The most prevalent Freeman-Sheldon Syndrome mutations in the embryonic myosin motor share functional defects

The embryonic myosin isoform is expressed during fetal development and rapidly down-regulated after birth. Freeman-Sheldon syndrome (FSS) is a disease associated with missense mutations in the motor domain of this myosin. It is the most severe form of distal arthrogryposis, leading to overcontraction of the hands, feet, and orofacial muscles and other joints of the body. Availability of human embryonic muscle tissue has been a limiting factor in investigating the properties of this isoform and its mutations. Using a recombinant expression system, we have studied homogeneous samples of human motors for the WT and three of the most common FSS mutants: R672H, R672C, and T178I. Our data suggest that the WT embryonic myosin motor is similar in contractile speed to the slow type I/β cardiac based on the rate constant for ADP release and ADP affinity for actin-myosin. All three FSS mutations show dramatic changes in kinetic properties, most notably the slowing of the apparent ATP hydrolysis step (reduced 5–9-fold), leading to a longer lived detached state and a slowed Vmax of the ATPase (2–35-fold), indicating a slower cycling time. These mutations therefore seriously disrupt myosin function.

1. Oldfors, A. (2007) Hereditary myosin myopathies. Neuromusclar Disorders. 17, 355-367
2. Hamady, M., Buvoli, M., Leinwand, L.A., and Knight, R. (2010) Estimate of the abundance of cardiomyopathic mutations in the β-myosin gene. International Journal of Cardiology. 144, 124-126
3. Clarke, N.F., Amburgey, K., Teener, J., Camelo-Piragua, S., Kesari, A., Punetha, J., Waddell, L.B., Davis, M., Laing, N.G., Monnier, N., North, K.N., Hoffman, E.P., and Dowling, J.J. (2013) A novel mutation expands the genetic and clinical spectrum of MYH7-related myopathies<br /> . Neuromusclar Disorders. 23, 432-436
4. Lamont, P.J., Wallefeld, W., Hilton-Jones, D., Udd, B., Argov, Z., Barboi, A.C., Bonneman, C., Boycott, K.M., Bushby, K., Connolly, A.M., Davies, N., Beggs, A.H., Cox, G.F., Dastgir, J., DeChene, E.T., Gooding, R., Jungbluth, H., Muelas, N., Palmio, J., Penttila, S., Schmedding, E., Suominen, T., Straub, V., Staples, C., Van den Bergh, P. Y. K., Vilchez, J.J., Wagner, K.R., Wheeler, P.G., Wraige, E., and Laing, N.G. (2014) Novel mutations widen the phenotypic spectrum of slow skeletal/β-cadiac myosin (MYH7) distal myopathy. Human Mutation. 35, 868-879
5. Yüceyar, N., Ayhan, Ö, Karasoy, H., and Tolun, A. (2015) HomozygousMYH7R1820W mutation results in recessive myosin storage myopathy: Scapuloperoneal and respiratory weakness with dilated cardiomyopathy. Neuromusclar Disorders. 25, 340-344
6. Schiaffino, S., Rossi, A.C., Smerdu, V., Leinwand, L.A., and Reggiani, C. (2015) Developmental myosins: expression patterns and functional significance. Skeletal Muscle. 5,
7. Fidzianska, A. (1980) Human ontogensis. I. Ultrastructural characteristics of developing human muscle. Journal of Neuropathology and experimental Neurology. 39, 476-486
8. Barbet, J.P., Thornell, L.E., and Butler-Browne, G.S. (1991) Immunocytochemical characterisation of two generations of fibers during the development of the human quadiceps muscle. Mechanisms of development. 35, 3-11
9. Draeger, A., Weeds, A.G., and Fitzsimons, R.B. (1987) Primary, secondary and tertiary myotubes in developing skeletal muscle: A new approach to the analysis of human myogenesis. Journal of the Neurological Sciences. 81, 19-43
10. Cho, M., Webster, S.G., and Blau, H.M. (1993) Evidence for myoblast-extrinsic regulation of slow myosin heavy chain expression during muscle fiber formation in embryonic development. The Journal of Cell Biology. 121, 795-810
11. Racca, A.W., Beck, A.E., Rao, V.S., Flint, G.V., Lundy, S.D., Born, D.E., Bamshad, M., and Regnier, M. (2013) Contractility of kinetics of human fetal and human adult skeletal muscle. The Journal of Physiology. 591, 3049-3061
12. Ecob-Prince, M., Hill, M., and Brown, W. (1989) Immunocytochemical demonstration of myosin heavy chain expression in human muscle. Journal of the Neurological Sciences. 91, 71-78
13. Racca, A.W., Beck, A.E., McMillin, M.J., Korte, F.S., Bamshad, M.J., and Regnier, M. (2015) The embryonic myosin R672C mutation that underlies Freeman-Sheldon syndrome impairs cross-bridge detachment and cycling in adult skeletal muscle. Human Molecular Genetics.
