PARP1-PKM2 Axis Mediates Right Ventricular Failure Associated With Pulmonary Arterial Hypertension

Figures

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  Figure 1. Increased Expression and Activity of PARP1 and PKM2 in dRV From Patients With PAH (A) Representative images and corresponding quantification of human right ventricular (RV) sections from control subjects (CTRL) (n = 5-8), compensated right ventricle (cRV; n = 7 or 8), and decompensated right ventricle (dRV; n = 11) patients with PAH stained with hematoxylin and eosin (cardiomyocyte surface area [CSA]), Masson’s trichrome (fibrosis), cluster of differentiation 68 (CD68) (infiltration of macrophages), or terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL; apoptosis) or double-labeled for troponin T (TnT) and either poly(adenosine diphosphate–ribose) polymerase 1 (PARP1) or poly(adenosine diphosphate–ribose) (PAR). (B) Representative western blots and quantification of PARP1, phosphorylated pyruvate kinase muscle isozyme 2 (pPKM2), PKM2, and pyruvate kinase muscle isozyme 1 (PKM1) in human right ventricle from control subjects (n = 20), cRV (n = 11), and dRV (n = 10 or 11) patients with PAH. Scale bars, 50 mm. Arrowheads denote positive cells. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scatter dot plots show individual values and mean ± SEM. Comparisons were made…

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  Figure 2. Increased Expression and Activity of PARP1 and PKM2 in Decompensated Right Ventricles From PAB Rats (A) Schematic representation of categorization of pulmonary artery banding (PAB) rats on the basis of hemodynamic status assessed using echocardiography and relative messenger RNA levels of Nppb. PAB rats underwent right heart catheterization at different time points over a period of 6 weeks, starting 2 weeks after PAB surgery. A group of sham-operated rats was used to define normal cardiac index (CO) and stroke volume (SV). PAB rats were subsequently classified into cRV or dRV groups on the basis of hemodynamic data. The relative expression levels of Nppb were used to validate the classification. (B) Representative images and corresponding quantification of RV sections from sham-operated (n = 7) and PAB-operated rats classified as cRV (n = 9) or dRV (n = 8) stained with hematoxylin and eosin (cardiomyocyte cell surface area), Masson’s trichrome (fibrosis), CD68 (infiltration of macrophages), or TUNEL (apoptosis) or double-labeled for TnT and either PARP1 or PAR. (C) Representative western blots and quantification of PARP1, pPKM2, PKM2, and PKM1 in right ventricles from rats…

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  Figure 3. Inhibition of PARP1 and Cytosolic Retention of PKM2 Prevent CM Dysfunction Under Exposure to Oxidative Damage (A) Schematic of the experimental design. (B) Representative immunofluorescent images of PARP1, PKM2, nuclear factor κB (NF-κB), TUNEL, and cytosolic 8-Oxo-2′-deoxyguanosine (8oxodG) in neonatal rat RV CMs (n = 3 or 4) exposed or not to either endothelin-1 (ET-1) or ET-1 + H2O2 in the presence or absence of ABT-888, TEPP-46, and DASA-58. (C) Representative western blots (of 3 independent studies) for PARP1, PKM2, NF-κB, α/β tubulin (Tub), and histone H3 in nuclear and cytosolic extracts from H9c2 cells exposed or not to either ET-1 or ET-1 + H2O2 in the presence or absence of ABT-888, TEPP-46, DASA-58, and siPARP1. (D) Representative immunofluorescent images of TnT, PKM2, NF-κB, and PARP1 in neonatal CMs isolated from wild-type (Parp1+/+, n = 3) and Parp1-knockout (Parp1−/−, n = 3) mice exposed or not to ET-1 + H2O2. Scale bars, 25 mm. NT = non-treated; siSCRM = scrambled siRNA; Veh = vehicle; other abbreviations as in Figure 1.

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  Figure 4. Inhibition of PARP1 and Cytosolic Retention of PKM2 Prevent CM Dysfunction Under Exposure to LPS (A) Schematic of the experimental design. (B) Representative immunofluorescent images of PARP1, NF-κB, PKM2, TnT, and TUNEL in adult rat CMs (n = 3) exposed or not to either ET-1 or ET-1 + lipopolysaccharide (LPS) in the presence or absence of ABT-888, olaparib, and TEPP-46. Quantification of the percentage of adult rat CMs exhibiting nuclear staining of PARP1, PKM2, NF-κB, and TUNEL is presented. (C) Relative messenger RNA levels of Ccl2, IL-1β, IL-6, and IL-8 in adult rat CMs (n = 4) exposed or not to either ET-1 or ET-1 + LPS in the presence or absence of ABT-888, olaparib, and TEPP-46. Scale bars, 50 mm. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. Scatter dot plots show individual values and mean ± SEM. Comparisons were made using 1-way analysis of variance followed by Tukey multiple-comparison tests. Abbreviations as in Figures 1 and 3.

