Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function

Abstract

Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including β-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human β-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human β-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human β-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.

Keywords: heart disease; optical trapping; single-molecule force measurements.

Fig. 1.

Actin-activated ATPase. (A) Actin-activated ATPase activities for purified human WT (black) and R453C mutant (red) β-cardiac S1 at 23 °C. The data points shown are the average of multiple experiments (n = 4–6), from three protein preparations. The error bars represent SEM. The data were fit to the Michaelis–Menten equation to obtain the k_cat and K_m values shown in Table S1. At 23 °C, WT has a k_cat of 7.4 ± 0.4 s⁻¹ and K_m of 38 ± 4 µM, whereas R453C has a k_cat of 5.0 ± 0.2 s⁻¹ and K_m of 28 ± 4 µM. (B) The ATPase cycle illustrates the basic myosin states along with the presumed position of the lever arm. Strong actin-binding states of myosin are indicated in red, the weakly associated states of myosin in yellow, and actin in gray. The time spent for one cycle (t_c) is equal to the sum of the times spent in the weakly (t_w) and the strongly bound states (t_s).

Fig. 2.

Length-dependent actin filament velocity at 23 °C and 30 °C. The average length and average velocity distributions are shown for WT in black (A and C) and for R453C in red (B and D) at 23 °C (A and B) and 30 °C (C and D). For WT, average filament and velocity data were combined from ≥3 independent motor preparations and ≥3,000 filament tracks. For R453C, average filament and velocity data were combined from ≥2 independent motor preparations and ≥2,000 filament tracks. The line in gray represents the fit of the experimental velocity (v_exp) and the actin length (l) to the following equation: (15). The d values are from our optical trap measurements (6 nm), and the t_c values are from our actin-activated ATPase measurements. The fitting parameters are k, which represents the number of motor heads per unit length of actin filament, and t_s. The maximum unloaded in vitro velocity (v₀) is equal to d/t_s.

Fig. 3.

Intrinsic and ensemble force measurements of purified human WT and R453C mutant β-cardiac S1 at 23 °C. WT is shown in black and R453C in red. (A) Single-molecule intrinsic force measurements. Each data point is an independent protein preparation, which is averaged over two or more molecules. Each single molecule measurement contained more than 50 events on average. The error bars represent SEM. The horizontal bar is the average of all of the measurements. For the R453C, the average intrinsic force produced by a single S1 molecule is 2.1 ± 0.1 pN (n = 10), and for the WT S1, it is 1.4 ± 0.1 pN (n = 14). (B) Loaded in vitro motility measurements. For both WT and R453C, percent actin filaments moving at varying α-actinin concentrations were examined for at least four motor preparations. Error bars represent SEM (n = 2–5 surfaces) (Materials and Methods).

Fig. 4.

Human β-cardiac myosin S1 motor domain bound to ATP. Residues 1–808 of human β-cardiac myosin S1, in gray, bound by the ventricular ELC in blue. As shown in the right panel, the R453C residue (red) binds in the linker between the α-helix (yellow) and strand five (dark green) of the β-pleated sheet (green). This region in the core of the motor domain is also referred to the transducer (22). This figure is a combination of Protein Data Bank structure 2MYS and 4DB1.