14. Beck, A.E., McMillin, M.J., Gildersleeve, H.I.S., Shively, K.M.B., Tang, A., and Bamshad, M.J. (2014) Genotype-phenotype relationships in Freeman-Sheldon syndrome. American Journal of Medical Genetics Part A. 164, 2808-2813
15. Tajsharghi, H., Kimber, E., Kroksmark, A.K., Jerre, R., Tulinius, M., and Oldfors, A. (2008) Embryonic Myosin Heavy-Chain Mutations Cause Distal Arthrogyposis and Developmental Myosin Myopathy That Persists Postnatally. Archives of Neurology. 65, 1083-1090
16. Toydemir, R.M., Rutherford, A., Whitby, F.G., Jorde, L.B., Carey, J.C., and Bamshad, M. (2006) Mutations in embryonic myosin heavy chain (MYH3) cause Freeman-sheldon syndrome and Sheldon-Hall syndrome. Nature Genetics. 38, 561-565
17. Tajsharghi, H., Kimber, E., Kroksmark, A.K., Jerre, R., Tulinius, M., and Oldfors, A. (2008) Embryonic Myosin Heavy-CHain Mutations Cause Distal Arthogryposis and Developmental Myosin Myopathy That Persists Postnatally. Archives of Neurology. 65, 1083-1090
18. Ontell, M.P., Sopper, M.M., Lyons, G., Buckingham, M., and Ontell, M. (1993) Modulation of contractile protein gene expression in fetal murine crural muscles: Emergence of muscle diversity. Developmental Dynamics. 198, 203-213
19. Preller, M., and Holmes, K.C. (2013) The myosin start-of-power stroke state and how actin binding drives the power stroke. Cytoskeleton. 70, 651-660
20. Sweeney, H.L., and Houdusse, A. (2010) Structural and functional insights into the myosin motor mechanism. Annual Reviews of Biophysics. 39, 539-557
21. Resnicow, D.I., Deacon, J.C., Warrick, H.M., Spudich, J.A., and Leinwand, L.A. (2010) Functional diversity among a family of human skeletal muscle myosin motors. Proceedings of the National Academy of Sciences of the United States of America. 107, 1053-1058
22. Wang, Q., Moncman, C.L., and Winkelmann, D.A. (2003) Mutations in the motor domain modulate myosin activity and myofibril organization<br /> . Journal of Cell Science. 116, 4227-4238
23. Bloemink, M.J., Deacon, J.C., Langer, S., Vera, C., Combs, A., Leinwand, L.A., and Geeves, M.A. (2014) The Hypertrophic Cardiomyopathy Myosin Mutation R453C alters ATP-binding and hydrolysis of human cardiac β-myosin . The Journal of Biological Chemistry. 289, 5158-5167
24. Sommese, R.F., Sung, J., Nag, S., Sutton, S., Deacon, J.C., Choe, E., Leinwand, L.A., Ruppel, K., and Spudich, J.A. (2013) Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human ß-cardiac myosin motor function. Proceedings of the National Academy of Sciences. 110, 12607-12612
25. Nag, S., Sommese, R.F., Ujfalusi, Z., Combs, A., Langer, S., Sutton, S., Leinwand, L.A., Geeves, M.A., Ruppel, K., and Spudich, J.A. (2015) Contractility parameters of human ß-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function. Science Advances. 1,
26. Bottinelli, R., and Reggiani, C. (2000) Human skeletal muscle fibres: molecular and functional diversity. Progress in Biophysics & Molecular Biology. 73, 195-262
27. Furch, M., Geeves, M.A., and Manstein, D.J. (1998) Modulation of actin affinity and actomyosin adenosine triphosphatase by charge changes in the myosin motor domain. Biochemistry. 37, 6317-6326
28. Bagshaw, C.R., and Trentham, D.R. (1973) The Reversibility of Adenosine Triphosphate Cleavage by Myosin. Biochemical Journal. 133, 323-328