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  Figure 5. Inhibition of PARP1 and Cytosolic Retention of PKM2 Attenuate Cardiac Fibroblast Activation (A) Schematic of the experimental design, representative western blots, and quantification of PARP1, pPKM2, PKM2, connective tissue growth factor (CTGF), fibronectin (FN), matrix metalloproteinase–2 (MMP-2) and alpha smooth muscle actin (αSMA) in adult rat cardiac fibroblasts (CFs; n = 4) exposed or not to transforming growth factor–β1 (TGF-β1) (5 ng/mL) in the presence or absence of ABT-888, olaparib (Olap), or TEPP-46. (B) Schematic of the experimental design, representative western blots, and quantification of PARP1, pPKM2, PKM2, CTGF, FN, MMP-2, and αSMA in adult CFs isolated from wild-type (WT; n = 5) of Parp1-knockout (KO; n = 5) mice treated or not with TGF-β1 (5 ng/mL). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Scatter dot plots show individual values and mean ± SEM. Comparisons were made using 1-way analysis of variance followed by Tukey multiple-comparison tests. Abbreviations as in Figures 1 and 3.

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  Figure 6. Cardioprotective Effects of PARP1 and PKM2 Modulators in Rats Subjected to PAB (A) Schematic representation of the experimental protocol for induction and therapeutic intervention in a PAB-induced RV dysfunction model in rats. (B) Pressure gradient (PG) at PAB site, SV, CO, tricuspid annular plane systolic excursion (TAPSE), S′ wave, RV fractional area change (FAC), and systolic and diastolic eccentricity index (EI) measured using echocardiography in PAB rats before and every week after treatment or not with olaparib or TEPP-46. (C) Representative echocardiographic images of the right and left ventricles obtained from a parasternal short-axis view in PAB rats treated with olaparib, TEPP-46, or Veh at 5 weeks after the initiation of the treatment. (D) RV systolic pressure (RVSP), CO, SV, and RV end-diastolic pressure (RVEDP) measured using right heart catheterization (RHC) at the end of the protocol in PAB rats treated or not with olaparib or TEPP-46. (E) RV hypertrophy by Fulton index and relative messenger RNA (mRNA) levels of Nppa and Nppb expression in PAB rats treated or not with olaparib or TEPP-46. (F) Representative images and corresponding quantifications of CD68, TUNEL,…

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  Figure 7. Parp1 Loss of Function Confers Protection Against PAB-Induced RV Dysfunction (A) Schematic of the experimental design. (B) CO, SV, TAPSE, and S′ wave measured using echocardiography in WT and Parp1-KO mice 7 weeks after PAB or sham surgery. (C) RV hypertrophy by Fulton index and relative mRNA levels of Nppa and Nppb expression in mice. (D) Representative images and quantification of Masson’s trichrome staining and relative mRNA levels of Col1α1 and Col3α1 expression in right ventricles from WT and Parp1-KO mice subjected to PAB or sham surgery. (E) Representative images of hematoxylin and eosin–stained RV sections and quantification of CSA. (F) Representative images and quantification of infiltrated macrophages stained with anti-CD68. Relative mRNA levels of Ccl2 and Socs3 in right ventricles of 7-week sham- or PAB-operated WT and Parp1-KO mice. (G) Representative images of TUNEL-stained RV sections and quantification of the percentage of positive cells. (H) Representative western blots and quantification of PARP1, pPKM2, PKM2, PKM1, phosphorylated signal transducer activator of transcription 3 (pSTAT3), STAT3, cMYC, hexokinase 2 (HK2), lactate dehydrogenase (LDH), and NUDT1 in…

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  Figure 8. CM-Targeted Parp1 Inactivation Improves RV Function in Mice Exposed to Su/Hx (A) Schematic of the experimental design. (B) TAPSE, S′ wave, SV, CO, RV FAC, and RV E/E′ ratio measured using echocardiography in Parp1flox/flox (WT) and Parp1flox/flox;Tg+/Myh6-Cre (conditional KO [cKO]) mice at baseline and after exposure to Sugen/hypoxia (Su/Hx). (C) RVSP, SV, CO, and RVEDP measured using RHC at the end of the protocol in Su/Hx-exposed WT and conditional mutant mice. (D) RV hypertrophy by Fulton index and relative mRNA levels of Nppa and Nppb expression in mice. (E) Representative images of hematoxylin and eosin–stained RV sections and quantification of CM surface area. (F) Representative images and quantification of Masson’s trichrome staining and relative mRNA levels of Col1α1 and Col3α1 expression in right ventricles from Su/Hx-treated WT and cKO mice. (G) Representative images and quantification of infiltrated macrophages stained with anti-CD68. Relative mRNA levels of IL-1β, IL-6, and Ccl2 in right ventricles of mice. (H) Representative images of TUNEL-stained RV sections and quantification of the percentage of positive cells. Scale bars, 50 mm. Arrowheads denote positive cells.…