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. 1998 Dec;75(6):3023–3030. doi: 10.1016/S0006-3495(98)77743-4

Functional analysis of myosin mutations that cause familial hypertrophic cardiomyopathy.

O Roopnarine 1, L A Leinwand 1
PMCID: PMC1299973  PMID: 9826622

Abstract

We have studied the actin-activated ATPase activities of three mutations in the motor domain of the myosin heavy chain that cause familial hypertrophic cardiomyopathy. We placed these mutations in rodent alpha-cardiac myosin to establish the relevance of using rodent systems for studying the biochemical mechanisms of the human disease. We also wished to determine whether the biochemical defects in these mutant alleles correlate with the severity of the clinical phenotype of patients with these alleles. We expressed histidine-tagged rat cardiac myosin motor domains along with rat ventricular light chain 1 in mammalian COS cells. Those myosins studied were wild-type alpha-cardiac and three mutations in the alpha-cardiac myosin heavy chain head (Arg249Gln, Arg403Gln, and Val606Met). These mutations in human beta-cardiac myosin heavy chain have predominantly moderate, severe, and mild clinical phenotypes, respectively. The crystal structure of the skeletal myosin head shows that the Arg249Gln mutation is near the ATP-binding site and the Arg403Gln and Val606Met mutations are in the actin-binding region. Expressed histidine-tagged alpha-motor domains retain physiological ATPase properties similar to those derived from cardiac tissue. All three myosin mutants show defects in the ATPase activity, with the degree of enzymatic impairment of the mutant myosins correlated with the clinical phenotype of patients with the disease caused by the corresponding mutation.

