Mutation that causes hypertrophic cardiomyopathy increases force production in human β-cardiac myosin

Myosin Mutant That Breaks Hearts

Genetically linked hypertrophic cardiomyopathy [HCM; also known as familial hypertrophic cardiomyopathy (FHC)] afflicts 1 in 500 people (1). The most prominent phenotypes of HCM include increased arrhythmias and sudden cardiac arrest, even in otherwise healthy adults and children. The HCM pathologies are associated with increased thickening of the ventricular wall and decreased ventricular cavity volume, accompanied by diastolic dysfunction. More than half (60%) of all HCM cases are caused by mutations in sarcomeric proteins (2). How these mutations trigger the disease phenotype is not clear (3). Several hypotheses have been proposed, including conflicting models where HCM mutations cause either decreased or increased force generation during contraction (3). In PNAS, Sommese et al. (4) provide critical insight, determining the mechanical and kinetic basis for one of the most malignant HCM-causing mutations, R453C, in the β-cardiac myosin II heavy chain.

Sommese et al. characterize the biochemical and biophysical effect of the R453C missense mutation engineered directly into the motor domain of recombinant human β-cardiac myosin, expressed and purified from a novel muscle cell-based protein expression system (5). This work opens the door for in vitro analysis of the more than 300 mutations in the cardiac myosin heavy chain that cause heart disease in humans (3). Stepping through that door is a first step toward development of targeted treatments for these devastating genetic disorders.

Sommese et al. find that the malignant R453C mutation (Fig. 1) results in subtle but significant changes in the actin-activated ATPase activity, a 30% decrease in V_max (activity at saturating actin) and a 30% decrease in K_m (actin concentration needed to achieve 1/2V_max), suggesting increased actin binding affinity during ATPase cycling. Mechanical measurements show a 50% increase in the intrinsic force generated by single myosin molecules per ATP hydrolyzed, resulting in an overall increase in force generated by an ensemble of myosins. The authors propose that these force increases cause the aberrant contractility seen in hearts with this disease.

Fig. 1.

HCM and dilated cardiomyopathy (DCM) causing β-cardiac myosin heavy chain mutations (A, magenta ribbons, R453C shown as magenta spheres) found in the myosin motor domain (4DB1 crystal structure) (3). The R453C HCM mutation is located between the HO helix (orange) and the 7-stranded β-sheet (green). Relay helix (cyan) and bound nucleotide (AMPPNP nucleotide, red spheres) shown for reference. (B) Crystal structure of β-cardiac myosin (4DB1) suggests potential hydrogen bonds between the R453 side chain and neighboring backbone oxygen atoms (Q451, and T449 shown), as well as the hydroxyl oxygen on S260 in the AMPPNP-bound state. These interactions would by altered in the R453C mutant (modeled in C), which could affect the flexibility of the β-sheet.

Intrinsic Force of a Myosin Mutant

Myosin generates force to power and control motility in all eukaryotic cells. Muscle cells, with their near crystalline sarcomeric arrays of myosin and actin filaments, are some of the most well-studied examples. Muscle cells orchestrate the mechanical work performed by individual myosins to power muscle contraction and movement. Muscle myosin performs mechanical work by converting the chemical free energy of ATP hydrolysis into force-generating changes in myosin structure (6), which cause the sarcomere to shorten. The authors view the total force of the ensemble of myosin motors in the muscle as being proportional to the average force generated by each individual myosin molecule and the amount of time an average molecule spends generating force, according to the equation . Here, F is the total ensemble force, f is the ensemble average intrinsic force generated by the individual myosins per ATP hydrolyzed, N is the total number of motors in the ensemble, and ts/tc is the duty ratio defining the fraction of time during the ATPase cycle that cycling myosins spend actively generating force.

The intrinsic force, f, reflects the mechanical properties of individual myosin molecules as they transition from a structural state that binds actin weakly (>1 μM affinity) to one that binds actin strongly (nM affinity). Single myosin molecules are often modeled as a simple spring following Hooke’s law: . Here, x is the ensemble average displacement, the change in myosin structure associated with force generation that is often attributed to the rotation of myosin’s catalytic and light chain-binding domains during force generation, and k is the ensemble-average spring constant of the force-sustaining actomyosin cross-bridge.

Using the sliding filament assay, Sommese et al. find that the R453C mutation causes an increase in the total force, F, produced by an ensemble of mutant myosin molecules. Although the total cycle time, tc, is increased by ∼40%, the time spent in the strong binding state, ts, is also increased by a similar amount, and thus the R453C mutation does not change the motor’s duty cycle—the mutant and WT motors spend the same fraction of their ATPase cycles generating force. Using single-molecule optical trapping, the authors show that the mutation increases the intrinsic force of a single cardiac myosin motor domain without changing the displacement step size, x. This implies that the mutation increases the intrinsic spring constant k of the motor. The authors suggest that this results from an increase in the stiffness of the myosin molecule in the region of the bend between a prominent α-helix in the upper 50-kDa actin-binding subdomain and the seven-stranded β-sheet (Fig. 1). This makes sense because the β-sheet is hypothesized to be part of the spring in the myosin catalytic domain that mediates force transduction (6).

This study relied on single-molecule and steady-state kinetics approaches, which required small amounts of protein. In the future, a more detailed characterization of the transient kinetics and mechanics, as well as molecular dynamics simulations, will reveal how the mutation affects critical steps in the ATPase cycle. Because the duty cycle did not change, even though the V_max decreased by 30%, the mutation most likely affects steps in both actin-bound and actin-detached states, potentially even the energetics of actomyosin binding as suggested by the decrease in ATPase K_m.

Treating HCM by Targeting Myosin

Determining how single residue missense mutations cause HCM has not been easy. Sommese et al. point out that earlier studies using animal models of HCM should be interpreted with caution. They comment, “observing the effects of such a mutation in a nonhuman protein background where there are many other residue differences from the human sequence is far from ideal.” The effect of a single mutation like R453C on myosin function is subtle, as indicated by the

Sommese et al. point out that earlier studies using animal models of HCM should be interpreted with caution.

30% decrease in V_max, 30% decrease in K_m, and 50% increase in force seen in this study. However, the mutation is strongly malignant, so these changes are far from trivial. Although mammalian striated muscle myosins are all highly homologous, a small number of polymorphisms between species cause subtle differences in biochemical and mechanical properties. Therefore, interpreting the effect of a substitution that causes human disease by assessing the phenotype that the mutation causes in an animal model is risky. To understand how genetic disorders in human proteins cause human diseases, we need experiments on human proteins and, in the long run, human cells.

So, how do we stop a genetic heartbreaker like R453C? The identification of a biochemical and biophysical phenotype is the first step. Now that we know that this mutation causes a subtle decrease in ATPase cycling and an increase in force production by changing the stiffness of the myosin motor domain, we can investigate how these changes affect heart function and development. We can also develop assays that specifically target these properties for drug discovery. The recent development of a drug that treats heart failure by modulating cardiac myosin (7) suggests this will be possible.