Ensemble force changes that result from human cardiac myosin mutations and a small-molecule effector

Abstract

Cardiomyopathies due to mutations in human β-cardiac myosin are a significant cause of heart failure, sudden death, and arrhythmia. To understand the underlying molecular basis of changes in the contractile system's force production due to such mutations and search for potential drugs that restore force generation, an in vitro assay is necessary to evaluate cardiac myosin's ensemble force using purified proteins. Here, we characterize the ensemble force of human α- and β-cardiac myosin isoforms and those of β-cardiac myosins carrying left ventricular non-compaction (M531R) and dilated cardiomyopathy (S532P) mutations using a utrophin-based loaded in vitro motility assay and new filament-tracking software. Our results show that human α- and β-cardiac myosin, as well as the mutants, show opposite mechanical and enzymatic phenotypes with respect to each other. We also show that omecamtiv mecarbil, a previously discovered cardiac-specific myosin activator, increases β-cardiac myosin force generation.

Figure 1. The FAST Output for an Unloaded Human β-Cardiac sS1 In Vitro Motility Experiment

For this and all unloaded velocity experiments, filaments tracked for at least ten consecutive frames were included in the analysis, velocity and filament lengths were averaged over five consecutive frames, a 20% tolerance filter was applied to eliminate intermittently moving filaments with velocities fluctuating higher than 20% of their mean velocity, and stuck filaments were excluded from the scatterplot analysis. (A) Scatterplot of filament length and velocities. Maximum velocity of each filament is in blue (triangle), and lower velocities are in gray (circles). The solid line is the fit to single exponential decay function to the maximum velocities (blue) to obtain the PLATEAU velocity. The dashed line marks the mean of the top 5% velocities (TOP5%). (B) A 20%tolerance-filtered filament velocity distribution excluding stuck filaments. The line marks the mean of the filtered velocity distribution (MVEL₂₀; subscript 20 is for the percent tolerance value). (C) The unfiltered filament velocity distribution excluding stuck filaments. The line marks the mean of the unfiltered distribution (MVEL). Filaments with a mean velocity less than one pixel length per second (80 nm/s) are classified as stuck. %STUCK represents the time-weighted fraction of stuck filaments. (D) Filament length distribution. The line marks the mean of the distribution. (E) Mean of the velocity distribution (solid line) and TOP5% velocity (dashed line) from the data subjected to different levels of tolerance filtering (see the Supplemental Experimental Procedures). Lower percent tolerance values indicate more stringent filtering allowing only filaments with smoother movement. Asterisk (*) is for the unfiltered velocity distribution. See also Figure S1.

Figure 2. Utrophin Causes Actin Filaments to Glide with Intermittent Stops in the Loaded In Vitro Motility Assay

(A) Cartoon representation of the loaded in vitro motility setup with human β-cardiac sS1 and utrophin on a surface coated with the anchoring protein SNAP-PDZ18 (light blue). Myosin (red) and utrophin (green) are anchored on the surface via the same attachment system involving binding of a C-terminal eight-residue peptide that specifically binds to the SNAP-PDZ18. Approximately 400-fold less utrophin (1 nM) than myosin (400 nM) is required to inhibit actin filament (yellow) movement. Smoothly moving actin filaments encounter utrophin at their front (minus) end and stop moving temporarily (see Movies S1, S2, S3, and S4). (B) Wild-type β-cardiac sS1 instantaneous velocity-time trajectories for an unloaded actin filament (blue), a stuck filament (red), and a transiently loaded filament (green) with utrophin. Dashed lines and error bars mark the mean velocity and the SD of instantaneous velocities, respectively, along the trajectories. Unloaded and stuck intervals of the transiently loaded trajectory are marked by blue and red, respectively, over the dashed mean velocity line. (C) Effect of utrophin on gliding filaments is explained by a model in which utrophin stops filament gliding at the maximal velocity (Supplemental Experimental Procedures). Stars on the curve are for the unloaded (blue), stuck (red), and transiently loaded (green) velocity trajectories in (B). (D) Heatmaps show the probability density for experimental measurements of filament mean velocity and velocity dispersion at different utrophin concentrations (upper left corner). The white solid line is for the best parabola fit to aggregated probability densities from all utrophin concentrations. The yellow vertical line marks the unloaded TOP5% velocity. See also Figure S2.

Figure 3. Percent Time Mobile Is the Measure of Actin-Gliding Inhibition by Utrophin

(A and B) Percent mobile filaments (A) and MVEL (B) as a function of utrophin concentration. (C) The stop model fits well to human β-cardiac sS1 loaded in vitro motility percent time mobile data. Percent time mobile is equal to the product of percent mobile filaments (A) and MVEL (B) normalized by unloaded TOP5% velocity. Blue solid and black dashed lines are for the best stop model and single exponential fits, respectively. K_S is indicated by the vertical dashed blue line. Each loaded in vitro motility point is the average from ≥1,000 filament tracks and ≥5,000 instantaneous velocities averaged over five frames. Confidence intervals are the SEM from at least four movie replicates. (D) Alternative representation of β-cardiac sS1 loaded motility data as stuck propensity, which is equivalent to percent time stuck (100% − percent time mobile) divided by percent time mobile. Unloaded stuck propensity is subtracted from each point. The line shows the best linear equation fit to the data points. See also Figure S3.

