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• W2977464806 abstract "Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the β-cardiac myosin heavy chain gene (β-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any β-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations. Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the β-cardiac myosin heavy chain gene (β-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any β-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations. The most common inherited cardiovascular disease is hypertrophic cardiomyopathy (HCM) 4The abbreviations used are: HCMhypertrophic cardiomyopathyMyBPCmyosin-binding protein CsS1short subfragment 1ELCessential light chainABassay bufferDCMDilated cardiomyopathyeGFPenhanced green fluorescent proteinpNpico NewtonDRduty ratio. with a disease prevalence of 1:250–500 (1Harris K.M. Spirito P. Maron M.S. Zenovich A.G. Formisano F. Lesser J.R. Mackey-Bojack S. Manning W.J. Udelson J.E. Maron B.J. Prevalence, clinical profile, and significance of left ventricular remodeling in the end-stage phase of hypertrophic cardiomyopathy.Circulation. 2006; 114 (16831987): 216-22510.1161/CIRCULATIONAHA.105.583500Crossref PubMed Scopus (496) Google Scholar, 2Hershberger R.E. Hedges D.J. Morales A. Dilated cardiomyopathy: the complexity of a diverse genetic architecture.Nat. Rev. Cardiol. 2013; 10 (23900355): 531-54710.1038/nrcardio.2013.105Crossref PubMed Scopus (575) Google Scholar). Excluding those with a history of hypertension, aortic stenosis, other pre-existing systemic diseases, or being a world-class athlete, HCM is diagnosed as unexplained left ventricular hypertrophy and is typically accompanied by diastolic dysfunction (3Stewart B. Mason D.T. Braunwald E. Impaired rate of left ventricular filling in idiopathic hypertrophic subaortic stenosis and valvular aortic stenosis.Circulation. 1968; 37 (5688694): 8-1410.1161/01.CIR.37.1.8Crossref PubMed Scopus (102) Google Scholar). The first gene identified to contain a mutation leading to HCM was MYH7 for the β-cardiac myosin heavy chain and was reported almost 30 years ago (4Jarcho J.A. McKenna W. Pare J.A. Solomon S.D. Holcombe R.F. Dickie S. Levi T. Donis-Keller H. Seidman J.G. Seidman C.E. Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14q1.N. Engl. J. Med. 1989; 321 (2811944): 1372-137810.1056/NEJM198911163212005Crossref PubMed Scopus (407) Google Scholar, 5Geisterfer-Lowrance A.A. Kass S. Tanigawa G. Vosberg H.P. McKenna W. Seidman C.E. Seidman J.G. A molecular basis for familial hypertrophic cardiomyopathy: a β cardiac myosin heavy chain gene missense mutation.Cell. 1990; 62 (1975517): 999-100610.1016/0092-8674(90)90274-IAbstract Full Text PDF PubMed Scopus (1042) Google Scholar). There are now thousands of mutations in genes that encode proteins of the cardiac sarcomere, and these account for 60–70% of HCM cases (6Teekakirikul P. Kelly M.A. Rehm H.L. Lakdawala N.K. Funke B.H. Inherited cardiomyopathies: molecular genetics and clinical genetic testing in the postgenomic era.J. Mol. Diagn. 2013; 15 (23274168): 158-17010.1016/j.jmoldx.2012.09.002Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). There are 8 major sarcomeric proteins that are implicated in the majority of HCM index cases and their families, with 60–70% found in either β-cardiac myosin or myosin-binding protein C (MyBPC). This suggests that myosin is an important target for therapeutic intervention (7Green E.M. Wakimoto H. Anderson R.L. Evanchik M.J. Gorham J.M. Harrison B.C. Henze M. Kawas R. Oslob J.D. Rodriguez H.M. Song Y. Wan W. Leinwand L.A. Spudich J.A. McDowell R.S. Seidman J.G. Seidman C.E. Heart disease: a small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice.Science. 