Protein Changes Contributing to Right Ventricular Cardiomyocyte Diastolic Dysfunction in Pulmonary Arterial Hypertension

<jats:sec xml:lang="en"> <jats:title>Background</jats:title> <jats:p xml:lang="en"> Right ventricular ( <jats:styled-content style="fixed-case">RV</jats:styled-content> ) diastolic function is impaired in patients with pulmonary arterial hypertension ( <jats:styled-content style="fixed-case">PAH</jats:styled-content> ). Our previous study showed that elevated cardiomyocyte stiffness and myofilament Ca <jats:sup>2+</jats:sup> sensitivity underlie diastolic dysfunction in <jats:styled-content style="fixed-case">PAH</jats:styled-content> . This study investigates protein modifications contributing to cellular diastolic dysfunction in <jats:styled-content style="fixed-case">PAH</jats:styled-content> . </jats:p> </jats:sec> <jats:sec xml:lang="en"> <jats:title>Methods and Results</jats:title> <jats:p xml:lang="en"> RV samples from PAH patients undergoing heart‐lung transplantation were compared to non‐failing donors (Don). <jats:italic>Titin stiffness</jats:italic> contribution to RV diastolic dysfunction was determined by Western‐blot analyses using antibodies to protein‐kinase‐A (PKA), Cα (PKCα) and Ca <jats:sup>2+</jats: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 ( <jats:italic>P</jats:italic> <0.0001). To test the functional relevance of PKA‐, PKCα‐, and CamK <jats:sub>IIδ</jats:sub> ‐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 <jats:sub>IIδ</jats:sub> had no major effect. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca <jats:sup>2+</jats:sup> sensitivity is mediated by sarcomeric <jats:italic>troponin I</jats:italic> ( <jats:styled-content style="fixed-case">cTnI</jats:styled-content> ) phosphorylation. We observed increased unphosphorylated <jats:styled-content style="fixed-case">cTnI</jats:styled-content> in PAH compared with donors ( <jats:italic>P</jats:italic> <0.05) and reduced PKA‐mediated <jats:styled-content style="fixed-case">cTnI</jats:styled-content> phosphorylation (Ser22/23) ( <jats:italic>P</jats:italic> <0.001). Finally, alterations in <jats:italic>Ca</jats:italic> <jats:sup> <jats:italic>2+</jats:italic> </jats:sup> <jats:italic>‐handling proteins</jats:italic> contribute to RV diastolic dysfunction due to insufficient diastolic Ca <jats:sup>2+</jats:sup> clearance. PAH SERCA2a levels and PLN phosphorylation were significantly reduced compared with donors ( <jats:italic>P</jats:italic> <0.05). </jats:p> </jats:sec> <jats:sec xml:lang="en"> <jats:title>Conclusions</jats:title> <jats:p xml:lang="en"> Increased titin stiffness, reduced <jats:styled-content style="fixed-case">cTnI</jats:styled-content> phosphorylation, and altered levels of phosphorylation of Ca <jats:sup>2+</jats:sup> handling proteins contribute to RV diastolic dysfunction in PAH. </jats:p> </jats:sec>