Journal of Muscle Research and Cell Motility

, Volume 33, Issue 6, pp 449–459 | Cite as

The extent of cardiac myosin binding protein-C phosphorylation modulates actomyosin function in a graded manner

  • Abbey E. Weith
  • Michael J. Previs
  • Gregory J. Hoeprich
  • Samantha Beck Previs
  • James Gulick
  • Jeffrey Robbins
  • David M. Warshaw
Original Paper


Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains, C0–C10, binds to the myosin rod via its C-terminus, while its N-terminus binds regions of the myosin head and actin. These N-terminal interactions can be attenuated by phosphorylation of serines in the C1–C2 motif linker. Within the sarcomere, cMyBP-C exists in a range of phosphorylation states, which may affect its ability to regulate actomyosin motion generation. To examine the functional importance of partial phosphorylation, we bacterially expressed N-terminal fragments of cMyBP-C (domains C0–C3) with three of its phosphorylatable serines (S273, S282, and S302) mutated in combinations to either aspartic acids or alanines, mimicking phosphorylation and dephosphorylation respectively. The effect of these C0–C3 constructs on actomyosin motility was characterized in both the unloaded in vitro motility assay and in the load-clamped laser trap assay where force:velocity (F:V) relations were obtained. In the motility assay, phosphomimetic replacement (i.e. aspartic acid) reduced the slowing of actin velocity observed in the presence of C0–C3 in proportion to the total number phosphomimetic replacements. Under load, C0–C3 depressed the F:V relationship without any effect on maximal force. Phosphomimetic replacement reversed the depression of F:V by C0–C3 in a graded manner with respect to the total number of replacements. Interestingly, the effect of C0–C3 on F:V was well fitted by a model that assumed C0–C3 acts as an effective viscous load against which myosin must operate. This study suggests that increasing phosphorylation of cMyBP-C incrementally reduces its modulation of actomyosin motion generation providing a tunable mechanism to regulate cardiac function.


Force–velocity Viscosity Motility assay Laser trap Protein kinase A Contractility 



We thank G. Kennedy, from the Instrumentation and Modeling Facility, for imaging expertise. National Institutes of Health funds supported AW (HL007944); MP (HL07647); JG, JR, and DW (HL059408). The Fondation Leducq supported JR.