2016; 351 (26912705): 617-62110.1126/science.aad3456Crossref PubMed Scopus (325) Google Scholar, 8Ashrafian H. McKenna W.J. Watkins H. Disease pathways and novel therapeutic targets in hypertrophic cardiomyopathy.Circ. Res. 2011; 109 (21700950): 86-9610.1161/CIRCRESAHA.111.242974Crossref PubMed Scopus (128) Google Scholar). hypertrophic cardiomyopathy myosin-binding protein C short subfragment 1 essential light chain assay buffer Dilated cardiomyopathy enhanced green fluorescent protein pico Newton duty ratio. We consider here the difference between myosin mutations causing early onset versus late onset HCM. We hypothesized that β-cardiac myosin mutations associated with early onset HCM would be more severe than those mutations seen more typically in individuals who are diagnosed later in life. Analysis of the biochemical and biophysical properties of these 2 classes of myosins should reveal the severity of the mutational changes. We compared the properties of known adult pathogenic HCM mutations (R719W, R723G, and G741R) with novel sporadic mutations that had appeared in recent cardiomyopathy screens as specific to early onset patients (H251N, D382Y, P710R, and V763M) (9Morita H. Rehm H.L. Menesses A. McDonough B. Roberts A.E. Kucherlapati R. Towbin J.A. Seidman J.G. Seidman C.E. Shared genetic causes of cardiac hypertrophy in children and adults.N. Engl. J. Med. 2008; 358 (18403758): 1899-190810.1056/NEJMoa075463Crossref PubMed Scopus (297) Google Scholar, 10Kaski J.P. Syrris P. Esteban M.T. Jenkins S. Pantazis A. Deanfield J.E. McKenna W.J. Elliott P.M. Prevalence of sarcomere protein gene mutations in preadolescent children with hypertrophic cardiomyopathy.Circ. Cardiovasc. Genet. 2009; 2 (20031618): 436-44110.1161/CIRCGENETICS.108.821314Crossref PubMed Scopus (137) Google Scholar). The locations of the 7 residues in the β-MyHC protein under consideration here are shown in Fig. 1A. The high degree of conservation at those positions underscores the importance of these sites (Fig. 1B). Myosin is very vulnerable to mutations and there are now >400 different mutations described in MYH7 (11Hamady M. Buvoli M. Leinwand L.A. Knight R. Estimate of the abundance of cardiomyopathic mutations in the β-myosin gene.Int. J. Cardiol. 2010; 144 (19174318): 124-12610.1016/j.ijcard.2008.12.199Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 12Colegrave M. Peckham M. Structural implications of β-cardiac myosin heavy chain mutations in human disease.Anat. Rec. 2014; 297: 1670-168010.1002/ar.22973Crossref Scopus (67) Google Scholar). A number of disease-causing MYH7 mutations have been studied in the context of recombinant human β-MyHC motors (13Bloemink M. Deacon J. Langer S. Vera C. Combs A. Leinwand L. Geeves M.A. The hypertrophic cardiomyopathy myosin mutation R453C alters ATP binding and hydrolysis of human cardiac β-myosin.J. Biol. Chem. 2014; 289 (24344137): 5158-516710.1074/jbc.M113.511204Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar14Nag S. Sommese R.F. Ujfalusi Z. Combs A. Langer S. Sutton S. Leinwand L.A. Geeves M.A. Ruppel K.M. Spudich J.A. Contractility parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function.Sci. Adv. 2015; 1 (26601291): e150051110.1126/sciadv.1500511Crossref PubMed Scopus (62) Google Scholar, 15Kawana M. Sarkar S.S. Sutton S. Ruppel K.M. Spudich J.A. Biophysical properties of human β-cardiac myosin with converter mutations that cause hypertrophic cardiomyopathy.Sci. Adv. 2017; 3 (28246639): e1601959Crossref PubMed Scopus (43) Google Scholar, 16Sommese R.F. Sung J. Nag S. Sutton S. Deacon J.C. Choe E. Leinwand L.A. Ruppel K. Spudich J.A. Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function.Proc. Natl. Acad. Sci. 2013; 110 (23798412): 12607-1261210.1073/pnas.1309493110Crossref PubMed Scopus (108) Google Scholar17Adhikari A.S. Kooiker K.B. Sarkar S.S. Liu C. Bernstein D. Spudich J.A. Ruppel K.M. Early-onset hypertrophic cardiomyopathy mutations significantly increase the velocity, force, and actin-activated ATPase activity of human β-cardiac myosin.Cell Rep. 2016; 17 (27974200): 2857-286410.1016/j.celrep.2016.11.040Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). R403Q, R453C, R719W, R723G, G741R, and D239N are mutations that are widely recognized as pathogenic and have been seen in multiple families (18Landrum M.J. Lee J.M. Benson M. Brown G.R. Chao C. Chitipiralla S. Gu B. Hart J. Hoffman D. Jang W. Karapetyan K. Katz K. Liu C. Maddipatla Z. Malheiro A. et al.ClinVar: improving access to variant interpretations and supporting evidence.Nucleic Acids Res. 2018; 46 (29165669): D1062-D106710.1093/nar/gkx1153Crossref PubMed Scopus (1511) Google Scholar). Less prevalent sporadic mutations like H251N, D382Y, P710R, and V763M appeared in genetic screens of early onset HCM patients (<15 years old) and their pathogenicity has not yet been clearly established (9Morita H. Rehm H.L. Menesses A. McDonough B. Roberts A.E. Kucherlapati R. Towbin J.A. Seidman J.G. Seidman C.E. Shared genetic causes of cardiac hypertrophy in children and adults.N. Engl. J. Med. 2008; 358 (18403758): 1899-190810.1056/NEJMoa075463Crossref PubMed Scopus (297) Google Scholar, 18Landrum M.J. Lee J.M. Benson M. Brown G.R. Chao C. Chitipiralla S. Gu B. Hart J. Hoffman D. Jang W. Karapetyan K. Katz K. Liu C. Maddipatla Z. Malheiro A. et al.ClinVar: improving access to variant interpretations and supporting evidence.Nucleic Acids Res. 2018; 46 (29165669): D1062-D106710.1093/nar/gkx1153Crossref PubMed Scopus (1511) Google Scholar, 19Smith D.A. A new mechanokinetic model for muscle contraction, where force and movement are triggered by phosphate release.J. Muscle Res. Cell Motil. 2014; 35 (25319769): 295-30610.1007/s10974-014-9391-zCrossref PubMed Scopus (19) Google Scholar). H251N is in the central β-sheet (Fig. 1A) that undergoes strain-induced twisting upon ATP-binding and communicates to the upper 50,000 domain to open and release actin. H251N was identified in a screen of 79 pre-adolescent children and later characterized biophysically (10Kaski J.P. Syrris P. Esteban M.T. Jenkins S. Pantazis A. Deanfield J.E. McKenna W.J. Elliott P.M. Prevalence of sarcomere protein gene mutations in preadolescent children with hypertrophic cardiomyopathy.Circ. Cardiovasc. Genet. 2009; 2 (20031618): 436-44110.1161/CIRCGENETICS.108.821314Crossref PubMed Scopus (137) Google Scholar, 17Adhikari A.S. Kooiker K.B. Sarkar S.S. Liu C. Bernstein D. Spudich J.A. Ruppel K.M. Early-onset hypertrophic cardiomyopathy mutations significantly increase the velocity, force, and actin-activated ATPase activity of human β-cardiac myosin.Cell Rep. 2016; 17 (27974200): 2857-286410.1016/j.celrep.2016.11.040Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Adhikari et al. (17Adhikari A.S. Kooiker K.B. Sarkar S.S. Liu C. Bernstein D. Spudich J.A. Ruppel K.M. Early-onset hypertrophic cardiomyopathy mutations significantly increase the velocity, force, and actin-activated ATPase activity of human β-cardiac myosin.Cell Rep. 2016; 17 (27974200): 2857-286410.1016/j.celrep.2016.11.040Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) found that this early onset mutation resulted in higher kcat, actin-gliding velocity, and intrinsic force. Located close to the cardiomyopathy loop of the actin-binding domain, D382Y has been categorized as a variant of unknown (or uncertain) significance (VUS) (10Kaski J.P. Syrris P. Esteban M.T. Jenkins S. Pantazis A. Deanfield J.E. McKenna W.J. Elliott P.M. Prevalence of sarcomere protein gene mutations in preadolescent children with hypertrophic cardiomyopathy.Circ. Cardiovasc. Genet. 