Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function Ruth F. Sommesea,1, Jongmin Sunga,b,1, Suman Naga,1, Shirley Suttona, John C. Deaconc, Elizabeth Choea,d, Leslie A. Leinwandc, Kathleen Ruppela,e,2, and James A. Spudicha,2 Departments of aBiochemistry, ePediatrics (Cardiology), and dCancer Biology Program, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Applied Physics, Stanford University, Stanford, CA 94305; and cBioFrontiers Institute, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309 Contributed by James A. Spudich, May 19, 2013 (sent for review April 26, 2013) Cardiovascular disorders are the leading cause of morbidity and activity (8). Lowey et al. subsequently showed that the effects of mortality in the developed world, and hypertrophic cardiomyopa- the R403Q mutation in mouse cardiac myosin depended on the thy (HCM) is among the most frequently occurring inherited cardiac isoform into which the mutation was introduced. In α-cardiac disorders. HCM is caused by mutations in the genes encoding the myosin, they saw an increase in both ATPase activity and ve- fundamental force-generating machinery of the cardiac muscle, locity, whereas in the β-cardiac myosin, there was no significant including β-cardiac myosin. Here, we present a biomechanical anal- change in the velocity and actually a slight decrease in the ATPase ysis of the HCM-causing mutation, R453C, in the context of human activity (9). β-cardiac myosin. We found that this mutation causes a ∼30% de- As the R403Q data illustrate, the difficulty in interpreting the crease in the maximum ATPase of the human β-cardiac subfragment effects of these mutations has largely been due to the limited 1, the motor domain of myosin, and a similar percent decrease in the availability of homogeneous and fully active human motor pro- in vitro velocity. The major change in the R453C human β-cardiac teins and the inadequacy of commonly used animal models. Given subfragment 1 is a 50% increase in the intrinsic force of the motor that the HCM mutant phenotype results from single-residue compared with wild type, with no appreciable change in the stroke substitutions in β-MHC, observing the effects of such a mutation BIOCHEMISTRY size, as observed with a dual-beam optical trap. These results predict in a nonhuman protein background where there are many other that the overall force of the ensemble of myosin molecules in the residue differences from the human sequence is far from ideal. muscle should be higher in the R453C mutant compared with wild The majority of animal protein studies are derived from rodent type. Loaded in vitro motility assay confirms that the net force in the models that have a different cardiac MHC isoform composition ensemble is indeed increased. Overall, this study suggests that (predominantly α-MHC instead of β-MHC). Both α- and β-MHC the R453C mutation should result in a hypercontractile state in isoforms from rodents have >30 residue differences compared the heart muscle. with human β-cardiac myosin, and HCM-causing myosin muta- tions in mice have different biochemical effects depending on heart disease | optical trapping | single-molecule force measurements their backbone isoform (9, 10). Until recently, expression of hu- man cardiac muscle myosin has proven recalcitrant to common ypertrophic cardiomyopathy (HCM) is among the most recombinant protein expression methods. Additionally, cardiac Hcommon of inherited cardiovascular disorders, affecting ∼1in biopsies yield limited amounts of enzymatically active motor and 500 individuals (1). HCM is characterized by left ventricular hy- are heterogeneous in the proportion of the mutated form. This pertrophy, cardiomyocyte disarray, and myocardial fibrosis. HCM problem has now been addressed by a recently developed muscle patients typically have left ventricles with small cavities and pre- cell expression system that provides a source of active recombi- β served or even enhanced global systolic function, but impaired nant human -cardiac muscle myosin (11). β relaxation (2). Clinically, HCM is the most common cause of In this study, we have focused on the HCM-causing -cardiac fi β sudden cardiac death in young adults (3). The first genetic cause of myosin mutation R453C, located next to strand ve of the - fi pleated sheet of myosin ∼5 nm from the actin-binding region and familial HCM was identi ed in 1990 (4). Missense mutations in the ∼ Discussion major proteins of the sarcomere have since been identified in HCM 3 nm from the nucleotide-binding site ( ). This muta- patients, and may account for up to 60% of all cases (2). Although tion is considered to be malignant and the small number of a number of these