COMMENTARY

Mutation that causes hypertrophiccardiomyopathy increases force productionin human β-cardiac myosinJoseph M. Muretta and David D. Thomas1

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota,Minneapolis, MN 55455

Myosin Mutant That Breaks HeartsGenetically linked hypertrophic cardiomy-opathy [HCM; also known as familial hy-pertrophic cardiomyopathy (FHC)] afflicts 1in 500 people (1). The most prominent phe-notypes of HCM include increased arrhyth-mias and sudden cardiac arrest, even inotherwise healthy adults and children. TheHCM pathologies are associated with in-creased thickening of the ventricular wall anddecreased ventricular cavity volume, accom-panied by diastolic dysfunction. More thanhalf (60%) of all HCM cases are caused bymutations in sarcomeric proteins (2). Howthese mutations trigger the disease phenotypeis not clear (3). Several hypotheses have beenproposed, including conflicting models whereHCM mutations cause either decreased orincreased force generation during contraction(3). In PNAS, Sommese et al. (4) providecritical insight, determining the mechanicaland kinetic basis for one of the most malig-nant HCM-causing mutations, R453C, in theβ-cardiac myosin II heavy chain.

Sommese et al. characterize the bio-chemical and biophysical effect of the R453Cmissense mutation engineered directly intothe motor domain of recombinant humanβ-cardiac myosin, expressed and purifiedfrom a novel muscle cell-based protein ex-pression system (5). This work opens the doorfor in vitro analysis of the more than 300mutations in the cardiac myosin heavy chainthat cause heart disease in humans (3). Step-ping through that door is a first step towarddevelopment of targeted treatments for thesedevastating genetic disorders.

Sommese et al. find that the malignantR453C mutation (Fig. 1) results in subtlebut significant changes in the actin-activatedATPase activity, a 30% decrease in Vmax (ac-tivity at saturating actin) and a 30% decreasein Km (actin concentration needed to achieve1/2Vmax), suggesting increased actin bindingaffinity during ATPase cycling. Mechanicalmeasurements show a 50% increase in the

intrinsic force generated by single myosinmolecules per ATP hydrolyzed, resulting inan overall increase in force generated by anensemble of myosins. The authors proposethat these force increases cause the aberrantcontractility seen in hearts with this disease.

Intrinsic Force of a Myosin MutantMyosin generates force to power and controlmotility in all eukaryotic cells. Muscle cells,with their near crystalline sarcomeric arraysof myosin and actin filaments, are some ofthe most well-studied examples. Muscle cellsorchestrate the mechanical work performedby individual myosins to power musclecontraction and movement. Muscle myosinperforms mechanical work by converting thechemical free energy of ATP hydrolysis intoforce-generating changes in myosin structure(6), which cause the sarcomere to shorten.The authors view the total force of the en-semble of myosin motors in the muscle asbeing proportional to the average force gen-erated by each individual myosin moleculeand the amount of time an average moleculespends generating force, according to theequation F = f ×Nðts=tcÞ. Here, F is thetotal ensemble force, f is the ensemble av-erage intrinsic force generated by the in-dividual myosins per ATP hydrolyzed, N isthe total number of motors in the ensemble,and ts/tc is the duty ratio defining thefraction of time during the ATPase cyclethat cycling myosins spend actively gener-ating force.

The intrinsic force, f, reflects the me-chanical properties of individual myosinmolecules as they transition from a structuralstate that binds actin weakly (>1 μM affinity)to one that binds actin strongly (nM affinity).Single myosin molecules are often modeledas a simple spring following Hooke’s law:f = kx. Here, x is the ensemble average dis-placement, the change in myosin structureassociated with force generation that is oftenattributed to the rotation of myosin’s catalytic

and light chain-binding domains duringforce generation, and k is the ensemble-average spring constant of the force-sustainingactomyosin cross-bridge.

Using the sliding filament assay, Sommeseet al. find that the R453C mutation causes anincrease in the total force, F, produced by anensemble of mutant myosin molecules. Al-though the total cycle time, tc, is increased by∼40%, the time spent in the strong bindingstate, ts, is also increased by a similar amount,and thus the R453C mutation does notchange the motor’s duty cycle—the mutantand WT motors spend the same fraction oftheir ATPase cycles generating force. Usingsingle-molecule optical trapping, the authorsshow that the mutation increases the intrinsicforce of a single cardiac myosin motor do-main without changing the displacement step

Fig. 1. HCM and dilated cardiomyopathy (DCM) caus-ing β-cardiac myosin heavy chain mutations (A, magentaribbons, R453C shown as magenta spheres) found in themyosin motor domain (4DB1 crystal structure) (3). TheR453C HCM mutation is located between the HO helix(orange) and the 7-stranded β-sheet (green). Relay helix(cyan) and bound nucleotide (AMPPNP nucleotide, redspheres) shown for reference. (B) Crystal structure ofβ-cardiac myosin (4DB1) suggests potential hydrogenbonds between the R453 side chain and neighboringbackbone oxygen atoms (Q451, and T449 shown), aswell as the hydroxyl oxygen on S260 in the AMPPNP-bound state. These interactions would by altered in theR453C mutant (modeled in C ), which could affect theflexibility of the β-sheet.

