSO (A) or 10 μm OM (B). C, representative fluorescence transients (average of 2 transients) in the presence of 25 μm ATP are shown fit to a two-exponential function (values for the fast phase rate constant and relative amplitude for WT DMSO, k = 61.95 ± 0.86 s⁻¹; A = 0.86; WT OM, k = 47.20 ± 0.78 s⁻¹; A = 0.92; F764L DMSO, k = 39.7 ± 0.69 s⁻¹, A = 0.83; F764L OM, k = 27.98 ± 0.60 s⁻¹, A = 0.82).

Figure 5.

ATP binding to acto-myosin. ATP-induced dissociation from pyrene actin was performed by mixing a complex of M2β-S1:pyrene actin (0.375 μm M2β-S1 and pyrene actin) with a series of ATP concentration (4 to 250 μm). The fluorescence transients were fit to a two-exponential function at all ATP concentrations. The observed fast rate constant was hyperbolically dependent on ATP concentrations in the presence of 0.1% DMSO (A) or 10 μm OM (B). C, representative fluorescence transients in the presence of 25 μm ATP (average of 2 transients) were fit to a two-exponential function (values for the fast phase rate constant and relative amplitude for WT DMSO, k = 222.6 ± 5.0 s⁻¹; A = 0.92; WT OM, k = 213.2 ± 7.3 s⁻¹; A = 0.94; F764L DMSO, k = 207.1 ± 4.8 s⁻¹, A = 0.91; F764L OM, k = 232 ± 8.8 s⁻¹, A = 0.88).

Figure 6.

Actin-activated phosphate release. The actin-activated phosphate release rate constants in the presence of OM were examined by mixing M2β-S1 (0.225–1 μm) with substoichiometric ATP, aged for 10 s, and mixed with varying actin concentrations. A, the MDCC-PBP fluorescence transients (average of 2–3 transients) in 10 μm actin were fit to a two-exponential function (values for the fast phase rate constant and amplitude for WT, k = 25.1 ± 0.9 s⁻¹, A = 0.32 ± 0.01; and F764L, k = 18.4 ± 0.8 s⁻¹, A = 0.21 ± 0.01) (inset shows the entire time course). B, the rate constant of the fast phase of the fluorescence transients were plot as a function of actin concentration and fit to a linear relationship to compare actin-activated phosphate release in F764L and WT in the presence of OM. The phosphate release rate constants (WT versus F764L) were also measured in the presence of a single actin concentration (30 μm) in the absence of OM (see Table 3).

Figure 7.

ADP release from acto-myosin. A, the ADP release rate constant was examined by mixing pyrene actomyosin·ADP (0.225 μm M2β-S1 and pyrene actin, 50 μm ADP) with varying ATP concentrations (10 to 750 μm) in the presence of 0.1% DMSO or 10 μm OM. Fluorescent transients were fit to a single exponential function. The rate constants were fit to hyperbolic function (k_obs = k_+D^′/(1 + K_app/[ATP])) to determine the ADP release rate constant (k_+D^′). B, the ADP release rate constant was also examined with mant-labeled ADP by mixing a complex of 0.5 μm M2β-S1, 1 μm actin, and 10 μm mant-ADP with 1 mm ATP. Representative fluorescence transients of mant-ADP release from actomyosin (average of 5 transients) fit to a single exponential function are shown (rate constant and amplitude at each condition; WT DMSO, k = 347.4 ± 7.6 s⁻¹, A = 0.38; WT OM, k = 348.9 ± 6.1 s⁻¹, A = 0.45; F764L DMSO, k = 232.1 ± 3.2 s⁻¹, A = 0.51; F764L OM, k = 251.6 ± 3.0 s⁻¹, A = 0.53).

Figure 8.

Structure of M2β-sS1 (PDB ID 5N69) highlighting the Phe-764 residue and OM-binding site. A, Phe-764 (F764) (green) is located at the interface of the relay helix (red) and converter domain (blue). B, F764 is involved in forming a hydrophobic interaction at the relay helix/converter domain interface. C, location of F764 in the M2β-sS1 OM-bound state. OM is shown in cyan, residues involved in direct OM binding interaction are shown in orange (27).