A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function

Sakthivel Sadayappan; James Gulick; Hanna Osinska; David Barefield; Friederike Cuello; Metin Avkiran; Valerie M. Lasko; John N. Lorenz; Marjorie Maillet; Jody Martin; Joan Heller Brown; Donald M. Bers; Jeffery D. Molkentin; Jeanne James; Jeffrey Robbins

doi:10.1161/CIRCRESAHA.111.242560

A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function

Sakthivel Sadayappan, James Gulick, Hanna Osinska, David Barefield, Friederike Cuello, Metin Avkiran, Valerie M. Lasko, John N. Lorenz, Marjorie Maillet, Jody Martin, Joan Heller Brown, Donald M. Bers, Jeffery D. Molkentin, Jeanne James, Jeffrey Robbins

Research output: Contribution to journal › Article › peer-review

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Abstract

Rationale: Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation at Ser-273, Ser-282, and Ser-302 regulates myocardial contractility. In vitro and in vivo experiments suggest the nonequivalence of these sites and the potential importance of Ser-282 phosphorylation in modulating the protein's overall phosphorylation and myocardial function. Objective: To determine whether complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation and to define its role in myocardial function. We hypothesized that Ser-282 regulates Ser-302 phosphorylation and cardiac function during beta-adrenergic stimulation. Methods and Results: Using recombinant human C1-M-C2 peptides in vitro, we determined that protein kinase A can phosphorylate Ser-273, Ser-282, and Ser-302. Protein kinase C can also phosphorylate Ser-273 and Ser-302. In contrast, Ca2+-calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca2+-calmodulin-activated kinase II was abolished by ablating the ability of Ser-282 to be phosphorylated via alanine substitution. To determine the functional roles of the sites in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-C-SAS), Ala-273-Asp-282-Ala-302 (cMyBP-C-ADA), or Asp-273-Ala-282-Asp-302 (cMyBP-C-DAD), were generated. Mutant protein was completely substituted for endogenous cMyBP-C by breeding each mouse line into a cMyBP-C null (t/t) background. Serine-to-alanine substitutions were used to ablate the abilities of the residues to be phosphorylated, whereas serine-to-aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control nontransgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-C-SAS(t/t), cMyBP-C-ADA(t/t), and cMyBP-C-DAD(t/t) mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C-(t/t) phenotype. The loss of cMyBP-C phosphorylation at Ser-282 led to an altered beta-adrenergic response. In vivo hemodynamic studies revealed that contractility was unaffected but that cMyBP-C-SAS(t/t) hearts showed decreased diastolic function at baseline. However, the normal increases in cardiac function (increased contractility/relaxation) as a result of infusion of beta-agonist was significantly decreased in all of the mutants, suggesting that competency for phosphorylation at multiple sites in cMyBP-C is a prerequisite for normal beta-adrenergic responsiveness. Conclusions: Ser-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent phosphorylation of Ser-302. However, each residue plays a role in regulating the contractile response to beta-agonist stimulation. (Circ Res. 2011;109:141-150.)

Original language	English
Pages (from-to)	141 - 150
Number of pages	10
Journal	Circulation Research
Volume	109
Issue number	2
Early online date	10 May 2011
DOIs	https://doi.org/10.1161/CIRCRESAHA.111.242560
Publication status	Published - 8 Jul 2011

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10.1161/CIRCRESAHA.111.242560

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Sadayappan, S., Gulick, J., Osinska, H., Barefield, D., Cuello, F., Avkiran, M., Lasko, V. M., Lorenz, J. N., Maillet, M., Martin, J., Brown, J. H., Bers, D. M., Molkentin, J. D., James, J., & Robbins, J. (2011). A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function. Circulation Research, 109(2), 141 - 150. https://doi.org/10.1161/CIRCRESAHA.111.242560

