A Critical Contribution of Cardiac Myofibroblasts in Right Ventricular Failure and the Role of UCP2 SNPs in the Predisposition to RV Decompensation in Pulmonary Arterial Hypertension

Figures

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  Figure 1.. Right ventricular performance is reduced in decompensated RV in vivo and ex vivo, whereas CM contractility remains unchanged across control, cRV, and dRV. A, Representative echocardiography pictures (short axis; left) show the size of RV chamber (red dotted line) in the 3 stages (normal RV, cRV, and dRV) of monocrotaline (MCT) rats. Parameters measured by echocardiography (cardiac output, RV free wall thickness, RVEDD, and TAPSE) are shown on the right, expressed as mean±SEM; n=10 animals for each group. ****P<0.0001. B, Representative tracings of RV systolic pressure (RVSP) and pulmonary artery pressures (PAPs) in CTRL, cRV, and dRV of MCT rats (left) performed by close-chest right heart catheterization. Parameters measured (mPAP, RVSP, and RAP) are shown on the right and expressed as mean±SEM; n=6 animals for each group. *P<0.05; **P<0.01. C, Representative tracings of dp/dt of RV pressures in CTRL, cRV, and dRV of MCT rats (left), with values (right) expressed as mean±SEM; n=6 animals for each group. *P<0.05; **P<0.01. D, Representative tracings (left) of RV pressures in ex vivo isolated hearts from CTRL, cRV, and dRV of MCT rats, with values (middle) expressed as mean±SEM;…

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  Figure 2.. Decompensated RVs have more myofibroblasts, which exhibit a metabolic cross-talk with CMs via lactate. A, Confocal microscopy (left) shows the number of myofibroblasts (positive with both α-SMA and vimentin) in right ventricle frozen sections from control, cRV, and dRV of MCT rats. White boxes in the low-magnification images mark the areas enlarged in the high-magnification panels below for each group. Quantification values (right) expressed as mean±SEM; n=9 rats per each group. ***P<0.001; ****P<0.0001. B, Confocal microscopy (left) shows the ratio of Ki67-positive cells (nuclear Ki67) in (myo)fibroblasts from normal RV and dRV of MCT rats. We studied 3 rats for each group (shown as 1, 2, and 3 on x axis), and for each rat, we measured 3 fields. The percent positive ratio for each field is shown in circles along with the median of all rats (right). n=9 fields (≈200 (myo)fibroblasts from 3 rats for each group). We initially used the The Mann-Whitney test to show a significant difference between the 2 groups (P=0.0002). To address potential random effects from variability within different fields from each rat, or among different rats within each group, we also performed a linear…

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  Figure 3.. Mitochondrial calcium levels are decreased and MICU1 methylation is increased in dRV myofibroblasts compared with normal RV and cRV fibroblasts. A, Confocal microscopy (left) shows mitochondrial calcium levels (Rhod-2 fluorescence) in cultured (myo)fibroblasts from normal RV, cRV and dRV of MCT rats. We studied 3 rats for each group (shown as 1, 2, and 3 on x axis) and for each rat, we measured 15–20 cells. The Rhod-2 fluorescence level in each cell is shown in circles along with mean±SEM (right). n=50 cells from 3 animals for each group. We initially used the Kruskal-Wallis test to show significant differences among groups (P<0.0001). To address potential random effects from variability from cells in each rat, or among different rats in each group, we also performed a linear mixed-effects model, which showed that the only difference was between groups (fixed effects): (F(1.413, 93.27)=35.95; P<0.0001) and not among different animals within the same group (F(2, 132)=0.3773; P=0.6865). Post hoc comparisons between groups were performed using Tukey’s multiple-comparisons test. *P<0.05; **** P<0.0001. B, PDH activity was measured using an nicotinamide adenine dinucleotide (reduced…

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  Figure 4.. UCP2 levels decrease from control to cRV to dRV (myo)fibroblasts, whereas TNF-α reduces UCP2 expression and mitochondrial calcium levels, promoting myofibroblast activation. A, Immunoblot shows the UCP2 protein levels in the myocardium of the left ventricles and right ventricles from control, cRV, and dRV MCT rats (red arrows point the right ventricles). UCP2 is the lower band with a molecular weight of 33kd (verified by Ucp2 WT and KO immunoblot). B, Immunoblot (left) shows the UCP2 protein levels in right ventricular (myo)fibroblasts from normal RV, cRV, and dRV of MCT rats. Quantification values (right) normalized by β-actin are expressed as mean±SEM; n=5 animals for each group. *P<0.05; **P<0.01. C, Confocal microscopy (left) shows the mitochondrial calcium levels (Rhod-2 fluorescence) in cultured right ventricular (myo)fibroblasts from WT, Ucp2 heterozygous, and Ucp2 knockout mice. We studied 3 mice from each group (shown as 1, 2, and 3 on x axis), and for each mouse, we measured 15–20 cells. The Rhod-2 fluorescence level of each cell is shown in circles along with mean±SEM (right). n=50 cells from 3 animals for each group. We initially used the Kruskal-Wallis test to show…