2009; 2 (20031618): 436-44110.1161/CIRCGENETICS.108.821314Crossref PubMed Scopus (137) Google Scholar, 18Landrum M.J. Lee J.M. Benson M. Brown G.R. Chao C. Chitipiralla S. Gu B. Hart J. Hoffman D. Jang W. Karapetyan K. Katz K. Liu C. Maddipatla Z. Malheiro A. et al.ClinVar: improving access to variant interpretations and supporting evidence.Nucleic Acids Res. 2018; 46 (29165669): D1062-D106710.1093/nar/gkx1153Crossref PubMed Scopus (1511) Google Scholar). Close to this residue is the well-studied pathogenic R403Q mutation that exhibits subtle biophysical changes compared with WT (14Nag S. Sommese R.F. Ujfalusi Z. Combs A. Langer S. Sutton S. Leinwand L.A. Geeves M.A. Ruppel K.M. Spudich J.A. Contractility parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function.Sci. Adv. 2015; 1 (26601291): e150051110.1126/sciadv.1500511Crossref PubMed Scopus (62) Google Scholar). Pro-710, a residue on the border between the SH1 helix and the converter, has been reported to be mutated to an arginine, leucine, or a histidine (10Kaski J.P. Syrris P. Esteban M.T. Jenkins S. Pantazis A. Deanfield J.E. McKenna W.J. Elliott P.M. Prevalence of sarcomere protein gene mutations in preadolescent children with hypertrophic cardiomyopathy.Circ. Cardiovasc. Genet. 2009; 2 (20031618): 436-44110.1161/CIRCGENETICS.108.821314Crossref PubMed Scopus (137) Google Scholar, 20Kindel S.J. Miller E.M. Gupta R. Cripe L.H. Hinton R.B. Spicer R.L. Towbin J.A. Ware S.M. Pediatric cardiomyopathy: importance of genetic and metabolic evaluation.J. Card. Fail. 2012; 18 (22555271): 396-403Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Unlike the arginine mutation, P710H and P710L have been seen multiple times, but the histidine is considered pathogenic, whereas the leucine and arginine mutations are rare and of unknown severity (10Kaski J.P. Syrris P. Esteban M.T. Jenkins S. Pantazis A. Deanfield J.E. McKenna W.J. Elliott P.M. Prevalence of sarcomere protein gene mutations in preadolescent children with hypertrophic cardiomyopathy.Circ. Cardiovasc. Genet. 2009; 2 (20031618): 436-44110.1161/CIRCGENETICS.108.821314Crossref PubMed Scopus (137) Google Scholar, 18Landrum M.J. Lee J.M. Benson M. Brown G.R. Chao C. Chitipiralla S. Gu B. Hart J. Hoffman D. Jang W. Karapetyan K. Katz K. Liu C. Maddipatla Z. Malheiro A. et al.ClinVar: improving access to variant interpretations and supporting evidence.Nucleic Acids Res. 2018; 46 (29165669): D1062-D106710.1093/nar/gkx1153Crossref PubMed Scopus (1511) Google Scholar, 20Kindel S.J. Miller E.M. Gupta R. Cripe L.H. Hinton R.B. Spicer R.L. Towbin J.A. Ware S.M. Pediatric cardiomyopathy: importance of genetic and metabolic evaluation.J. Card. Fail. 2012; 18 (22555271): 396-403Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The myosin converter is well-known to be enriched for HCM-causing mutations with a range of adverse effects (21García-Giustiniani D. Arad M. Ortíz-Genga M. Barriales-Villa R. Fernández X. Rodríguez-García I. Mazzanti A. Veira E. Maneiro E. Rebolo P. Lesende I. Cazón L. Freimark D. Gimeno-Blanes J.R. et al.Phenotype and prognostic correlations of the converter region mutations affecting the β myosin heavy chain.Heart. 2015; 101 (25935763): 1047-105310.1136/heartjnl-2014-307205Crossref PubMed Scopus (37) Google Scholar, 22Homburger J.R. Green E.M. Caleshu C. Sunitha M.S. Taylor R.E. Ruppel K.M. Metpally R.P. Colan S.D. Michels M. Day S.M. Olivotto I. Bustamante C.D. Dewey F.E. Ho C.Y. Spudich J.A. Ashley E.A. Multidimensional structure-function relationships in human β-cardiac myosin from population-scale genetic variation.Proc. Natl. Acad. Sci. 2016; 113 (27247418): 6701-670610.1073/pnas.1606950113Crossref PubMed Scopus (63) Google Scholar). Converter mutations studied here include the late onset mutations R719W, R723G, and G741R and the early onset mutation, V763M. Muscle biopsy and skinned fiber studies have shown the converter domain late onset mutations have increased fiber stiffness with subtle changes in cross-bridge