AMP-Activated protein kinase phosphorylates cardiac troponin i and alters contractility of murine ventricular myocytes

Sandra Marisa Oliveira, Yin Hua Zhang, Raquel Sancho Solis, Henrik Isackson, Mohamed Bellahcene, Arash Yavari, Katalin Pinter, Joanna K. Davies, Ying Ge, Houman Ashrafian, Jeffery W. Walker, David Carling, Hugh Watkins, Barbara Casadei, Charles Redwood

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Abstract

Rationale: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory γ2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. Objective: The γ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of γ2 may be involved in conferring substrate specificity or in determining intracellular localization. Methods and Results: A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of γ2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca transient and prolonged relaxation despite shortening the time constant of Ca transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca sensitivity. Conclusions: We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.

Original languageEnglish
Pages (from-to)1192-1201
Number of pages10
JournalCirculation Research
Volume110
Issue number9
DOIs
StatePublished - 27 Apr 2012

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AMP-Activated Protein Kinases
Troponin
Muscle Cells
Troponin I
Myofibrils
Myosins
Substrate Specificity
Gene Library
Cardiac Myocytes
Hypertrophy
Protein Isoforms
Ischemia
Yeasts
Phosphorylation
Phenotype
Mutation

Keywords

  • Familial hypertrophic cardiomyopathy
  • Myocardial contractility
  • Phosphorylation

Cite this

Oliveira, Sandra Marisa ; Zhang, Yin Hua ; Solis, Raquel Sancho ; Isackson, Henrik ; Bellahcene, Mohamed ; Yavari, Arash ; Pinter, Katalin ; Davies, Joanna K. ; Ge, Ying ; Ashrafian, Houman ; Walker, Jeffery W. ; Carling, David ; Watkins, Hugh ; Casadei, Barbara ; Redwood, Charles. / AMP-Activated protein kinase phosphorylates cardiac troponin i and alters contractility of murine ventricular myocytes. In: Circulation Research. 2012 ; Vol. 110, No. 9. pp. 1192-1201.
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abstract = "Rationale: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory γ2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. Objective: The γ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of γ2 may be involved in conferring substrate specificity or in determining intracellular localization. Methods and Results: A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of γ2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca transient and prolonged relaxation despite shortening the time constant of Ca transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca sensitivity. Conclusions: We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.",
keywords = "Familial hypertrophic cardiomyopathy, Myocardial contractility, Phosphorylation",
author = "Oliveira, {Sandra Marisa} and Zhang, {Yin Hua} and Solis, {Raquel Sancho} and Henrik Isackson and Mohamed Bellahcene and Arash Yavari and Katalin Pinter and Davies, {Joanna K.} and Ying Ge and Houman Ashrafian and Walker, {Jeffery W.} and David Carling and Hugh Watkins and Barbara Casadei and Charles Redwood",
year = "2012",
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Oliveira, SM, Zhang, YH, Solis, RS, Isackson, H, Bellahcene, M, Yavari, A, Pinter, K, Davies, JK, Ge, Y, Ashrafian, H, Walker, JW, Carling, D, Watkins, H, Casadei, B & Redwood, C 2012, 'AMP-Activated protein kinase phosphorylates cardiac troponin i and alters contractility of murine ventricular myocytes', Circulation Research, vol. 110, no. 9, pp. 1192-1201. https://doi.org/10.1161/CIRCRESAHA.111.259952

AMP-Activated protein kinase phosphorylates cardiac troponin i and alters contractility of murine ventricular myocytes. / Oliveira, Sandra Marisa; Zhang, Yin Hua; Solis, Raquel Sancho; Isackson, Henrik; Bellahcene, Mohamed; Yavari, Arash; Pinter, Katalin; Davies, Joanna K.; Ge, Ying; Ashrafian, Houman; Walker, Jeffery W.; Carling, David; Watkins, Hugh; Casadei, Barbara; Redwood, Charles.

In: Circulation Research, Vol. 110, No. 9, 27.04.2012, p. 1192-1201.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - AMP-Activated protein kinase phosphorylates cardiac troponin i and alters contractility of murine ventricular myocytes

AU - Oliveira, Sandra Marisa

AU - Zhang, Yin Hua

AU - Solis, Raquel Sancho

AU - Isackson, Henrik

AU - Bellahcene, Mohamed

AU - Yavari, Arash

AU - Pinter, Katalin

AU - Davies, Joanna K.

AU - Ge, Ying

AU - Ashrafian, Houman

AU - Walker, Jeffery W.

AU - Carling, David

AU - Watkins, Hugh

AU - Casadei, Barbara

AU - Redwood, Charles

PY - 2012/4/27

Y1 - 2012/4/27

N2 - Rationale: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory γ2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. Objective: The γ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of γ2 may be involved in conferring substrate specificity or in determining intracellular localization. Methods and Results: A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of γ2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca transient and prolonged relaxation despite shortening the time constant of Ca transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca sensitivity. Conclusions: We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.

AB - Rationale: AMP-activated protein kinase (AMPK) is an important regulator of energy balance and signaling in the heart. Mutations affecting the regulatory γ2 subunit have been shown to cause an essentially cardiac-restricted phenotype of hypertrophy and conduction disease, suggesting a specific role for this subunit in the heart. Objective: The γ isoforms are highly conserved at their C-termini but have unique N-terminal sequences, and we hypothesized that the N-terminus of γ2 may be involved in conferring substrate specificity or in determining intracellular localization. Methods and Results: A yeast 2-hybrid screen of a human heart cDNA library using the N-terminal 273 residues of γ2 as bait identified cardiac troponin I (cTnI) as a putative interactor. In vitro studies showed that cTnI is a good AMPK substrate and that Ser150 is the principal residue phosphorylated. Furthermore, on AMPK activation during ischemia, Ser150 is phosphorylated in whole hearts. Using phosphomimics, measurements of actomyosin ATPase in vitro and force generation in demembraneated trabeculae showed that modification at Ser150 resulted in increased Ca sensitivity of contractile regulation. Treatment of cardiomyocytes with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) resulted in increased myocyte contractility without changing the amplitude of Ca transient and prolonged relaxation despite shortening the time constant of Ca transient decay (tau). Compound C prevented the effect of AICAR on myocyte function. These results suggest that AMPK activation increases myocyte contraction and prolongs relaxation by increasing myofilament Ca sensitivity. Conclusions: We conclude that cTnI phosphorylation by AMPK may represent a novel mechanism of regulation of cardiac function.

KW - Familial hypertrophic cardiomyopathy

KW - Myocardial contractility

KW - Phosphorylation

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U2 - 10.1161/CIRCRESAHA.111.259952

DO - 10.1161/CIRCRESAHA.111.259952

M3 - Article

VL - 110

SP - 1192

EP - 1201

JO - Circulation research

JF - Circulation research

SN - 0009-7330

IS - 9

ER -