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title = "A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function",

abstract = "Rationale: Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation at Ser-273, Ser-282, and Ser-302 regulates myocardial contractility. In vitro and in vivo experiments suggest the nonequivalence of these sites and the potential importance of Ser-282 phosphorylation in modulating the protein's overall phosphorylation and myocardial function. Objective: To determine whether complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation and to define its role in myocardial function. We hypothesized that Ser-282 regulates Ser-302 phosphorylation and cardiac function during beta-adrenergic stimulation. Methods and Results: Using recombinant human C1-M-C2 peptides in vitro, we determined that protein kinase A can phosphorylate Ser-273, Ser-282, and Ser-302. Protein kinase C can also phosphorylate Ser-273 and Ser-302. In contrast, Ca2+-calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca2+-calmodulin-activated kinase II was abolished by ablating the ability of Ser-282 to be phosphorylated via alanine substitution. To determine the functional roles of the sites in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-C-SAS), Ala-273-Asp-282-Ala-302 (cMyBP-C-ADA), or Asp-273-Ala-282-Asp-302 (cMyBP-C-DAD), were generated. Mutant protein was completely substituted for endogenous cMyBP-C by breeding each mouse line into a cMyBP-C null (t/t) background. Serine-to-alanine substitutions were used to ablate the abilities of the residues to be phosphorylated, whereas serine-to-aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control nontransgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-C-SAS(t/t), cMyBP-C-ADA(t/t), and cMyBP-C-DAD(t/t) mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C-(t/t) phenotype. The loss of cMyBP-C phosphorylation at Ser-282 led to an altered beta-adrenergic response. In vivo hemodynamic studies revealed that contractility was unaffected but that cMyBP-C-SAS(t/t) hearts showed decreased diastolic function at baseline. However, the normal increases in cardiac function (increased contractility/relaxation) as a result of infusion of beta-agonist was significantly decreased in all of the mutants, suggesting that competency for phosphorylation at multiple sites in cMyBP-C is a prerequisite for normal beta-adrenergic responsiveness. Conclusions: Ser-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent phosphorylation of Ser-302. However, each residue plays a role in regulating the contractile response to beta-agonist stimulation. (Circ Res. 2011;109:141-150.)",

author = "Sakthivel Sadayappan and James Gulick and Hanna Osinska and David Barefield and Friederike Cuello and Metin Avkiran and Lasko, {Valerie M.} and Lorenz, {John N.} and Marjorie Maillet and Jody Martin and Brown, {Joan Heller} and Bers, {Donald M.} and Molkentin, {Jeffery D.} and Jeanne James and Jeffrey Robbins",

year = "2011",

month = jul,

day = "8",

doi = "10.1161/CIRCRESAHA.111.242560",

language = "English",

volume = "109",

pages = "141 -- 150",

journal = "Circulation Research",

publisher = "Lippincott Williams and Wilkins",

number = "2",

}

Sadayappan, S, Gulick, J, Osinska, H, Barefield, D, Cuello, F , Avkiran, M, Lasko, VM, Lorenz, JN, Maillet, M, Martin, J, 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', Circulation Research, vol. 109, no. 2, pp. 141 - 150. https://doi.org/10.1161/CIRCRESAHA.111.242560

TY - JOUR

T1 - A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function

AU - Sadayappan, Sakthivel

AU - Gulick, James

AU - Osinska, Hanna

AU - Barefield, David

AU - Cuello, Friederike

AU - Avkiran, Metin

AU - Lasko, Valerie M.

AU - Lorenz, John N.

AU - Maillet, Marjorie

AU - Martin, Jody

AU - Brown, Joan Heller

AU - Bers, Donald M.

AU - Molkentin, Jeffery D.

AU - James, Jeanne

AU - Robbins, Jeffrey

PY - 2011/7/8

Y1 - 2011/7/8

N2 - Rationale: Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation at Ser-273, Ser-282, and Ser-302 regulates myocardial contractility. In vitro and in vivo experiments suggest the nonequivalence of these sites and the potential importance of Ser-282 phosphorylation in modulating the protein's overall phosphorylation and myocardial function. Objective: To determine whether complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation and to define its role in myocardial function. We hypothesized that Ser-282 regulates Ser-302 phosphorylation and cardiac function during beta-adrenergic stimulation. Methods and Results: Using recombinant human C1-M-C2 peptides in vitro, we determined that protein kinase A can phosphorylate Ser-273, Ser-282, and Ser-302. Protein kinase C can also phosphorylate Ser-273 and Ser-302. In contrast, Ca2+-calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca2+-calmodulin-activated kinase II was abolished by ablating the ability of Ser-282 to be phosphorylated via alanine substitution. To determine the functional roles of the sites in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-C-SAS), Ala-273-Asp-282-Ala-302 (cMyBP-C-ADA), or Asp-273-Ala-282-Asp-302 (cMyBP-C-DAD), were generated. Mutant protein was completely substituted for endogenous cMyBP-C by breeding each mouse line into a cMyBP-C null (t/t) background. Serine-to-alanine substitutions were used to ablate the abilities of the residues to be phosphorylated, whereas serine-to-aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control nontransgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-C-SAS(t/t), cMyBP-C-ADA(t/t), and cMyBP-C-DAD(t/t) mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C-(t/t) phenotype. The loss of cMyBP-C phosphorylation at Ser-282 led to an altered beta-adrenergic response. In vivo hemodynamic studies revealed that contractility was unaffected but that cMyBP-C-SAS(t/t) hearts showed decreased diastolic function at baseline. However, the normal increases in cardiac function (increased contractility/relaxation) as a result of infusion of beta-agonist was significantly decreased in all of the mutants, suggesting that competency for phosphorylation at multiple sites in cMyBP-C is a prerequisite for normal beta-adrenergic responsiveness. Conclusions: Ser-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent phosphorylation of Ser-302. However, each residue plays a role in regulating the contractile response to beta-agonist stimulation. (Circ Res. 2011;109:141-150.)