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Selected References

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  1. Becker K. D., Gottshall K. R., Hickey R., Perriard J. C., Chien K. R. Point mutations in human beta cardiac myosin heavy chain have differential effects on sarcomeric structure and assembly: an ATP binding site change disrupts both thick and thin filaments, whereas hypertrophic cardiomyopathy mutations display normal assembly. J Cell Biol. 1997 Apr 7;137(1):131–140. doi: 10.1083/jcb.137.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bonne G., Carrier L., Bercovici J., Cruaud C., Richard P., Hainque B., Gautel M., Labeit S., James M., Beckmann J. Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy. Nat Genet. 1995 Dec;11(4):438–440. doi: 10.1038/ng1295-438. [DOI] [PubMed] [Google Scholar]
  3. Cuda G., Fananapazir L., Zhu W. S., Sellers J. R., Epstein N. D. Skeletal muscle expression and abnormal function of beta-myosin in hypertrophic cardiomyopathy. J Clin Invest. 1993 Jun;91(6):2861–2865. doi: 10.1172/JCI116530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dausse E., Komajda M., Fetler L., Dubourg O., Dufour C., Carrier L., Wisnewsky C., Bercovici J., Hengstenberg C., al-Mahdawi S. Familial hypertrophic cardiomyopathy. Microsatellite haplotyping and identification of a hot spot for mutations in the beta-myosin heavy chain gene. J Clin Invest. 1993 Dec;92(6):2807–2813. doi: 10.1172/JCI116900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dausse E., Schwartz K. Genetic heterogeneity of familial hypertrophic cardiomyopathy. Neuromuscul Disord. 1993 Sep-Nov;3(5-6):483–486. doi: 10.1016/0960-8966(93)90101-o. [DOI] [PubMed] [Google Scholar]
  6. Davies M. J. The current status of myocardial disarray in hypertrophic cardiomyopathy. Br Heart J. 1984 Apr;51(4):361–363. doi: 10.1136/hrt.51.4.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Epstein N. D., Cohn G. M., Cyran F., Fananapazir L. Differences in clinical expression of hypertrophic cardiomyopathy associated with two distinct mutations in the beta-myosin heavy chain gene. A 908Leu----Val mutation and a 403Arg----Gln mutation. Circulation. 1992 Aug;86(2):345–352. doi: 10.1161/01.cir.86.2.345. [DOI] [PubMed] [Google Scholar]
  8. Fananapazir L., Epstein N. D. Genotype-phenotype correlations in hypertrophic cardiomyopathy. Insights provided by comparisons of kindreds with distinct and identical beta-myosin heavy chain gene mutations. Circulation. 1994 Jan;89(1):22–32. doi: 10.1161/01.cir.89.1.22. [DOI] [PubMed] [Google Scholar]
  9. Fisher A. J., Smith C. A., Thoden J. B., Smith R., Sutoh K., Holden H. M., Rayment I. X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP.BeFx and MgADP.AlF4-. Biochemistry. 1995 Jul 18;34(28):8960–8972. doi: 10.1021/bi00028a004. [DOI] [PubMed] [Google Scholar]
  10. Fujita H., Sugiura S., Momomura S., Omata M., Sugi H., Sutoh K. Characterization of mutant myosins of Dictyostelium discoideum equivalent to human familial hypertrophic cardiomyopathy mutants. Molecular force level of mutant myosins may have a prognostic implication. J Clin Invest. 1997 Mar 1;99(5):1010–1015. doi: 10.1172/JCI119228. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Geisterfer-Lowrance A. A., Christe M., Conner D. A., Ingwall J. S., Schoen F. J., Seidman C. E., Seidman J. G. A mouse model of familial hypertrophic cardiomyopathy. Science. 1996 May 3;272(5262):731–734. doi: 10.1126/science.272.5262.731. [DOI] [PubMed] [Google Scholar]
  12. Geisterfer-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 beta cardiac myosin heavy chain gene missense mutation. Cell. 1990 Sep 7;62(5):999–1006. doi: 10.1016/0092-8674(90)90274-i. [DOI] [PubMed] [Google Scholar]
  13. Kimura A., Harada H., Park J. E., Nishi H., Satoh M., Takahashi M., Hiroi S., Sasaoka T., Ohbuchi N., Nakamura T. Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy. Nat Genet. 1997 Aug;16(4):379–382. doi: 10.1038/ng0897-379. [DOI] [PubMed] [Google Scholar]
  14. L'Ecuyer T. J., Fulton A. B. Specific and quantitative immunoprecipitation of tropomyosin and other cytoskeletal proteins by magnetic separation. Biotechniques. 1993 Mar;14(3):436–441. [PubMed] [Google Scholar]
  15. Lankford E. B., Epstein N. D., Fananapazir L., Sweeney H. L. Abnormal contractile properties of muscle fibers expressing beta-myosin heavy chain gene mutations in patients with hypertrophic cardiomyopathy. J Clin Invest. 1995 Mar;95(3):1409–1414. doi: 10.1172/JCI117795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lanzetta P. A., Alvarez L. J., Reinach P. S., Candia O. A. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem. 1979 Nov 15;100(1):95–97. doi: 10.1016/0003-2697(79)90115-5. [DOI] [PubMed] [Google Scholar]
