Journal of Muscle Research and Cell Motility

, Volume 33, Issue 6, pp 449–459

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

DOI: 10.1007/s10974-012-9312-y

Cite this article as:
Weith, A.E., Previs, M.J., Hoeprich, G.J. et al. J Muscle Res Cell Motil (2012) 33: 449. doi:10.1007/s10974-012-9312-y

Abstract

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.

Keywords

Force–velocityViscosityMotility assayLaser trapProtein kinase AContractility

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