Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension

Silvia Rain, Denielli Silva Goncalves Bos, M. Louis Handoko, Nico Westerhof, Ger Stienen, Coen Ottenheijm, Max Goebel, Peter Dorfmüller, Christophe Guignabert, Marc Humbert, Harm Jan Bogaard, Cris Dos Remedios, Chandra Saripalli, Carlos G. Hidalgo, Hendrikus "Henk" Granzier, Anton Vonk-Noordegraaf, Jolanda Van der Velden, Frances S. De Man

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Background-Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca<sup>2+</sup> sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH. Methods and Results-RV samples from PAH patients undergoing heart-lung transplantation were compared to non-failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western-blot analyses using antibodies to proteinkinase- A (PKA), Cα (PKCα) and Ca<sup>2+</sup>/calmoduling-dependent-kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P < 0.0001). To test the functional relevance of PKA-, PKCα-, and CamK<inf>IIδ</inf>-mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamK<inf>IIδ</inf> had no major effect. CamKIId activation was determined indirectly by measuring PLN Thr 17phosphorylation level. No significant changes were found between the groups. Myofilament Ca<sup>2+</sup> sensitivity is mediated by sarcomeric troponin/(cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P < 0.05) and reduced PKA-mediated cTnI phosphorylation (Ser22/23) (P < 0.001). Finally, alterations in Ca<sup>2+</sup>-handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca<sup>2+</sup> clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P < 0.05). Conclusions-Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca<sup>2+</sup> handling proteins contribute to RV diastolic dysfunction in PAH.

Original languageEnglish (US)
Article number000716
JournalJournal of the American Heart Association
Volume3
Issue number3
DOIs
StatePublished - 2014

Fingerprint

Pulmonary Hypertension
Cardiac Myocytes
Connectin
Right Ventricular Dysfunction
Proteins
Phosphorylation
Tissue Donors
Myofibrils
Heart-Lung Transplantation
Right Ventricular Function
Troponin
Phosphotransferases
Western Blotting
Antibodies

Keywords

  • Diastole
  • Pulmonary heart disease

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

Rain, S., Goncalves Bos, D. S., Handoko, M. L., Westerhof, N., Stienen, G., Ottenheijm, C., ... De Man, F. S. (2014). Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension. Journal of the American Heart Association, 3(3), [000716]. https://doi.org/10.1161/JAHA.113.000716

Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension. / Rain, Silvia; Goncalves Bos, Denielli Silva; Handoko, M. Louis; Westerhof, Nico; Stienen, Ger; Ottenheijm, Coen; Goebel, Max; Dorfmüller, Peter; Guignabert, Christophe; Humbert, Marc; Bogaard, Harm Jan; Dos Remedios, Cris; Saripalli, Chandra; Hidalgo, Carlos G.; Granzier, Hendrikus "Henk"; Vonk-Noordegraaf, Anton; Van der Velden, Jolanda; De Man, Frances S.

In: Journal of the American Heart Association, Vol. 3, No. 3, 000716, 2014.

Research output: Contribution to journalArticle

Rain, S, Goncalves Bos, DS, Handoko, ML, Westerhof, N, Stienen, G, Ottenheijm, C, Goebel, M, Dorfmüller, P, Guignabert, C, Humbert, M, Bogaard, HJ, Dos Remedios, C, Saripalli, C, Hidalgo, CG, Granzier, HH, Vonk-Noordegraaf, A, Van der Velden, J & De Man, FS 2014, 'Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension', Journal of the American Heart Association, vol. 3, no. 3, 000716. https://doi.org/10.1161/JAHA.113.000716
Rain, Silvia ; Goncalves Bos, Denielli Silva ; Handoko, M. Louis ; Westerhof, Nico ; Stienen, Ger ; Ottenheijm, Coen ; Goebel, Max ; Dorfmüller, Peter ; Guignabert, Christophe ; Humbert, Marc ; Bogaard, Harm Jan ; Dos Remedios, Cris ; Saripalli, Chandra ; Hidalgo, Carlos G. ; Granzier, Hendrikus "Henk" ; Vonk-Noordegraaf, Anton ; Van der Velden, Jolanda ; De Man, Frances S. / Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension. In: Journal of the American Heart Association. 2014 ; Vol. 3, No. 3.
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title = "Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension",
abstract = "Background-Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca2+ sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH. Methods and Results-RV samples from PAH patients undergoing heart-lung transplantation were compared to non-failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western-blot analyses using antibodies to proteinkinase- A (PKA), Cα (PKCα) and Ca2+/calmoduling-dependent-kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P < 0.0001). To test the functional relevance of PKA-, PKCα-, and CamKIIδ-mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIId activation was determined indirectly by measuring PLN Thr 17phosphorylation level. No significant changes were found between the groups. Myofilament Ca2+ sensitivity is mediated by sarcomeric troponin/(cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P < 0.05) and reduced PKA-mediated cTnI phosphorylation (Ser22/23) (P < 0.001). Finally, alterations in Ca2+-handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca2+ clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P < 0.05). Conclusions-Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca2+ handling proteins contribute to RV diastolic dysfunction in PAH.",
keywords = "Diastole, Pulmonary heart disease",
author = "Silvia Rain and {Goncalves Bos}, {Denielli Silva} and Handoko, {M. Louis} and Nico Westerhof and Ger Stienen and Coen Ottenheijm and Max Goebel and Peter Dorfm{\"u}ller and Christophe Guignabert and Marc Humbert and Bogaard, {Harm Jan} and {Dos Remedios}, Cris and Chandra Saripalli and Hidalgo, {Carlos G.} and Granzier, {Hendrikus {"}Henk{"}} and Anton Vonk-Noordegraaf and {Van der Velden}, Jolanda and {De Man}, {Frances S.}",
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T1 - Protein changes contributing to right ventricular cardiomyocyte diastolic dysfunction in pulmonary arterial hypertension

