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

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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.

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Acknowledgments

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.

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  1. Abbey E. Weith

    Present address: Department of Physiology, Pennsylvania Muscle Institute, University of Pennsylvania School of Medicine, 3620 Hamilton Walk B40 Anatomy/Chemistry, Philadelphia, PA, 19104, USA

Authors and Affiliations

  1. Department of Molecular Physiology & Biophysics, University of Vermont, HSRF, Room 116, 149 Beaumont Ave., Burlington, VT, 05405, USA

    Abbey E. Weith, Michael J. Previs, Gregory J. Hoeprich, Samantha Beck Previs & David M. Warshaw

  2. Department of Pediatrics and The Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, USA

    James Gulick & Jeffrey Robbins

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  1. Abbey E. Weith
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Correspondence to David M. Warshaw.

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Abbey E. Weith and Michael J. Previs contributed equally to this study.

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Weith, A.E., Previs, M.J., Hoeprich, G.J. et al. The extent of cardiac myosin binding protein-C phosphorylation modulates actomyosin function in a graded manner. J Muscle Res Cell Motil 33, 449–459 (2012). https://doi.org/10.1007/s10974-012-9312-y

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  • DOI: https://doi.org/10.1007/s10974-012-9312-y

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