Figure 4. α-Cardiac sS1 Has Nearly 3-Fold Higher Actin-Activated Enzymatic Activity and Unloaded Gliding Velocity but Generates Less Force Than β-Cardiac sS1

(A) Representative unloaded velocity scatterplots for α- (left) and β-human cardiac sS1 (right), as described in Figure 1. For description of lines, see Figure 1A (a 20% tolerance filter was applied). (B) Actin-activated ATPase rates for α (red) and β (blue) human cardiac sS1. Solid lines show the best Michaelis-Menten fits for the ATPase rates (rate = k_cat[actin]/(K_m+[actin]). Dashed lines mark the K_m and half maximal ATPase rates (0.5 k_cat). Confidence intervals are the SEM from three replicate measurements for each of three myosin preparations. (C) Representative loaded in vitro motility percent time mobile data for α (red) and β (blue) human cardiac sS1. Lines show the best stop model fit to percent time mobile data. Dashed lines mark K_S values determined from the fit. Confidence intervals are the SEM from at least four movie replicates. (D) Normalized α-cardiac sS1 motility parameters (TOP5%, PLATEAU, K_S; filled bars) and ATPase (k_cat, K_m; empty bars) values with respect to β-cardiac sS1. For unloaded motility parameters, 95% confidence intervals are the SEM from at least four replicate movies from three different preparations of sS1. For K_S, the 95% confidence interval is estimated from 100 iterations of stop model fitting to bootstrapped data (Supplemental Experimental Procedures). For ATPase values, rates are the average of at least three replicates from three different preparations of sS1 (Table S1). See also Figure S4.

Figure 5. M531R and S532P Have Opposite Impact on WT β-Cardiac sS1 Enzymatic and Mechanical Activity

(A) M531 (green) and S532 (orange) are side by side on the actin-binding interface. The inset shows a close-up of the mutation positions (for details of the structural model, rendered using Pymol, see the Supplemental Experimental Procedures). (B) Representative unloaded velocity scatter plots comparing PLATEAU and TOP5% velocities for WT (blue), M531R (green), and S532P (orange). For description of lines, see Figure 1A (a 20% tolerance filter was applied). (C) Actin-activated ATPase rates for WT β-cardiac sS1 (blue), M531R (green), and S532P (orange). For description of lines and estimation of confidence intervals, see Figure 4B and Table S1 legend. (D) Representative unfiltered velocity distributions comparing M531R β-cardiac sS1 (green) and WT (blue). MVEL values for M531R and WT are marked on the velocity axis (inverted triangles). Velocity distributions are from four movies for WT and M531R collected on the same slide. (E) Lines show the best stop model fit to unfiltered percent time mobile loaded motility data. Dashed lines indicate the K_S determined from the fit. Confidence intervals are the SEM from at least four movie replicates. (F) M531R (green) and S532 (orange) motility (TOP5%, PLATEAU, K_S; filled bars) and ATPase (k_cat, K_m; empty bars) values normalized to WT values. For estimation of confidence intervals, see Figure 4D (legend) and Table S1.

Figure 6. Omecamtiv Mecarbil Binding to Cardiac sS1 Increases Ensemble Force Generation but Decreases Its Unloaded Velocity

(A) MVEL decreases with increasing concentration of omecamtiv mecarbil for both α- (red, inset) and β-cardiac (blue) sS1. Lines show the best binding isotherm fit to data (Supplemental Experimental Procedures). Dashed lines mark the KD determined from the fits. Confidence intervals are the SEM from at least four movie replicates. (B) Representative loaded in vitro motility data for β-cardiac sS1 at different concentrations of omecamtiv mecarbil (●, 0 µM; :▲, 0.25 µM; ;▼, 0.5 µM; ■, 2 µM). Increase in color intensity indicates increase in omecamtiv mecarbil concentration. Solid lines are for the best stop model fits to percent time mobile data. Dashed lines mark K_S determined from the fits. Confidence intervals are the SEM from at least four movie replicates. (C) K_S determined from stop model fits to β-cardiac sS1 loaded motility data. 95% confidence intervals are estimated from 100 iterations of stop model fitting to bootstrapped data (Supplemental Experimental Procedures).

Figure 7. The Mutations M531R and S532P Are Likely to Cause Local Structural Changes with a Major Impact on the Actin-Myosin Interaction

Local environments of M531 (green) and S532 (orange) correlate with the sequence conservation profiles in Figure S6. Both positions are on an α helix in close proximity to a patch of negatively charged residues (red) on actin. M531 (green) is buried in the hydrophobic core, and S532 (orange) is exposed to solvent. See also Figure S5.