mutations have been studied using a variety of investigations into the functional biochemical and biophysical effects of this mutation have yielded contradictory results (12–14). approaches, there is no clear consensus as to the mechanism(s) by fi which these mutations give rise to the disease state. We have puri ed a Subfragment 1 (S1) construct of human β-cardiac myosin containing a truncated human MHC (residues One of the most commonly mutated genes is myosin heavy – chain 7 or MYH7, which encodes β-cardiac myosin heavy chain 1 808) and the human ventricular essential light chain (ELC) (β-MHC), the major cardiac muscle myosin isoform in the human (Fig. S1), followed by a short linker and a carboxy terminal eGFP heart. In general, HCM resulting from β-MHC mutations are characterized by early onset and severe left ventricular hyper- trophy (5). Although several mutations in β-MHC have been Author contributions: R.F.S., J.S., S.N., K.R., and J.A.S. designed research; R.F.S., J.S., S.N., fi S.S., E.C., and K.R. performed research; J.C.D., E.C., L.A.L., and K.R. contributed new studied over the past 15 y, there has been signi cant disagreement reagents/analytic tools; R.F.S., J.S., S.N., and J.A.S. analyzed data; and R.F.S., J.S., S.N., K.R., in the literature as to the effects of these mutations on the bio- and J.A.S. wrote the paper. chemical and biophysical properties of β-cardiac myosin (reviewed The authors declare no conflict of interest. in ref. 6). For example, the first cardiomyopathy-causing mutation Freely available online through the PNAS open access option. β fi in -cardiac myosin to be identi ed was R403Q. Initial studies on 1R.F.S., J.S., and S.N. contributed equally to this work. this mutant isolated from cardiac and soleus muscle biopsies 2To whom correspondence may be addressed. E-mail: [email protected] or kruppel@ reported a decrease in in vitro sliding velocity (7). Later studies stanford.edu. α using mouse -cardiac myosin containing the R403Q mutation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. found an increase in velocity and also an increase in the ATPase 1073/pnas.1309493110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1309493110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 moiety (SI Materials and Methods). We refer to this construct as saturating ATP concentration, or the strongly bound state time (ts) simply human β-cardiac S1 throughout. Here we present a de- (Fig. S2). For the R453C mutant and the WT, ts was ∼16 ms. These scription of the effects of this mutation on human β-cardiac S1 values are, however, only estimates given the difficulty of accu- function at both the single-molecule and ensemble levels. Sin- rately detecting and selecting short events in the trap, which are gle-molecule analysis revealed a significant increase in intrinsic challenging to distinguish from Brownian noise of the trapped force of the mutant motor by 50%, such that the net ensemble beads and instrument noise. Hence, a small change in the ts is force is higher than the wild-type (WT) motor, as confirmed by currently difficult to measure with our instrument. a loaded in vitro motility assay. Together, the data support a model whereby heart muscle containing this mutation would R453C Human β-Cardiac S1 Has a Lower Velocity than WT. We mea- be hypercontractile. sured the velocity of motor-driven actin filaments in methylcel- lulose as a function of actin filament length for both WT and Results R453C human β-cardiac S1. The average maximum velocity (vo) − R453C Human β-Cardiac S1 Has a Lower Maximum Actin-Activated for R453C at 23 °C for human β-cardiac S1 (610 ± 30 nm·s 1) −1 ATPase than WT. To measure the maximal rate of ATP turnover was significantly slower than that of WT (800 ± 40 nm·s )(P < (kcat) by the acto–myosin complex, we used an actin-activated 0.0001). The decrease in velocity for R453C compared with WT myosin ATPase assay at 23 °C. The kcat of R453C human was also observed at 30 °C (Fig. 2, Table S1). −1 β-cardiac S1 (5.0 ± 0.2 s ) was significantly lower (30%; P < The maximum velocity vo in the in vitro motility assay is related − 0.0002) than that of the WT motor (7.4 ± 0.4 s 1) (Fig. 1A, Table to the displacement generated by the myosin power stroke (i.e., S1). This difference was also seen with human β-cardiac S1 that the stroke size d) and the duration that myosin remains strongly was freshly recycled by binding to actin in the absence of ATP attached to actin (ts), such that vo= d/ts (15). Besides the estimates and then releasing active heads from the actin in 2 mM ATP (this from the trap measurements, the ts of S1 in saturating ATP process eliminates “dead heads” from the population).