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 Ca(2+) 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 protein-kinase-A (PKA), Cα (PKCα) and Ca(2+)/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. CamKIIδ activation was determined indirectly by measuring PLN Thr17phosphorylation level. No significant changes were found between the groups. Myofilament Ca(2+) sensitivity is mediated by sarcomeric troponin I (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(2+)-handling proteins contribute to RV diastolic dysfunction due to insufficient diastolic Ca(2+) 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(2+) handling proteins contribute to RV diastolic dysfunction in PAH.

Keywords: diastole; pulmonary heart disease.

Figure 1.

PKA, PKCα, and CaMK_IIδ treatment effect on diastolic stiffness mediated by titin phosphorylation. A‐A1. Titin N2B domain serine 469 PKΑ‐dependent phosphorylation. Typical example of the 2 titin isoforms (upper band: N2BA; lower band N2B) immunostained with phosphospecific antibody against serine 469 site on titin N2B domain and the corresponding Ponceau S staining for total titin (n_Don=7, n_PAH=5). A2. Donor and PAH cardiomyocyte stiffness was measured in relaxing solution at increasing sarcomere length (1.8 to 2.4 μm)—continuous line. The same cardiomyocyte was further incubated with PKA active subunit and stiffness measurements were repeated—dotted line (n_Don=4, n_PAH=4). B‐B1. Titin PEVK domain serine 170 PKCα‐dependent phosphorylation. Typical example of the 2 titin isoforms (upper band: N2BA; lower band N2B) immunostained with phosphospecific antibody against serine 170 site on titin PEVK domain and the corresponding Ponceau S staining for total titin (n_Don=7, n_PAH=5). B2.Titin PEVK domain serine 26 PKCα‐dependent phosphorylation. Typical example of the 2 titin isoforms (upper band: N2BA; lower band N2B) immunostained with phosphospecific antibody against serine 26 site on titin PEVK domain and the corresponding Ponceau S staining for total titin (n_Don=7, n_PAH=5). B3. Donor and PAH cardiomyocyte stiffness was measured in relaxing solution at increasing sarcomere length (1.8 to 2.4 μm)—continuous line. The same cardiomyocyte was further incubated with PKCα active subunit and stiffness measurements were repeated—dotted line (n_Don=3, n_PAH=3). C‐C1. Phospholamban threonine 17 CamK_IIδ‐dependent phosphorylation was used as an indirect measurement of titin CamK_IIδ phosphorylation. The phosphorylation level was normalized to the total amount of Phospholamban present in the sample (n_Don=7, n_PAH=11). C2. Donor and PAH cardiomyocyte stiffness was measured in relaxing solution at increasing sarcomere length (1.8 to 2.4 μm)—continuous line. The same cardiomyocyte was further incubated with CamK_IIδ and stiffness measurements were repeated—dotted line (n_Don=3, n_PAH=3). Data presented as mean±SEM. CamKIIδ indicates calmoduling‐dependent‐kinase; PAH, pulmonary arterial hypertension; PKA, protein‐kinase‐A; PLN, phospholamban.

Figure 2.

Sarcomeric protein phosphorylation. A, cTnI total phosphorylation was determined by ProQ‐Diamond staining, while total protein content was determined by SYPRO‐Ruby staining (n_Don=10, n_PAH=10). B, MyBPC total phosphorylation was determined by ProQ staining, while total protein content was determined by SYPRO staining (n_Don=10, n_PAH=10). C, Typical example of donor and PAH samples ProQ and SYPRO staining and the relative location of specific sarcomeric protein. ProQ staining annotations—ov, ovalbumin; β, β‐casein; MyBPC, myosin binding protein C; cTnT, cardiac troponin T; cTnI, cardiac troponin I; MLC₂, myosin light chain 2. SYPRO staining annotations—MHC, myosin heavy chain. Data presented as mean±SEM. PAH inidcates pulmonary arterial hypertension.

Figure 3.

cTnI phosphorylation. A, Typical example of donor and PAH samples immunostained against the unphosphorylated form of cTnI and normalized for Ponceau‐S stained actin. cTnI total phosphorylation was determined by Western blot analysis in donor and PAH samples (n_Don=8, n_PAH=9). B, Typical example of donor and PAH samples immunostained against cTnI serine 22/23 residue and normalized for Ponceau‐S stained actin. cTnI serine 22/23 phosphorylation was determined by Western blot analysis in donor and PAH samples (n_Don=9, n_PAH=11). C, Typical example of donor and PAH samples immunostained against unphosphorylated (P₀) cTnI, mono‐phosphorylated (P₁) cTnI and bis‐phosphorylated (P₂) cTnI. cTnI relative phosphorylation distribution was determined by Phos‐Tag analysis in donor and PAH samples (n_Don=5, n_PAH=9). Data presented as mean±SEM. cTnI indicates cardiac troponin I; cTnT, cardiac troponin T; PAH, pulmonary arterial hypertension.

Figure 4.

Ca²⁺‐handling proteins expression and phosphorylation. Typical example of donor and PAH samples immunostained against the corresponding Ca²⁺‐handeling protein and normalized for Ponceau‐S stained actin. A, SERCA2a: Sarco/Endoplasmic Reticulum Ca²⁺‐ATPase 2a (cardiac isoform) expression level (n_Don=9, n_PAH=11). B, Total PLN: phospholamban expression level (n_Don=7, n_PAH=10). C, PLN (Ser16)/Total PKA‐dependent phosphorylation level of serine 16 residue on PLN normalized to total PLN protein content (n_Don=7, n_PAH=9). D, PLN/SERCA2a inhibitory effect of PLN on SERCA2a quantified by the ratio between 2 (n_Don=8, n_PAH=9). E, NCX1, Na⁺/Ca²⁺ exchanger 1 (cardiac isoform) expression level (n_Don=9, n_PAH=11). F, RyR2, ryanodine receptor 2 (cardiac isoform) expression level (n_Don=9, n_PAH=11). Data presented as mean±SEM. PAH indicates pulmonary arterial hypertension; PLA, protein‐kinase‐A; PLN, phospholamban.