  17. Lauer B., Van Thiem N., Swynghedauw B. ATPase activity of the cross-linked complex between cardiac myosin subfragment 1 and actin in several models of chronic overloading. A new approach to the biochemistry of contractility. Circ Res. 1989 Jun;64(6):1106–1115. doi: 10.1161/01.res.64.6.1106. [DOI] [PubMed] [Google Scholar]
  18. Marian A. J., Mares A., Jr, Kelly D. P., Yu Q. T., Abchee A. B., Hill R., Roberts R. Sudden cardiac death in hypertrophic cardiomyopathy. Variability in phenotypic expression of beta-myosin heavy chain mutations. Eur Heart J. 1995 Mar;16(3):368–376. doi: 10.1093/oxfordjournals.eurheartj.a060920. [DOI] [PubMed] [Google Scholar]
  19. Marian A. J., Yu Q. T., Mann D. L., Graham F. L., Roberts R. Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes. Circ Res. 1995 Jul;77(1):98–106. doi: 10.1161/01.res.77.1.98. [DOI] [PubMed] [Google Scholar]
  20. Marian A. J., Yu Q. T., Mares A., Jr, Hill R., Roberts R., Perryman M. B. Detection of a new mutation in the beta-myosin heavy chain gene in an individual with hypertrophic cardiomyopathy. J Clin Invest. 1992 Dec;90(6):2156–2165. doi: 10.1172/JCI116101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maron B. J., Bonow R. O., Cannon R. O., 3rd, Leon M. B., Epstein S. E. Hypertrophic cardiomyopathy. Interrelations of clinical manifestations, pathophysiology, and therapy (1). N Engl J Med. 1987 Mar 26;316(13):780–789. doi: 10.1056/NEJM198703263161305. [DOI] [PubMed] [Google Scholar]
  22. Maron B. J., Epstein S. E., Roberts W. C. Causes of sudden death in competitive athletes. J Am Coll Cardiol. 1986 Jan;7(1):204–214. doi: 10.1016/s0735-1097(86)80283-2. [DOI] [PubMed] [Google Scholar]
  23. Maron B. J., Roberts W. C., Edwards J. E., McAllister H. A., Jr, Foley D. D., Epstein S. E. Sudden death in patients with hypertrophic cardiomyopathy: characterization of 26 patients with functional limitation. Am J Cardiol. 1978 May 1;41(5):803–810. doi: 10.1016/0002-9149(78)90717-8. [DOI] [PubMed] [Google Scholar]
  24. McNally E. M., Bravo-Zehnder M. M., Leinwand L. A. Identification of sequences necessary for the association of cardiac myosin subunits. J Cell Biol. 1991 May;113(3):585–590. doi: 10.1083/jcb.113.3.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Milligan R. A. Protein-protein interactions in the rigor actomyosin complex. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):21–26. doi: 10.1073/pnas.93.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Poetter K., Jiang H., Hassanzadeh S., Master S. R., Chang A., Dalakas M. C., Rayment I., Sellers J. R., Fananapazir L., Epstein N. D. Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle. Nat Genet. 1996 May;13(1):63–69. doi: 10.1038/ng0596-63. [DOI] [PubMed] [Google Scholar]
  27. Posen B. M., Moolman J. C., Corfield V. A., Brink P. A. Clinical and prognostic evaluation of familial hypertrophic cardiomyopathy in two South African families with different cardiac beta myosin heavy chain gene mutations. Br Heart J. 1995 Jul;74(1):40–46. doi: 10.1136/hrt.74.1.40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rayment I., Holden H. M., Sellers J. R., Fananapazir L., Epstein N. D. Structural interpretation of the mutations in the beta-cardiac myosin that have been implicated in familial hypertrophic cardiomyopathy. Proc Natl Acad Sci U S A. 1995 Apr 25;92(9):3864–3868. doi: 10.1073/pnas.92.9.3864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rayment I., Holden H. M., Whittaker M., Yohn C. B., Lorenz M., Holmes K. C., Milligan R. A. Structure of the actin-myosin complex and its implications for muscle contraction. Science. 1993 Jul 2;261(5117):58–65. doi: 10.1126/science.8316858. [DOI] [PubMed] [Google Scholar]
  30. Rayment I., Smith C., Yount R. G. The active site of myosin. Annu Rev Physiol. 1996;58:671–702. doi: 10.1146/annurev.ph.58.030196.003323. [DOI] [PubMed] [Google Scholar]
  31. Sata M., Ikebe M. Functional analysis of the mutations in the human cardiac beta-myosin that are responsible for familial hypertrophic cardiomyopathy. Implication for the clinical outcome. J Clin Invest. 1996 Dec 15;98(12):2866–2873. doi: 10.1172/JCI119115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schröder R. R., Manstein D. J., Jahn W., Holden H., Rayment I., Holmes K. C., Spudich J. A. Three-dimensional atomic model of F-actin decorated with Dictyostelium myosin S1. Nature. 1993 Jul 8;364(6433):171–174. doi: 10.1038/364171a0. [DOI] [PubMed] [Google Scholar]
