Impact of site-specific phosphorylation of protein kinase a sites ser23 and ser24 of cardiac troponin i in human cardiomyocytes

Paul J M Wijnker, Darren Brian Foster, Allison L. Tsao, Aisha H. Frazier, Cristobal G. dos Remedios, Anne M Murphy, Ger J M Stienen, Jolanda van der Velden

Research output: Contribution to journalArticle

Abstract

PKA-mediated phosphorylation of contractile proteins upon β-adrenergic stimulation plays an important role in the regulation of cardiac performance. Phosphorylation of the PKA sites (Ser23/Ser24) of cardiac troponin (cTn)I results in a decrease in myofilament Ca2+ sensitivity and an increase in the rate of relaxation. However, the relation between the level of phosphorylation of the sites and the functional effects in the human myocardium is unknown. Therefore, site-directed mutagenesis was used to study the effects of phosphorylation at Ser23 and Ser24 of cTnI on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnI-DD mimics both sites phosphorylated, cTnI-AD mimics Ser23 unphosphorylated and Ser24 phosphorylated, cTnI-DA mimics Ser23 phosphorylated and Ser24 unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca2+ concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca2+ sensitivity (pCa50) was significantly lower in cTnI-DD (pCa50: 5.39 ± 0.01) compared with cTnI-AA (pCa50: 5.50 ± 0.01), cTnI-AD (pCa50: 5.48 ± 0.01), and cTnI-DA (pCa50: 5.51 ± 0.01) at ~70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa50 with cTnI-DD content was observed, saturating at ~55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca2+ sensitivity, which is maximal at ~55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the DISCUSSION in this article.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume304
Issue number2
DOIs
StatePublished - Jan 15 2013

Fingerprint

Troponin
Cardiac Myocytes
Protein Kinases
Myofibrils
Phosphorylation
Myocardium
D-Aspartic Acid
Contractile Proteins
Troponin I
Site-Directed Mutagenesis
Alanine
Adrenergic Agents
Serine
Health

Keywords

  • Cardiomyocyte
  • Myofilament function
  • Protein phosphorylation
  • Troponin I

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)
  • Cardiology and Cardiovascular Medicine

Cite this

Impact of site-specific phosphorylation of protein kinase a sites ser23 and ser24 of cardiac troponin i in human cardiomyocytes. / Wijnker, Paul J M; Foster, Darren Brian; Tsao, Allison L.; Frazier, Aisha H.; dos Remedios, Cristobal G.; Murphy, Anne M; Stienen, Ger J M; van der Velden, Jolanda.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 304, No. 2, 15.01.2013.

Research output: Contribution to journalArticle

Wijnker, Paul J M ; Foster, Darren Brian ; Tsao, Allison L. ; Frazier, Aisha H. ; dos Remedios, Cristobal G. ; Murphy, Anne M ; Stienen, Ger J M ; van der Velden, Jolanda. / Impact of site-specific phosphorylation of protein kinase a sites ser23 and ser24 of cardiac troponin i in human cardiomyocytes. In: American Journal of Physiology - Heart and Circulatory Physiology. 2013 ; Vol. 304, No. 2.
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AU - Foster, Darren Brian

AU - Tsao, Allison L.

AU - Frazier, Aisha H.

AU - dos Remedios, Cristobal G.

AU - Murphy, Anne M

AU - Stienen, Ger J M

AU - van der Velden, Jolanda

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N2 - PKA-mediated phosphorylation of contractile proteins upon β-adrenergic stimulation plays an important role in the regulation of cardiac performance. Phosphorylation of the PKA sites (Ser23/Ser24) of cardiac troponin (cTn)I results in a decrease in myofilament Ca2+ sensitivity and an increase in the rate of relaxation. However, the relation between the level of phosphorylation of the sites and the functional effects in the human myocardium is unknown. Therefore, site-directed mutagenesis was used to study the effects of phosphorylation at Ser23 and Ser24 of cTnI on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnI-DD mimics both sites phosphorylated, cTnI-AD mimics Ser23 unphosphorylated and Ser24 phosphorylated, cTnI-DA mimics Ser23 phosphorylated and Ser24 unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca2+ concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca2+ sensitivity (pCa50) was significantly lower in cTnI-DD (pCa50: 5.39 ± 0.01) compared with cTnI-AA (pCa50: 5.50 ± 0.01), cTnI-AD (pCa50: 5.48 ± 0.01), and cTnI-DA (pCa50: 5.51 ± 0.01) at ~70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa50 with cTnI-DD content was observed, saturating at ~55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca2+ sensitivity, which is maximal at ~55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the DISCUSSION in this article.

AB - PKA-mediated phosphorylation of contractile proteins upon β-adrenergic stimulation plays an important role in the regulation of cardiac performance. Phosphorylation of the PKA sites (Ser23/Ser24) of cardiac troponin (cTn)I results in a decrease in myofilament Ca2+ sensitivity and an increase in the rate of relaxation. However, the relation between the level of phosphorylation of the sites and the functional effects in the human myocardium is unknown. Therefore, site-directed mutagenesis was used to study the effects of phosphorylation at Ser23 and Ser24 of cTnI on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnI-DD mimics both sites phosphorylated, cTnI-AD mimics Ser23 unphosphorylated and Ser24 phosphorylated, cTnI-DA mimics Ser23 phosphorylated and Ser24 unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca2+ concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca2+ sensitivity (pCa50) was significantly lower in cTnI-DD (pCa50: 5.39 ± 0.01) compared with cTnI-AA (pCa50: 5.50 ± 0.01), cTnI-AD (pCa50: 5.48 ± 0.01), and cTnI-DA (pCa50: 5.51 ± 0.01) at ~70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa50 with cTnI-DD content was observed, saturating at ~55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca2+ sensitivity, which is maximal at ~55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the DISCUSSION in this article.

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