kinetics (23Köhler J. Winkler G. Schulte I. Scholz T. McKenna W. Brenner B. Kraft T. Mutation of the myosin converter domain alters cross-bridge elasticity.Proc. Natl. Acad. Sci. U.S.A. 2002; 99 (11904418): 3557-356210.1073/pnas.062415899Crossref PubMed Scopus (73) Google Scholar, 24Fananapazir L. Dalakas M.C. Cyran F. Cohn G. Epstein N.D. Missense mutations in the β-myosin heavy-chain gene cause central core disease in hypertrophic cardiomyopathy.Proc. Natl. Acad. Sci. U.S.A. 1993; 90 (8483915): 3993-399710.1073/pnas.90.9.3993Crossref PubMed Scopus (178) Google Scholar25Seebohm B. Matinmehr F. Köhler J. Francino A. Navarro-Lopéz F. Perrot A. Ozcelik C. McKenna W.J. Brenner B. Kraft T. Cardiomyopathy mutations reveal variable region of myosin converter as major element of cross-bridge compliance.Biophys. J. 2009; 97 (19651039): 806-82410.1016/j.bpj.2009.05.023Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The steady-state kcat, the actin-gliding velocities, and the intrinsic force measurements of the myosin motor domain for these late onset pathogenic HCM mutations did not vary much when compared with WT (15Kawana M. Sarkar S.S. Sutton S. Ruppel K.M. Spudich J.A. Biophysical properties of human β-cardiac myosin with converter mutations that cause hypertrophic cardiomyopathy.Sci. Adv. 2017; 3 (28246639): e1601959Crossref PubMed Scopus (43) Google Scholar). V763M has been reported in an early onset HCM screen and in a separate HCM cohort (10Kaski J.P. Syrris P. Esteban M.T. Jenkins S. Pantazis A. Deanfield J.E. McKenna W.J. Elliott P.M. Prevalence of sarcomere protein gene mutations in preadolescent children with hypertrophic cardiomyopathy.Circ. Cardiovasc. Genet. 2009; 2 (20031618): 436-44110.1161/CIRCGENETICS.108.821314Crossref PubMed Scopus (137) Google Scholar, 26Mohiddin S.A. Begley D.A. McLam E. Cardoso J.P. Winkler J.B. Sellers J.R. Fananapazir L. Utility of genetic screening in hypertrophic cardiomyopathy: prevalence and significance of novel and double (homozygous and heterozygous) β-myosin mutations.Genet. Test. 2003; 7 (12820698): 21-2710.1089/109065703321560895Crossref PubMed Scopus (32) Google Scholar). Both pathogenicity and age of symptom emergence are unclear for V763M (18Landrum M.J. Lee J.M. Benson M. Brown G.R. Chao C. Chitipiralla S. Gu B. Hart J. Hoffman D. Jang W. Karapetyan K. Katz K. Liu C. Maddipatla Z. Malheiro A. et al.ClinVar: improving access to variant interpretations and supporting evidence.Nucleic Acids Res. 2018; 46 (29165669): D1062-D106710.1093/nar/gkx1153Crossref PubMed Scopus (1511) Google Scholar). Here, for the early and late onset HCM mutations, we show that the kinetic parameters of mutations also do not have a “unifying” disruption of the cycle. Contrary to our hypothesis, we did not detect any strong differences in kinetic parameters between mutations seen in late onset versus early onset patients, or among mutations in the different structural domains of the motor that account for the difference in the timing of pathological manifestation. We produced recombinant mutant and WT human myosin motors in differentiated C2C12 muscle cells, performed extensive kinetic analysis, and evaluated the severity of these mutations based on alterations to the cross-bridge cycle. We specifically focused on the cross-bridge kinetics of the motor domain region utilizing the short subfragment 1 (sS1, corresponding to residues 1–808, Fig. S1). Steady-state ATPase data for 4 of the mutations examined here H251N, R719W, R723G, and G741R, have already been published (15Kawana M. Sarkar S.S. Sutton S. Ruppel K.M. Spudich J.A. Biophysical properties of human β-cardiac myosin with converter mutations that cause hypertrophic cardiomyopathy.Sci. Adv. 2017; 3 (28246639): e1601959Crossref PubMed Scopus (43) Google Scholar, 17Adhikari A.S. Kooiker K.B. Sarkar S.S. Liu