Author contributions: J.M.M. and D.D.T. wrote the paper.

The authors declare no conflict of interest.

See companion article on page 12607.

1To whom correspondence should be addressed. E-mail: ddt@umn.edu.

www.pnas.org/cgi/doi/10.1073/pnas.1310669110 PNAS | July 30, 2013 | vol. 110 | no. 31 | 12507–12508

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size, x. This implies that the mutation in-creases the intrinsic spring constant k of themotor. The authors suggest that this resultsfrom an increase in the stiffness of the my-osin molecule in the region of the bend be-tween a prominent α-helix in the upper 50-kDa actin-binding subdomain and the seven-stranded β-sheet (Fig. 1). This makes sensebecause the β-sheet is hypothesized to be partof the spring in the myosin catalytic domainthat mediates force transduction (6).

This study relied on single-molecule andsteady-state kinetics approaches, which re-quired small amounts of protein. In the fu-ture, a more detailed characterization of thetransient kinetics and mechanics, as well asmolecular dynamics simulations, will revealhow the mutation affects critical steps in theATPase cycle. Because the duty cycle did notchange, even though the Vmax decreased by30%, the mutation most likely affects steps inboth actin-bound and actin-detached states,potentially even the energetics of actomyosinbinding as suggested by the decrease inATPase Km.

Treating HCM by Targeting MyosinDetermining how single residue missensemutations cause HCM has not been easy.Sommese et al. point out that earlier studiesusing animal models of HCM should beinterpreted with caution. They comment,

“observing the effects of such a mutation ina nonhuman protein background where thereare many other residue differences from thehuman sequence is far from ideal.” Theeffect of a single mutation like R453C onmyosin function is subtle, as indicated by the

Sommese et al. pointout that earlier studiesusing animal models ofHCM should be inter-preted with caution.

30% decrease in Vmax, 30% decrease in Km,and 50% increase in force seen in this study.However, the mutation is strongly malignant,so these changes are far from trivial. Al-though mammalian striated muscle myosinsare all highly homologous, a small number ofpolymorphisms between species cause subtledifferences in biochemical and mechanical

properties. Therefore, interpreting the effectof a substitution that causes human diseaseby assessing the phenotype that the mutationcauses in an animal model is risky. To un-derstand how genetic disorders in humanproteins cause human diseases, we needexperiments on human proteins and, in thelong run, human cells.So, how do we stop a genetic heartbreaker

like R453C? The identification of a bio-chemical and biophysical phenotype is thefirst step. Now that we know that this mu-tation causes a subtle decrease in ATPasecycling and an increase in force productionby changing the stiffness of the myosin mo-tor domain, we can investigate how thesechanges affect heart function and develop-ment. We can also develop assays that spe-cifically target these properties for drugdiscovery. The recent development of a drugthat treats heart failure by modulating cardiacmyosin (7) suggests this will be possible.

1 Liew CC, Dzau VJ (2004) Molecular genetics and genomics ofheart failure. Nat Rev Genet 5(11):811–825.2 Ramaraj R (2008) Hypertrophic cardiomyopathy: Etiology,diagnosis, and treatment. Cardiol Rev 16(4):172–180.3 Moore JR, Leinwand L, Warshaw DM (2012) Understandingcardiomyopathy phenotypes based on the functional impact ofmutations in the myosin motor. Circ Res 111(3):375–385.4 Sommese RF, et al. (2013) Molecular consequences of the R453Chypertrophic cardiomyopathy mutation on human β-cardiac myosinmotor function. Proc Natl Acad Sci USA 110:12607–12612.

5 Deacon JC, Bloemink MJ, Rezavandi H, Geeves MA, Leinwand LA(2012) Erratum to: Identification of functional differences betweenrecombinant human α and β cardiac myosin motors. Cell Mol Life Sci69(24):4239–4255.6 Sweeney HL, Houdusse A (2010) Structural and functionalinsights into the myosin motor mechanism. Annu Rev Biophys39:539–557.7 Malik FI, et al. (2011) Cardiac myosin activation: A potentialtherapeutic approach for systolic heart failure. Science 331(6023):1439–1443.

12508 | www.pnas.org/cgi/doi/10.1073/pnas.1310669110 Muretta and Thomas