AB - Rationale: Cardiac myosin-binding protein-C (cMyBP-C) phosphorylation at Ser-273, Ser-282, and Ser-302 regulates myocardial contractility. In vitro and in vivo experiments suggest the nonequivalence of these sites and the potential importance of Ser-282 phosphorylation in modulating the protein's overall phosphorylation and myocardial function. Objective: To determine whether complete cMyBP-C phosphorylation is dependent on Ser-282 phosphorylation and to define its role in myocardial function. We hypothesized that Ser-282 regulates Ser-302 phosphorylation and cardiac function during beta-adrenergic stimulation. Methods and Results: Using recombinant human C1-M-C2 peptides in vitro, we determined that protein kinase A can phosphorylate Ser-273, Ser-282, and Ser-302. Protein kinase C can also phosphorylate Ser-273 and Ser-302. In contrast, Ca2+-calmodulin-activated kinase II targets Ser-302 but can also target Ser-282 at nonphysiological calcium concentrations. Strikingly, Ser-302 phosphorylation by Ca2+-calmodulin-activated kinase II was abolished by ablating the ability of Ser-282 to be phosphorylated via alanine substitution. To determine the functional roles of the sites in vivo, three transgenic lines, which expressed cMyBP-C containing either Ser-273-Ala-282-Ser-302 (cMyBP-C-SAS), Ala-273-Asp-282-Ala-302 (cMyBP-C-ADA), or Asp-273-Ala-282-Asp-302 (cMyBP-C-DAD), were generated. Mutant protein was completely substituted for endogenous cMyBP-C by breeding each mouse line into a cMyBP-C null (t/t) background. Serine-to-alanine substitutions were used to ablate the abilities of the residues to be phosphorylated, whereas serine-to-aspartate substitutions were used to mimic the charged state conferred by phosphorylation. Compared to control nontransgenic mice, as well as transgenic mice expressing wild-type cMyBP-C, the transgenic cMyBP-C-SAS(t/t), cMyBP-C-ADA(t/t), and cMyBP-C-DAD(t/t) mice showed no increases in morbidity and mortality and partially rescued the cMyBP-C-(t/t) phenotype. The loss of cMyBP-C phosphorylation at Ser-282 led to an altered beta-adrenergic response. In vivo hemodynamic studies revealed that contractility was unaffected but that cMyBP-C-SAS(t/t) hearts showed decreased diastolic function at baseline. However, the normal increases in cardiac function (increased contractility/relaxation) as a result of infusion of beta-agonist was significantly decreased in all of the mutants, suggesting that competency for phosphorylation at multiple sites in cMyBP-C is a prerequisite for normal beta-adrenergic responsiveness. Conclusions: Ser-282 has a unique regulatory role in that its phosphorylation is critical for the subsequent phosphorylation of Ser-302. However, each residue plays a role in regulating the contractile response to beta-agonist stimulation. (Circ Res. 2011;109:141-150.)

U2 - 10.1161/CIRCRESAHA.111.242560

DO - 10.1161/CIRCRESAHA.111.242560

M3 - Article

VL - 109

SP - 141

EP - 150

JO - Circulation Research

JF - Circulation Research

IS - 2

ER -

A Critical Function for Ser-282 in Cardiac Myosin Binding Protein-C Phosphorylation and Cardiac Function

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