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GSK3? phosphorylates newly identified site in the proline-alanine-rich region of cardiac myosin-binding protein C and alters cross-bridge cycling kinetics in human: short communication.

ABSTRACT: Cardiac myosin-binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C.To identify and characterize additional phosphorylation sites in human cMyBP-C.Cardiac MyBP-C was semipurified from human heart tissue. Tandem mass spectrometry analysis identified a novel phosphorylation site on serine 133 in the proline-alanine-rich linker sequence between the C0 and C1 domains of cMyBP-C. Unlike the known sites, Ser133 was not a target of protein kinase A. In silico kinase prediction revealed glycogen synthase kinase 3? (GSK3?) as the most likely kinase to phosphorylate Ser133. In vitro incubation of the C0C2 fragment of cMyBP-C with GSK3? showed phosphorylation on Ser133. In addition, GSK3? phosphorylated Ser304, although the degree of phosphorylation was less compared with protein kinase A-induced phosphorylation at Ser304. GSK3? treatment of single membrane-permeabilized human cardiomyocytes significantly enhanced the maximal rate of tension redevelopment.GSK3? phosphorylates cMyBP-C on a novel site, which is positioned in the proline-alanine-rich region and increases kinetics of force development, suggesting a noncanonical role for GSK3? at the sarcomere level. Phosphorylation of Ser133 in the linker domain of cMyBP-C may be a novel mechanism to regulate sarcomere kinetics.

SUBMITTER: Kuster DW

PROVIDER: S-EPMC3595322 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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GSK3β phosphorylates newly identified site in the proline-alanine-rich region of cardiac myosin-binding protein C and alters cross-bridge cycling kinetics in human: short communication.

Kuster Diederik W D DW Sequeira Vasco V Najafi Aref A Boontje Nicky M NM Wijnker Paul J M PJ Witjas-Paalberends E Rosalie ER Marston Steven B SB Dos Remedios Cristobal G CG Carrier Lucie L Demmers Jeroen A A JA Redwood Charles C Sadayappan Sakthivel S van der Velden Jolanda J

Circulation research 20121231 4

<h4>Rationale</h4>Cardiac myosin-binding protein C (cMyBP-C) regulates cross-bridge cycling kinetics and, thereby, fine-tunes the rate of cardiac muscle contraction and relaxation. Its effects on cardiac kinetics are modified by phosphorylation. Three phosphorylation sites (Ser275, Ser284, and Ser304) have been identified in vivo, all located in the cardiac-specific M-domain of cMyBP-C. However, recent work has shown that up to 4 phosphate groups are present in human cMyBP-C.<h4>Objective</h4>To ...[more]

PMID: 23277198

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Project description:Cardiac myosin binding protein C (cMyBP-C) is an essential regulator of cross bridge cycling. Through mechanisms that are incompletely understood the N-terminal domains (NTDs) of cMyBP-C can activate contraction even in the absence of calcium and can also inhibit cross bridge kinetics in the presence of calcium. In vitro studies indicated that the proline-alanine rich (p/a) region and C1 domain are involved in these processes, although effects were greater using human proteins compared to murine proteins (Shaffer et al. J Biomed Biotechnol 2010, 2010: 789798). We hypothesized that the p/a and C1 region are critical for the timing of contraction. In this study we tested this hypothesis using a mouse model lacking the p/a and C1 region (p/a-C1(-/-) mice) to investigate the in vivo relevance of these regions on cardiac performance. Surprisingly, hearts of adult p/a-C1(-/-) mice functioned normally both on a cellular and whole organ level. Force measurements in permeabilized cardiomyocytes from adult p/a-C1(-/-) mice and wild type (Wt) littermate controls demonstrated similar rates of force redevelopment both at submaximal and maximal activation. Maximal and passive force and calcium sensitivity of force were comparable between groups as well. Echocardiograms showed normal isovolumetric contraction times, fractional shortening and ejection fraction, indicating proper systolic function in p/a-C1(-/-) mouse hearts. p/a-C1(-/-) mice showed a slight but significant reduction in isovolumetric relaxation time compared to Wt littermates, yet this difference disappeared in older mice (7-8months of age). Moreover, stroke volume was preserved in p/a-C1(-/-) mice, corroborating sufficient time for normal filling of the heart. Overall, the hearts of p/a-C1(-/-) mice showed no signs of dysfunction even after chronic stress with an adrenergic agonist. Together, these results indicate that the p/a region and the C1 domain of cMyBP-C are not critical for normal cardiac contraction in mice and that these domains have little if any impact on cross bridge kinetics in mice. These results thus contrast with in vitro studies utilizing proteins encoding the human p/a region and C1 domain. More detailed insight in how individual domains of cMyBP-C function and interact, across species and over the wide spectrum of conditions in which the heart has to function, will be essential to a better understanding of how cMyBP-C tunes cardiac contraction.

Dataset Information

GSK3? phosphorylates newly identified site in the proline-alanine-rich region of cardiac myosin-binding protein C and alters cross-bridge cycling kinetics in human: short communication.

Publications

GSK3β phosphorylates newly identified site in the proline-alanine-rich region of cardiac myosin-binding protein C and alters cross-bridge cycling kinetics in human: short communication.

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