  1. Bárány M (1967) ATPase activity of myosin correlated with speed of muscle shortening. J Gen Physiol 50(Suppl):197–218PubMedCrossRefGoogle Scholar
  2. Bárány M, Bárány K (1980) Phosphorylation of the myofibrillar proteins. Annu Rev Physiol 42:275–292PubMedCrossRefGoogle Scholar
  3. Barefield D, Sadayappan S (2011) Phosphorylation and function of cardiac myosin binding protein-C in health and disease. J Mol Cell Cardiol 48:866–875CrossRefGoogle Scholar
  4. Calaghan SC, Trinick J, Knight PJ, White E (2000) A role for C-protein in the regulation of contraction and intracellular Ca2+ in intact rat ventricular myocytes. J Physiol 528(Pt 1):151–156PubMedCrossRefGoogle Scholar
  5. Copeland O, Sadayappan S, Messer AE, Steinen GJ, van der Velden J, Marston SB (2010) Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle. J Mol Cell Cardiol 49:1003–1011PubMedCrossRefGoogle Scholar
  6. Craig R, Offer G (1976) The location of C-protein in rabbit skeletal muscle. Proc R Soc Lond B Biol Sci 192:451–461PubMedCrossRefGoogle Scholar
  7. Debold EP, Patlak JB, Warshaw DM (2005) Slip sliding away: load-dependence of velocity generated by skeletal muscle myosin molecules in the laser trap. Biophys J 89:L34–L36PubMedCrossRefGoogle Scholar
  8. Debold EP, Schmitt JP, Patlak JB, Beck SE, Moore JR, Seidman JG, Seidman C, Warshaw DM (2007) Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse alpha-cardiac myosin in the laser trap assay. Am J Physiol Heart Circ Physiol 293:H284–H291PubMedCrossRefGoogle Scholar
  9. Gautel M, Zuffardi O, Freiburg A, Labeit S (1995) Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction? EMBO J 14:1952–1960PubMedGoogle Scholar
  10. Greenberg MJ, Moore JR (2010) The molecular basis of frictional loads in the in vitro motility assay with applications to the study of the loaded mechanochemistry of molecular motors. Cytoskeleton 67:273–285PubMedCrossRefGoogle Scholar
  11. Gruen M, Gautel M (1999) Mutations in beta-myosin S2 that cause familial hypertrophic cardiomyopathy (FHC) abolish the interaction with the regulatory domain of myosin-binding protein-C. J Mol Biol 286:933–949PubMedCrossRefGoogle Scholar
  12. Gruen M, Prinz H, Gautel M (1999) cAPK-phosphorylation controls the interaction of the regulatory domain of cardiac myosin binding protein C with myosin-S2 in an on–off fashion. FEBS Lett 453:254–259PubMedCrossRefGoogle Scholar
  13. Guilford WH, Dupuis DE, Kennedy G, Wu J, Patlak JB, Warshaw DM (1997) Smooth muscle and skeletal muscle myosins produce similar unitary forces and displacements in the laser trap. Biophys J 72:1006–1021PubMedCrossRefGoogle Scholar
  14. Harris DE, Work SS, Wright RK, Alpert NR, Warshaw DM (1994) Smooth, cardiac and skeletal muscle myosin force and motion generation assessed by cross-bridge mechanical interactions in vitro. J Muscle Res Cell Motil 15:11–19PubMedCrossRefGoogle Scholar
  15. Harris SP, Bartley CR, Hacker TA, McDonald KS, Douglas PS, Greaser ML, Powers PA, Moss RL (2002) Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice. Circ Res 90:594–601PubMedCrossRefGoogle Scholar
  16. Harris SP, Rostkova E, Gautel M, Moss RL (2004) Binding of myosin binding protein-C to myosin subfragment S2 affects contractility independent of a tether mechanism. Circ Res 95:930–936PubMedCrossRefGoogle Scholar
  17. Harris SP, Lyons RG, Bezold KL (2011) In the thick of it: HCM-causing mutations in myosin binding proteins of the thick filament. Circ Res 108:751–764PubMedCrossRefGoogle Scholar
  18. Hill AV (1938) The heat of shortening and the dynamic constants of muscle. Proc R Soc London B Biol Sci 126:136–195CrossRefGoogle Scholar
  19. Hofmann PA, Greaser ML, Moss RL (1991a) C-protein limits shortening velocity of rabbit skeletal muscle fibres at low levels of Ca2+ activation. J Physiol 439:701–715PubMedGoogle Scholar
  20. Hofmann PA, Hartzell HC, Moss RL (1991b) Alterations in Ca2+ sensitive tension due to partial extraction of C-protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers. J Gen Physiol 97:1141–1163PubMedCrossRefGoogle Scholar
  21. Howarth JW, Ramisetti S, Nolan K, Sadayappan S, Rosevear PR (2012) Structural insight into unique cardiac myosin-binding protein-C motif: a partially folded domain. J Biol Chem 287:8254–8262PubMedCrossRefGoogle Scholar