C. Bernstein D. Spudich J.A. Ruppel K.M. Early-onset hypertrophic cardiomyopathy mutations significantly increase the velocity, force, and actin-activated ATPase activity of human β-cardiac myosin.Cell Rep. 2016; 17 (27974200): 2857-286410.1016/j.celrep.2016.11.040Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). These are listed in Table 1 together with the kcat values for the remaining 4 mutations. The error on these measurements is of the order of 10% and 3 of the mutations, the early onset V763M and the late onset R719W and R723G, differ from WT by less than 5%. The kcat for G741R is less than 10% lower than WT. Three other mutations, H251N, D382Y, and P710R, differ significantly from WT. The early onset H251N and D382Y are higher than WT by 24 and 20%, respectively, whereas P710R is 39% lower than WT. Thus, there is no common pattern of change in the kcat values for these HCM mutations and no difference in pattern between early and late onset groups. This lack of common patterns led us to focus on the transient kinetic analysis that can reveal more detail about how mutations change the ATPase cycle.Table 1Kinetic parameters for β-WT and 8 HCM mutations Open table in a new tab Considerable amounts of pre–steady-state kinetic data have been collected for a number of DCM and HCM mutations in the β-MyHC motor domain. The descriptions of methods, analysis tools, model assumptions, data quality, and details of the measurements have been presented in our earlier works (13Bloemink M. Deacon J. Langer S. Vera C. Combs A. Leinwand L. Geeves M.A. The hypertrophic cardiomyopathy myosin mutation R453C alters ATP binding and hydrolysis of human cardiac β-myosin.J. Biol. Chem. 2014; 289 (24344137): 5158-516710.1074/jbc.M113.511204Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Nag S. Sommese R.F. Ujfalusi Z. Combs A. Langer S. Sutton S. Leinwand L.A. Geeves M.A. Ruppel K.M. Spudich J.A. Contractility parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function.Sci. Adv. 2015; 1 (26601291): e150051110.1126/sciadv.1500511Crossref PubMed Scopus (62) Google Scholar, 27Ujfalusi Z. Vera C.D. Mijailovich S.M. Svicevic M. Yu E.C. Kawana M. Ruppel K.M. Spudich J.A. Geeves M.A. Leinwand L.A. Dilated cardiomyopathy myosin mutants have reduced force-generating capacity.J. Biol. Chem. 2018; 293 (29666183): 9017-902910.1074/jbc.RA118.001938Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 28Deacon J.C. Bloemink M.J. Rezavandi H. Geeves M.A. Leinwand L.A. Identification of functional differences between recombinant human α and β cardiac myosin motors.Cell. Mol. Life Sci. 2012; 69 (22349210): 2261-227710.1007/s00018-012-0927-3Crossref PubMed Scopus (59) Google Scholar29Mijailovich S.M. Nedic D. Svicevic M. Stojanovic B. Walklate J. Ujfalusi Z. Geeves M.A. Modeling the actin-myosin ATPase cross-bridge cycle for skeletal and cardiac muscle myosin isoforms.Biophys. J. 2017; 112 (28297657): 984-99610.1016/j.bpj.2017.01.021Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). We have taken the same approach to understand the impact of this set of HCM mutations and to compare with the results of previous studies. Details of individual measurements and the evaluation of data quality are presented in the supporting information (Fig. S2–S6). Here we focus on what effect each mutation has on the behavior of the cycle. For every mutant, each measurement was made at least 3 times with a minimum of 2 independent protein preparations. In general, all parameters were measured to a precision of better than 20% and in most cases better than 10%. Given this level of sensitivity, we assume any change of less than 20% is not significant. The data are interpreted in terms of the 8-step ATP-driven cross-bridge cycle that we have used previously (Fig. 2). Red shades indicate detached cross-bridges, yellow shades