  33. Smith C. A., Rayment I. Active site comparisons highlight structural similarities between myosin and other P-loop proteins. Biophys J. 1996 Apr;70(4):1590–1602. doi: 10.1016/S0006-3495(96)79745-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Solomon S. D., Geisterfer-Lowrance A. A., Vosberg H. P., Hiller G., Jarcho J. A., Morton C. C., McBride W. O., Mitchell A. L., Bale A. E., McKenna W. J. A locus for familial hypertrophic cardiomyopathy is closely linked to the cardiac myosin heavy chain genes, CRI-L436, and CRI-L329 on chromosome 14 at q11-q12. Am J Hum Genet. 1990 Sep;47(3):389–394. [PMC free article] [PubMed] [Google Scholar]
  35. Straceski A. J., Geisterfer-Lowrance A., Seidman C. E., Seidman J. G., Leinwand L. A. Functional analysis of myosin missense mutations in familial hypertrophic cardiomyopathy. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):589–593. doi: 10.1073/pnas.91.2.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sweeney H. L., Straceski A. J., Leinwand L. A., Tikunov B. A., Faust L. Heterologous expression of a cardiomyopathic myosin that is defective in its actin interaction. J Biol Chem. 1994 Jan 21;269(3):1603–1605. [PubMed] [Google Scholar]
  37. Thierfelder L., Watkins H., MacRae C., Lamas R., McKenna W., Vosberg H. P., Seidman J. G., Seidman C. E. Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere. Cell. 1994 Jun 3;77(5):701–712. doi: 10.1016/0092-8674(94)90054-x. [DOI] [PubMed] [Google Scholar]
  38. Thomas D. D., Seidel J. C., Gergely J. Rotational dynamics of spin-labeled F-actin in the sub-millisecond time range. J Mol Biol. 1979 Aug 15;132(3):257–273. doi: 10.1016/0022-2836(79)90259-6. [DOI] [PubMed] [Google Scholar]
  39. Vale R. D. Switches, latches, and amplifiers: common themes of G proteins and molecular motors. J Cell Biol. 1996 Oct;135(2):291–302. doi: 10.1083/jcb.135.2.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vikstrom K. L., Factor S. M., Leinwand L. A. Mice expressing mutant myosin heavy chains are a model for familial hypertrophic cardiomyopathy. Mol Med. 1996 Sep;2(5):556–567. [PMC free article] [PubMed] [Google Scholar]
  41. Vikstrom K. L., Leinwand L. A. Contractile protein mutations and heart disease. Curr Opin Cell Biol. 1996 Feb;8(1):97–105. doi: 10.1016/s0955-0674(96)80053-6. [DOI] [PubMed] [Google Scholar]
  42. Vikstrom K. L., Rovner A. S., Saez C. G., Bravo-Zehnder M., Straceski A. J., Leinwand L. A. Sarcomeric myosin heavy chain expressed in nonmuscle cells forms thick filaments in the presence of substoichiometric amounts of light chains. Cell Motil Cytoskeleton. 1993;26(3):192–204. doi: 10.1002/cm.970260303. [DOI] [PubMed] [Google Scholar]
  43. Vybiral T., Deitiker P. R., Roberts R., Epstein H. F. Accumulation and assembly of myosin in hypertrophic cardiomyopathy with the 403 Arg to Gln beta-myosin heavy chain mutation. Circ Res. 1992 Dec;71(6):1404–1409. doi: 10.1161/01.res.71.6.1404. [DOI] [PubMed] [Google Scholar]
  44. Watkins H., Conner D., Thierfelder L., Jarcho J. A., MacRae C., McKenna W. J., Maron B. J., Seidman J. G., Seidman C. E. Mutations in the cardiac myosin binding protein-C gene on chromosome 11 cause familial hypertrophic cardiomyopathy. Nat Genet. 1995 Dec;11(4):434–437. doi: 10.1038/ng1295-434. [DOI] [PubMed] [Google Scholar]
  45. Watkins H., McKenna W. J., Thierfelder L., Suk H. J., Anan R., O'Donoghue A., Spirito P., Matsumori A., Moravec C. S., Seidman J. G. Mutations in the genes for cardiac troponin T and alpha-tropomyosin in hypertrophic cardiomyopathy. N Engl J Med. 1995 Apr 20;332(16):1058–1064. doi: 10.1056/NEJM199504203321603. [DOI] [PubMed] [Google Scholar]
  46. Watkins H., Rosenzweig A., Hwang D. S., Levi T., McKenna W., Seidman C. E., Seidman J. G. Characteristics and prognostic implications of myosin missense mutations in familial hypertrophic cardiomyopathy. N Engl J Med. 1992 Apr 23;326(17):1108–1114. doi: 10.1056/NEJM199204233261703. [DOI] [PubMed] [Google Scholar]
  47. Watkins H., Seidman J. G., Seidman C. E. Familial hypertrophic cardiomyopathy: a genetic model of cardiac hypertrophy. Hum Mol Genet. 1995;4(Spec No):1721–1727. doi: 10.1093/hmg/4.suppl_1.1721. [DOI] [PubMed] [Google Scholar]
  48. Watkins H., Thierfelder L., Anan R., Jarcho J., Matsumori A., McKenna W., Seidman J. G., Seidman C. E. Independent origin of identical beta cardiac myosin heavy-chain mutations in hypertrophic cardiomyopathy. Am J Hum Genet. 1993 Dec;53(6):1180–1185. [PMC free article] [PubMed] [Google Scholar]

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