  22. Jacques AM, Copeland O, Messer AE, Gallon CE, King K, McKenna WJ, Tsang VT, Marston SB (2008) Myosin binding protein C phosphorylation in normal, hypertrophic and failing human heart muscle. J Mol Cell Cardiol 45:209–216PubMedCrossRefGoogle Scholar
  23. Jia W, Shaffer JF, Harris SP, Leary JA (2010) Identification of novel protein kinase A phosphorylation sites in the M-domain of human and murine cardiac myosin binding protein-C using mass spectrometry analysis. J Proteome Res 9:1843–1853PubMedCrossRefGoogle Scholar
  24. Korte FS, McDonald KS, Harris SP, Moss RL (2003) Loaded shortening, power output, and rate of force redevelopment are increased with knockout of cardiac myosin binding protein-C. Circ Res 93:752–758PubMedCrossRefGoogle Scholar
  25. Lu Y, Kwan AH, Trewhella J, Jeffries CM (2011) The C0–C1 fragment of human cardiac myosin binding protein C has common binding determinants for both actin and myosin. J Mol Biol 413:908–913PubMedCrossRefGoogle Scholar
  26. Luther PK, Winkler H, Taylor K, Zoghbi ME, Craig R, Padron R, Squire JM, Liu J (2011) Direct visualization of myosin-binding protein C bridging myosin and actin filaments in intact muscle. Proc Natl Acad Sci USA 108:11423–11428PubMedCrossRefGoogle Scholar
  27. Margossian SS, Lowey S (1982) Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol 85 (Pt B): 55–71Google Scholar
  28. McClellan G, Kulikovskaya I, Winegrad S (2001) Changes in cardiac contractility related to calcium-mediated changes in phosphorylation of myosin-binding protein C. Biophys J 81:1083–1092PubMedCrossRefGoogle Scholar
  29. Moos C, Offer G, Starr R, Bennett P (1975) Interaction of C-protein with myosin, myosin rod and light meromyosin. J Mol Biol 97:1–9PubMedCrossRefGoogle Scholar
  30. Moos C, Mason CM, Besterman JM, Feng IN, Dubin JH (1978) The binding of skeletal muscle C-protein to F-actin, and its relation to the interaction of actin with myosin subfragment-1. J Mol Biol 124:571–586PubMedCrossRefGoogle Scholar
  31. Mun JY, Gulick J, Robbins J, Woodhead J, Lehman W, Craig R (2011) Electron microscopy and 3D reconstruction of F-actin decorated with cardiac myosin-binding protein C (cMyBP-C). J Mol Biol 410:214–225PubMedCrossRefGoogle Scholar
  32. Nagayama T, Takimoto E, Sadayappan S, Mudd JO, Seidman JG, Robbins J, Kass DA (2007) Control of in vivo left ventricular contraction/relaxation kinetics by myosin binding protein C: protein kinase A phosphorylation dependent and independent regulation. Circulation 116:2399–2408PubMedCrossRefGoogle Scholar
  33. Palmer BM, Noguchi T, Wang Y, Heim JR, Alpert NR, Burgon PG, Seidman CE, Seidman JG, Maughan DW, LeWinter MM (2004) Effect of cardiac myosin binding protein-C on mechanoenergetics in mouse myocardium. Circ Res 94:1615–1622PubMedCrossRefGoogle Scholar
  34. Palmiter KA, Tyska MJ, Haeberle JR, Alpert NR, Fananapazir L, Warshaw DM (2000) R403Q and L908 V mutant beta-cardiac myosin from patients with familial hypertrophic cardiomyopathy exhibit enhanced mechanical performance at the single molecule level. J Muscle Res Cell Motil 21:609–620PubMedCrossRefGoogle Scholar
  35. Pardee JD, Spudich JA (1982) Purification of muscle actin. Methods Cell Biol 24:271–289PubMedCrossRefGoogle Scholar
  36. Ratti J, Rostkova E, Gautel M, Pfuhl M (2011) Structure and interactions of myosin-binding protein C domain C0: cardiac-specific regulation of myosin at its neck? J Biol Chem 286:12650–12658PubMedCrossRefGoogle Scholar
  37. Razumova MV, Shaffer JF, Tu AY, Flint GV, Regnier M, Harris SP (2006) Effects of the N-terminal domains of myosin binding protein-C in an in vitro motility assay: evidence for long-lived cross-bridges. J Biol Chem 281:35846–35854PubMedCrossRefGoogle Scholar
  38. Razumova MV, Bezold KL, Tu AY, Regnier M, Harris SP (2008) Contribution of the myosin binding protein C motif to functional effects in permeabilized rat trabeculae. J Gen Physiol 132:575–585PubMedCrossRefGoogle Scholar
  39. Rybakova IN, Greaser ML, Moss RL (2011) Myosin binding protein C interaction with actin: characterization and mapping of the binding site. J Biol Chem 286:2008–2016PubMedCrossRefGoogle Scholar
  40. Saber W, Begin KJ, Warshaw DM, VanBuren P (2008) Cardiac myosin binding protein-C modulates actomyosin binding and kinetics in the in vitro motility assay. J Mol Cell Cardiol 44:1053–1061PubMedCrossRefGoogle Scholar
  41. Sadayappan S, Gulick J, Osinska H, Martin LA, Hahn HS, Dorn GW 2nd, Klevitsky R, Seidman CE, Seidman JG, Robbins J (2005) Cardiac myosin-binding protein-C phosphorylation and cardiac function. Circ Res 97:1156–1163PubMedCrossRefGoogle Scholar