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  Figure 5.. Myofibroblasts are activated in the human decompensated RV of PAH patients, whereas arginine methylation in (myo)fibroblasts increases in the compensated RV but does not further increase in the decompensated RV compared with controls (normal RV). A, Confocal microscopy (left) shows the number of myofibroblasts (positive with both α-SMA and vimentin) from paraffin-embedded right ventricle sections from CTRL (n=9), cRV (n=10), and dRV (n=11) cohort 1 patients with PAH. White boxes in the low-magnification images mark the areas enlarged in the high-magnification panels below for each group. Quantification values (right) are expressed as mean±SEM. Scatter dot plots represent individual values. ****P<0.0001. B, Spearman correlation test shows the correlation between number of myofibroblasts and TAPSE in cohort 1 patients with PAH. Scatter dot plots represent individual values. *P<0.05. C, Spearman correlation test shows the correlation between number of myofibroblasts and cardiac index in cohort 1 patients with PAH. Scatter dot plots represent individual values. **P<0.01. D, Confocal microscopy (left) shows the cytoplasmic PRMT1 levels in (myo)fibroblasts (vimentin-positive cells)…

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  Figure 6.. UCP2 levels correlate negatively with the number of cardiac myofibroblasts and positively with TAPSE and cardiac index in patients with PAH. A, Confocal microscopy (left) shows the UCP2 levels in (myo)fibroblasts (vimentin-positive cells) from paraffin-embedded right ventricle sections from CTRL (n=9), cRV (n=10), and dRV (n=11) cohort 1 patients with PAH. White boxes in the low-magnification images mark the areas enlarged in the high-magnification panels below for each group. Quantification values (right) are expressed as mean±SEM. Scatter dot plots represent individual values. *P<0.05. This comparison was made using Kruskal-Wallis tests followed by pairwise Mann-Whitney U tests. B, Spearman correlation test shows the correlation between UCP2 immunofluorescence intensity in (myo)fibroblasts and number of myofibroblasts/field in cohort 1 patients with PAH. Scatter dot plots represent individual values. *P<0.05. C, Spearman correlation test shows the correlation between UCP2 immunofluorescence intensity in (myo)fibroblasts and TAPSE cohort 1 patients with PAH. Scatter dot plots represent individual values. *P<0.05. D, Spearman correlation test shows the correlation between UCP2…

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  Figure 7.. UCP2 SNP, reflecting UCP2 mRNA and protein levels, correlates with TAPSE and cardiac index in patients with PAH but not in those with group 2 PHT (PHT-2). A, qPCR shows the Ucp2 mRNA levels in human RV (from cohort 1and 3) genotyped with WT, UCP2 HET, and HOM SNP. Quantification values are expressed as mean±SEM. Scatter dot plots represent individual values. **P<0.01; ***P<0.001. B, Western blot (left) shows the UCP2 protein levels in human RV genotyped with WT, UCP2 HET, and HOM SNP. Quantification values (right) are expressed as mean±SEM. Scatter dot plots represent individual values. n=11 for WT, n=10 for UCP2 HET SNP, and n=4 for UCP2 HOM SNP. *P<0.05; **P<0.01. C, UCP2 genotyping and echocardiography (left) show the UCP2 genotype and TAPSE in patients with PAH (mPAP >20 mm Hg from cohort 1). Patients were genotyped for UCP2 and categorized into WT (n=6), UCP2 HET SNP (n=5), and UCP2 HOM SNP (n=4). Quantification values (right) are expressed as mean±SEM. Scatter dot plots represent individual values. *P<0.05; **P<0.01. D, UCP2 genotyping and echocardiography (left) show the UCP2 genotype and cardiac index (CI) in patients with PAH (mPAP>20mmHg from cohort 1). Patients were…

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  Figure 8.. Proposed mechanism of RV cardiac myofibroblast activation and its contribution to right ventricular decompensation in PAH. In PAH, the RV undergoes afterload induced remodeling in an inflammatory environment, leading to mitochondrial UCP2 downregulation in cardiac fibroblasts (cFBs) leading to their differentiation into myofibroblasts (cMFBs). UCP2 is essential to regulate mitochondrial calcium when the MICU1 is methylated. In the RV cFBs from PAH subjects, increased methylation of MICU1 (mediated by PRMT1) and suppression of UCP2 expression by tumor necrosis factor alpha (TNF-α), which is further exacerbated by UCP2 loss-of-function SNPs, reduce mitochondrial calcium uptake. This reduction inhibits pyruvate dehydrogenase (PDH) activity and impairs glucose oxidation, limiting the production of diffusible metabolites (eg, citrates) required for nuclear epigenetic signaling (eg, histone acetylation). These metabolic disruptions (marked by red crosses and arrows) facilitate the transition from cFBs to cMFBs, thereby facilitating the transition from compensated RV (cRV) to decompensated RV (dRV) and RV failure (bottom). Notably, MICU1 methylation increases primarily during the…