29. Kurzawa, S.E., and Geeves, M.A. (1996) A novel stopped-flow method for measuring the affinity of actin for myosin head freagment using microgram quantities of protein. Journal of Muscle Research and Cell Motility. 17, 669-676
30. Biasini, M., Bienert, S., Waterhouse, A., Arnold, K., Struder, G., Schmidt, T., Kiefer, F., Cassarino, T.G., Bertoni, M., Bordoli, L., and Schwede, T. (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research. 42, 252-258
31. Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006) The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics. 22, 195-201
32. Guex, N., Peitsch, M.C., and Schwede, T. (2009) Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis. 30, 162-173
33. Kiefer, F., Arnold, K., Künzil, M., Bordoli, L., and Schwede, T. (2009) The SWISS-MODEL Repository and associated resources. Nucleic Acids Research. 37, 387-382
34. Bloemink, M.J., Adamek, N., Reggiani, C., and Geeves, M.A. (2007) Kinetic analysis of the slow skeletal myosin MHC-1 isoform from Bovine masseter muscle. Journal of Molecular Biology. 373, 1184-1197
35. Cooke, R., and Pate, E. (1985) The effects of ADP and Phosphate on the contraction of muscle fibers. Biophysical Journal. 48, 789-798
36. Siemankowski, R.F., Wiseman, M.O., and White, H.D. (1985) ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle. Proceedings of the National Academy of Sciences of the United States of America. 82, 658-662
37. Deacon, J.C., Bloemink, M.J., Rezavandi, H., Geeves, M.A., and Leinwand, L.A. (2012) Erratum to: Identification of functional differences between recombinant human α and β cardiac myosin motors. Cellular and Molecular Life Sciences. 69, 4239-4255
38. Bloemink, M.J., and Geeves, M.A. (2011) Shaking the myosin family tree: Biochemical kinetics defines four types of myosin motor. Seminars in Cell & Developmental Biology. 22, 961-967
39. Geeves, M.A., and Holmes, K.C. (1999) Structural mechanism of muscle contraction. Annual Review of Biochemistry. 68, 687-728
40. Málnási-Csizmadia, A., Pearson, D.S., Kovács, M., Woolley, R.J., Geeves, M.A., and Bagshaw, C.R. (2001) Kinetic Resolution of a Conformational Transition and the ATP Hydrolysis Step Using Relaxation Methods with a Dictyostelium Myosin II Mutant Containing a Single Tryptophan Residue. Biochemistry. 40, 12727-12737
41. Bloemink, M.J., Deacon, J.C., Resnicow, D.I., Leinwand, L.A., and Geeves, M.A. (2013) The Superfast Human Extraocular Myosin Is Kinetically Distinct from the Fast Skeletal IIa, IIb, and IId Isoforms. The Journal of Biological Chemistry. 288, 27469-27479
42. Wheeler, S.E., and Bloom, W.G. (2014) Toward a More Complete Understanding of Noncovalent Interactions Involving Aromatic Rings<br /> . The Journal of Pyhsical Chemistry A. 118, 6133-6147
43. Dougherty, D.A. (1996) Cation-π Interaction in Chemistry and Biology: A New View of Benzene, Phe, Tyr, and Trp. Science. 271, 163-168
44. Fischer, S., Windschügel, B., Horak, D., Holmes, K.C., and Smith, J.C. (2005) Structural mechanism of the recovery stroke in the Myosin molecular motor. Proceedings of the National Academy of Sciences. 102, 6873-6878
45. Mijailovich, S.M., Stojanovic, B., Djordje, N., and Geeves, M.A. (2015) Activation and releaxation kinetics in skeletal and cardiac muscles. Biophysical Journal. 108, 337a-338a
46. Mijailovich, S.M., Djordje, N., Svicevic, M., Stojanovic, B., and Geeves, M.A. (2015) Modelling the calcium dependent actin-myosin ATP-ase cycle in solution. Biophysical Journal. 108, 594a