  42. Sadayappan S, Osinska H, Klevitsky R, Lorenz JN, Sargent M, Molkentin JD, Seidman CE, Seidman JG, Robbins J (2006) Cardiac myosin binding protein C phosphorylation is cardioprotective. Proc Natl Acad Sci USA 103:16918–16923PubMedCrossRefGoogle Scholar
  43. Sadayappan S, Gulick J, Klevitsky R, Lorenz JN, Sargent M, Molkentin JD, Robbins J (2009) Cardiac myosin binding protein-C phosphorylation in a {beta}-myosin heavy chain background. Circulation 119:1253–1262PubMedCrossRefGoogle Scholar
  44. Sadayappan S, Gulick J, Osinska H, Barefield D, Cuello F, Avkiran M, Lasko VM, Lorenz JN, Maillet M, Martin JL, Brown JH, Bers DM, Molkentin JD, James J, Robbins J (2011) A critical function for Ser-282 in cardiac myosin binding protein-C phosphorylation and cardiac function. Circ Res 109:141–150PubMedCrossRefGoogle Scholar
  45. Shaffer JF, Kensler RW, Harris SP (2009) The myosin-binding protein C motif binds to F-actin in a phosphorylation-sensitive manner. J Biol Chem 284:12318–12327PubMedCrossRefGoogle Scholar
  46. Shchepkin DV, Kopylova GV, Nikitina LV, Katsnelson LB, Bershitsky SY (2010) Effects of cardiac myosin binding protein-C on the regulation of interaction of cardiac myosin with thin filament in an in vitro motility assay. Biochem Biophys Res Commun 401:159–163PubMedCrossRefGoogle Scholar
  47. Stelzer JE, Dunning SB, Moss RL (2006a) Ablation of cardiac myosin-binding protein-C accelerates stretch activation in murine skinned myocardium. Circ Res 98:1212–1218PubMedCrossRefGoogle Scholar
  48. Stelzer JE, Patel JR, Moss RL (2006b) Protein kinase A-mediated acceleration of the stretch activation response in murine skinned myocardium is eliminated by ablation of cMyBP-C. Circ Res 99:884–890PubMedCrossRefGoogle Scholar
  49. Tong CW, Stelzer JE, Greaser ML, Powers PA, Moss RL (2008) Acceleration of cross-bridge kinetics by protein kinase A phosphorylation of cardiac myosin binding protein C modulates cardiac function. Circ Res 103:974–982PubMedCrossRefGoogle Scholar
  50. van Dijk SJ, Dooijes D, dos Remedios C, Michels M, Lamers JM, Winegrad S, Schlossarek S, Carrier L, ten Cate FJ, Stienen GJ, van der Velden J (2009) Cardiac myosin-binding protein C mutations and hypertrophic cardiomyopathy: haploinsufficiency, deranged phosphorylation, and cardiomyocyte dysfunction. Circulation 119:1473–1483PubMedCrossRefGoogle Scholar
  51. Walcott S, Fagnant PM, Trybus KM, Warshaw DM (2009) Smooth muscle heavy meromyosin phosphorylated on one of its two heads supports force and motion. J Biol Chem 284:18244–18251PubMedCrossRefGoogle Scholar
  52. Warshaw DM, Desrosiers JM, Work SS, Trybus KM (1990) Smooth muscle myosin cross–bridge interactions modulate actin filament sliding velocity in vitro. J Cell Biol 111:453–463PubMedCrossRefGoogle Scholar
  53. Weith A, Sadayappan S, Gulick J, Previs MJ, Vanburen P, Robbins J, Warshaw DM (2012) Unique single molecule binding of cardiac myosin binding protein-C to actin and phosphorylation-dependent inhibition of actomyosin motility requires 17 amino acids of the motif domain. J Mol Cell Cardiol 52:219–227PubMedCrossRefGoogle Scholar
  54. Witt CC, Gerull B, Davies MJ, Centner, Linke WA, Thierfelder L ((2001)) Hypercontractile properties of cardiac muscle fibers in a knock-in mouse model of cardiac myosin-binding protein-C. J Biol Chem 276:5353–5359CrossRefGoogle Scholar
  55. Woledge RC, Curtin NA, Homsher E (1985) Energetic aspects of muscle contraction. Monogr Physiol Soc 41:1–357PubMedGoogle Scholar
  56. Yadid M, Sela G, Amiad Pavlov D, Landesberg A (2011) Adaptive control of cardiac contraction to changes in loading: from theory of sarcomere dynamics to whole-heart function. Pflugers Arch 462:49–60PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Abbey E. Weith
    • 1
    • 3
  • Michael J. Previs
    • 1
  • Gregory J. Hoeprich
    • 1
  • Samantha Beck Previs
    • 1
  • James Gulick
    • 2
  • Jeffrey Robbins
    • 2
  • David M. Warshaw
    • 1
  1. 1.Department of Molecular Physiology & BiophysicsUniversity of VermontBurlingtonUSA
  2. 2.Department of Pediatrics and The Heart InstituteCincinnati Children’s Hospital Medical CenterCincinnatiUSA
  3. 3.Department of Physiology, Pennsylvania Muscle InstituteUniversity of Pennsylvania School of MedicinePhiladelphiaUSA

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