AU - Rain, Silvia

AU - Goncalves Bos, Denielli Silva

AU - Handoko, M. Louis

AU - Westerhof, Nico

AU - Stienen, Ger

AU - Ottenheijm, Coen

AU - Goebel, Max

AU - Dorfmüller, Peter

AU - Guignabert, Christophe

AU - Humbert, Marc

AU - Bogaard, Harm Jan

AU - Dos Remedios, Cris

AU - Saripalli, Chandra

AU - Hidalgo, Carlos G.

AU - Granzier, Hendrikus "Henk"

AU - Vonk-Noordegraaf, Anton

AU - Van der Velden, Jolanda

AU - De Man, Frances S.

PY - 2014

Y1 - 2014

N2 - Background-Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca2+ sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH. Methods and Results-RV samples from PAH patients undergoing heart-lung transplantation were compared to non-failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western-blot analyses using antibodies to proteinkinase- A (PKA), Cα (PKCα) and Ca2+/calmoduling-dependent-kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P < 0.0001). To test the functional relevance of PKA-, PKCα-, and CamKIIδ-mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIId activation was determined indirectly by measuring PLN Thr 17phosphorylation level. No significant changes were found between the groups. Myofilament Ca2+ sensitivity is mediated by sarcomeric troponin/(cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P < 0.05) and reduced PKA-mediated cTnI phosphorylation (Ser22/23) (P < 0.001). Finally, alterations in Ca2+-handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca2+ clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P < 0.05). Conclusions-Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca2+ handling proteins contribute to RV diastolic dysfunction in PAH.

AB - Background-Right ventricular (RV) diastolic function is impaired in patients with pulmonary arterial hypertension (PAH). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca2+ sensitivity underlie diastolic dysfunction in PAH. This study investigates protein modifications contributing to cellular diastolic dysfunction in PAH. Methods and Results-RV samples from PAH patients undergoing heart-lung transplantation were compared to non-failing donors (Don). Titin stiffness contribution to RV diastolic dysfunction was determined by Western-blot analyses using antibodies to proteinkinase- A (PKA), Cα (PKCα) and Ca2+/calmoduling-dependent-kinase (CamKIIδ) titin and phospholamban (PLN) phosphorylation sites: N2B (Ser469), PEVK (Ser170 and Ser26), and PLN (Thr17), respectively. PKA and PKCα sites were significantly less phosphorylated in PAH compared with donors (P < 0.0001). To test the functional relevance of PKA-, PKCα-, and CamKIIδ-mediated titin phosphorylation, we measured the stiffness of single RV cardiomyocytes before and after kinase incubation. PKA significantly decreased PAH RV cardiomyocyte diastolic stiffness, PKCα further increased stiffness while CamKIIδ had no major effect. CamKIId activation was determined indirectly by measuring PLN Thr 17phosphorylation level. No significant changes were found between the groups. Myofilament Ca2+ sensitivity is mediated by sarcomeric troponin/(cTnI) phosphorylation. We observed increased unphosphorylated cTnI in PAH compared with donors (P < 0.05) and reduced PKA-mediated cTnI phosphorylation (Ser22/23) (P < 0.001). Finally, alterations in Ca2+-handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca2+ clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors (P < 0.05). Conclusions-Increased titin stiffness, reduced cTnI phosphorylation, and altered levels of phosphorylation of Ca2+ handling proteins contribute to RV diastolic dysfunction in PAH.

KW - Diastole

KW - Pulmonary heart disease

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