Autophagy mechanism of right ventricular remodeling in murine model of pulmonary artery constriction

Abstract

Although right ventricular failure (RVF) is the hallmark of pulmonary arterial hypertension (PAH), the mechanism of RVF is unclear. Development of PAH-induced RVF is associated with an increased reactive oxygen species (ROS) production. Increases in oxidative stress lead to generation of nitro-tyrosine residues in tissue inhibitor of metalloproteinase (TIMPs) and liberate active matrix metalloproteinase (MMPs). To test the hypothesis that an imbalance in MMP-to-TIMP ratio leads to interstitial fibrosis and RVF and whether the treatment with folic acid (FA) alleviates ROS generation, maintains MMP/TIMP balance, and regresses interstitial fibrosis, we used a mouse model of pulmonary artery constriction (PAC). After surgery mice were given FA in their drinking water (0.03 g/l) for 4 wk. Production of ROS in the right ventricle (RV) was measured using oxidative fluorescent dye. The level of MMP-2, -9, and -13 and TIMP-4, autophagy marker (p62), mitophagy marker (LC3A/B), collagen interstitial fibrosis, and ROS in the RV wall was measured. RV function was measured by Millar catheter. Treatment with FA decreased the pressure to 35 mmHg from 50 mmHg in PAC mice. Similarly, RV volume in PAC mice was increased compared with the Sham group. A robust increase of ROS was observed in RV of PAC mice, which was decreased by treatment with FA. The protein level of MMP-2, -9, and -13 was increased in RV of PAC mice in comparison with that in the sham-operated mice, whereas supplementation with FA abolished this effect and mitigated MMPs levels. The protein level of TIMP-4 was decreased in RV of PAC mice compared with the Sham group. Treatment with FA helped PAC mice to improve the level of TIMP-4. To further support the claim of mitophagy occurrence during RVF, the levels of LC3A/B and p62 were measured by Western blot and immunohistochemistry. LC3A/B was increased in RV of PAC mice. Similarly, increased p62 protein level was observed in RV of PAC mice. Treatment with FA abolished this effect in PAC mice. These results suggest that FA treatment improves MMP/TIMP balance and ameliorates mitochondrial dysfunction that results in protection of RV failure during pulmonary hypertension.

Fig. 1.

Pulmonary artery constriction (PAC)-induced collagen deposition in the right ventricle (RV). A: heart sections from untreated sham-operated mice (Sham), mice with 4 wk of PAC 4 wk (PAC), and sham-operated mice with Pac treated with folic acid (FA; Sham + FA and PAC + FA, respectively). B: results of quantitative analyses of collagen (blue staining) in the samples from above mentioned mice groups. Data are presented as means ± SE; n = 6 for all groups. *P < 0.05 vs. Sham, Sham + FA; #P < 0.05 vs. PAC.

Fig. 2.

PAC-induced myocyte contractility changes. A: examples of myocytes isolated from Sham, Sham + FA, PAC, and PAC + FA mice. B: examples of cell shortening traces in myocytes from the above mentioned groups. C: changes in percent peak shortening presented as changes in baseline percent peak (bl % peak) and rates of contraction (+dL/dt) and relaxation (-dL/dt) of cardiomyocytes. The values are the means of measurements of at least 5 myocytes from each animal in each experimental group. The mean value of contractility was calculated from at least 5 contractions of each cardiomyocyte analyzed. *P < 0.05 vs. Sham, Sham + FA; #P < 0.05 vs. PAC; n = 5 for all groups.

Fig. 3.

PAC-induced superoxide production in mice RV. Superoxide production was detected in situ by staining heart tissue with the superoxide sensitive dye DHE (red fluorescence). A: examples of RV images in samples from wild-type (WT), WT + FA, PAC, and PAC + FA. B: summary of fluorescence intensity changes in RVs from the above mentioned groups. Quantification of fluorescence was done with the help proplus software. *P < 0.05 vs. Sham, Sham + FA; #P < 0.05 vs. PAC; n = 8 for all groups.

Fig. 4.

Effect of FA on matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinases (TIMP) ration mice. A: examples of Western blot analyses for MMP-2, -9, and -13 protein levels in RV of WT, WT + FA, PAC, and PAC + FA mice. B: example of Western blot analyses for TIMP-4 protein level in RV of above mentioned mice groups; β-actin was used as a loading control. C and D: relative protein expression is reported as a ratio of integrated optical density (IOD) of each band to the IOD of the respective β-actin band. *P < 0.05 vs. Sham, Sham + FA; #P < 0.05 vs. PAC; n = 5 for all groups.

Fig. 5.

Effect of FA on p62 in mice. A: RV immunohistochemical staining and colocalization of p62. Cryocut frozen sections of (8–10 μm) were stained and secondarily conjugated with FITC. B: bar diagrams depicted the intensity quantification of p62 in Sham, Sham + FA, PAC, and PAC + FA mice. C: Western blot analysis for p62 protein levels in RV of the above mentioned mice groups. β-Actin was used as a loading control. D: relative protein expression is reported as a ratio of IOD of each band to the IOD of the respective β-actin band. *P < 0.05 vs. Sham, Sham + FA; #P < 0.05 vs. PAC; n = 5 for all groups.

Fig. 6.

Effect of FA on LC3A/B in mice. A: RV immunohistochemical staining and colocalization of LC3A/B. Cryocut frozen sections of (8–10 μm) were stained and secondarily conjugated with FITC. B: bar diagrams depicted the intensity quantification of LC3A/B in Sham, Sham + FA, PAC, and PAC + FA mice. C: Western blot analysis for LC3A/B protein levels in RV of the above mentioned mice groups. β-Actin was used as a loading control. D: relative protein expression is reported as a ratio of IOD of each band to the IOD of the respective β-actin band. *P < 0.05 vs. Sham, Sham + FA; #P < 0.05 vs. PAC; n = 5 for all groups.

Fig. 7.

Schematic presentation of possible mechanism involved in FA induced cardiac function during PAC. A: PAC caused reactive oxygen species (ROS) production in RV, leading to mitophagy and MMP/TIMP imbalance. That caused RV failure (RVF). B: treatment with FA, which decreased ROS production, ameliorates mitophagy and MMP/TIMP imbalance and protects the heart from RVF.