are weakly-attached, and blue shades represent strongly-attached force-holding cross-bridges. Table 1 shows the mean values and errors of the actin and nucleotide-binding experiments for the steps in the cycle that are accessible, together with the ATPase parameters. To make the overall pattern of the induced changes clearer, the percent changes relative to WT are color coded in Table 1. The data are also summarized in Fig. 3, where the percentage differences in each parameter relative to WT are plotted.Figure 3Summary of percentage differences in kinetic parameters of HCM mutations relative to WT. A, % change for measured dissociation or equilibrium constants. Also included is the Kapp value from ATPase analysis. B, % change of several measured rate constants. Color-coded to match the parameter to each HCM mutation. The dashed lines represent ±20% change in the parameter considered to be the precision of each measurement.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Even though the reactions are not part of the normal cycle, nucleotide binding to the motor in the absence of actin is measured to understand how the nucleotide-binding pocket and the weakly-attached actin states might be affected by mutations. This process can be important to understand the cycle of the second myosin head, whereas the other can be attached to actin. The affinity of ATP for sS1 is weakened >2-fold for 2 mutations (P710R and G741R). All other mutations were 27–73% weaker except R719W, which was indistinguishable from WT. In contrast, most mutants bound ADP >2-fold tighter, with the exception of V763M (just less than 2-fold tighter) and P710R (6-fold weaker). Thus, no simple pattern of behavior was apparent for the sS1 in the absence of actin, but most mutations significantly affected nucleotide binding. The darker color coding of the data in Table 1 shows the parameters that change >2-fold (dark blue decrease, dark orange increase) and indicate that a large number of parameters have been affected, and at least one parameter for each mutant. Thus, the changes observed are not minor, despite relatively small changes in the value of the steady-state kcat. Fig. 3A shows that in general, the value of KT (the affinity of ATP for acto·sS1) measured at 10 °C where it is well-defined, KA (the affinity of actin for sS1), and KDA (the affinity of actin for sS1·ADP) have tighter binding. For KT this is >20% tighter, but none is as much as 2-fold tighter. For KDA the affinities are mostly >2-fold tighter with V763M, G741R, and P710R >4-fold tighter, whereas the data for KA is much more variable. The affinity of ADP for acto·sS1 is >2-fold weaker in 5 cases. R723G is an exception for both the KA and ADP affinity for acto·sS1 parameters, but there is a general pattern. Fig. 3B shows changes in measured rate constants. There is some consistent behavior, but it is not uniform for all mutations. The maximum rate constant for sS1 detachment from actin upon binding ATP (k+T*) is generally slower (20–70%) and the rate constant for ADP binding to acto·sS1 is also generally slower (30–70%) with the exception of R723G, which is increased by 70%. All other rate constants had variable changes or none at all. This highly variable pattern was also seen for the set of DCM mutations we previously reported (27Ujfalusi Z. Vera C.D. Mijailovich S.M. Svicevic M. Yu E.C. Kawana M. Ruppel K.M. Spudich J.A. Geev" @default.
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• W2977464806 date "2019-11-01" @default.
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• W2977464806 title "Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties" @default.
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• W2977464806 doi "https://doi.org/10.1074/jbc.ra119.010563" @default.
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