CYP2J2 metabolites, epoxyeicosatrienoic acids, attenuate Ang II-induced cardiac fibrotic response by targeting Gα12/13

Abstract

The arachidonic acid-cytochrome P450 2J2-epoxyeicosatrienoic acid (AA-CYP2J2-EET) metabolic pathway has been identified to be protective in the cardiovascular system. This study explored the effects of the AA-CYP2J2-EET metabolic pathway on cardiac fibrosis from the perspective of cardiac fibroblasts and underlying mechanisms. In in vivo studies, 8-week-old male CYP2J2 transgenic mice (aMHC-CYP2J2-Tr) and littermates were infused with angiotensin II (Ang II) or saline for 2 weeks. Results showed that CYP2J2 overexpression increased EET production. Meanwhile, impairment of cardiac function and fibrotic response were attenuated by CYP2J2 overexpression. The effects of CYP2J2 were associated with reduced activation of the α subunits of G12 family G proteins (Gα_12/13)/RhoA/Rho kinase (ROCK) cascade and elevation of the NO/cyclic guanosine monophosphate (cGMP) level in cardiac tissue. In in vitro studies, cardiac fibroblast activation, proliferation, migration, and collagen production induced by Ang II were associated with activation of the Gα_12/13/RhoA/ROCK pathway, which was inhibited by exogenous 11,12-EET. Moreover, silencing of Gα_12/13 or RhoA exerted similar effects as 11,12-EET. Furthermore, inhibitory effects of 11,12-EET on Gα_12/13 were blocked by NO/cGMP pathway inhibitors. Our findings indicate that enhancement of the AA-CYP2J2-EET metabolic pathway by CYP2J2 overexpression attenuates Ang II-induced cardiac dysfunction and fibrosis by reducing the fibrotic response of cardiac fibroblasts by targeting the Gα_12/13/RhoA/ROCK pathway via NO/cGMP signaling.

Keywords: angiotensin II; cardiac fibroblast; cardiac fibrosis; cytochrome P450 2J2; α subunits of G12 family G proteins.

Fig. 1.

Expression of CYP2J2 in cardiomyocytes prevents the development of Ang II-induced cardiac fibrosis. A: Evaluation of collagen deposition by picrosirius red staining, histological analysis of cardiac fibroblast transformation and collagen secretion by immunohistochemical staining of α-SMA and COL I, respectively. B: Quantitation of cardiac fibrosis. C: Quantitation of COLI-positive area. D: Quantitation of α-SMA-positive area. E: Representative immunoblots for CYP2J2, COL I, and α-SMA. F: Quantitation of COL I and α-SMA expression in cardiac tissue. G: Representative immunoblots for nuclear PCNA and MRTF-A in cardiac tissue. H: Quantitation of nuclear PCNA and MRTF-A in cardiac tissue. **P < 0.05 versus WT-saline; ^&P < 0.05 versus WT-Ang II (n = 10 for each group).

Fig. 2.

Inhibition of the Gα_12/13/RhoA/ROCK pathway is involved in the anti-fibrotic effects of CYP2J2. A: Representative immunoblots of G12 (TPR), G12 (Total), G13 (TPR), and G13 (Total) in cardiac tissue. B: Quantitation of Gα₁₂ activity and Gα₁₃ activity by determining the ratio of G12 (TPR)/G12 (Total) and G13 (TPR)/G13 (Total), respectively. C: Representative immunoblot of RhoA (RBD) and RhoA in cardiac tissue. D: Quantitation of RhoA activity by the ratio between RhoA (RBD) and RhoA (Total). E: Representative immunoblot of p-MYPT and MYPT in cardiac tissue. F: ROCK activity was evaluated by the ratio between p-MYPT and MYPT. **P < 0.05 versus WT-saline; ^&P < 0.05 versus WT-Ang II (n = 10 for each group).

Fig. 3.

Treatment of neonatal rat cardiac fibroblasts with 11,12-EET inhibits Ang II-induced fibrotic response in cardiac fibroblasts, including cardiac fibroblast transformation, proliferation, migration, and collagen secretion. A: Representative immunofluorescent images of α-SMA-positive cells and PCNA-positive cells in cultured neonatal rat cardiac fibroblasts treated with various reagents for 24 h. B: Quantitation of α-SMA-positive cells in (A). C: Quantitation of PCNA-positive cells in (A). D: Representative images of migrated fibroblasts treated with various reagents for 6 h. E: Quantitation of migrated fibroblasts in (D). F: Representative immunoblots for COL I and α-SMA in cultured neonatal rat cardiac fibroblasts treated with various reagents for 24 h. G: Quantitation of COL I and α-SMA in cardiac fibroblasts. H: Representative immunoblots for nuclear PCNA and MRTF-A in cultured neonatal rat cardiac fibroblasts treated with various reagents for 24 h. I: Quantitation of nuclear PCNA and MRTF-A in cardiac fibroblasts. **P < 0.05 versus DMSO; ^&P < 0.05 versus Ang II; ^#P < 0.05 versus Ang II + 11,12-EET (n = 5 for each experiment).

Fig. 4.

Inhibition of the Gα_12/13/RhoA/ROCK pathway is involved in the anti-fibrotic effects of EETs. A: Representative immunoblots of G12 (TPR), G12 (Total), G13 (TPR), and G13 (Total) in cardiac fibroblasts treated with various reagents for 5 min. B: Quantitation of Gα₁₂ activity and Gα₁₃ activity by determining the ratio of G12 (TPR)/G12 (Total) and G13 (TPR)/G13 (Total), respectively. C: Representative immunoblot of RhoA (RBD) and RhoA (Total) and quantitation of RhoA activity by the ratio between RhoA (RBD) and RhoA (Total) in cardiac fibroblasts treated with various reagents for 5 min. D: Representative immunoblot of p-MYPT and MYPT and quantitation of ROCK activity by the ratio between p-MYPT and MYPT in cardiac fibroblasts treated with various reagents for 5 min. **P < 0.05 versus DMSO; ^&P < 0.05 versus Ang II; ^#P < 0.05 versus Ang II + 11,12-EET (n = 5 for each experiment).

Fig. 5.

siRNA mediated knockdown of Gα_12/13 or RhoA inhibits Ang II-induced fibrotic response in cardiac fibroblasts, including cardiac fibroblast transformation, proliferation, migration, and collagen secretion. A: Representative immunofluorescent images of α-SMA-positive cells and PCNA-positive cells in cultured neonatal rat cardiac fibroblasts treated with and without Ang II or various siRNAs for 24 h. B: Representative image of migrated fibroblasts treated with and without Ang II or various siRNAs for 6 h. C: Quantitation of α-SMA-positive cells. D: Quantitation of PCNA-positive cells. E: Quantitation of migrated fibroblasts. F: Representative immunoblots for Gα₁₂, Gα₁₃, RhoA, COL I, and α-SMA in cardiac fibroblasts treated with and without Ang II or various siRNA for 24 h. G: Quantitation of COL I and α-SMA in cardiac fibroblasts. H: Representative immunoblots for nuclear PCNA and MRTF-A in cardiac fibroblasts treated with and without Ang II or various siRNAs for 24 h. I: Quantitation of nuclear PCNA and MRTF-A in cardiac fibroblasts. J: Representative immunoblot of p-MYPT and MYPT and quantitation of ROCK activity by the ratio between p-MYPT and MYPT in cardiac fibroblasts treated with and without Ang II or various siRNAs for 5 min. **P < 0.05 versus siControl; ^&P < 0.05 versus Ang II + siControl (n = 5 for each experiment).

Fig. 6.

Cardiomyocyte specific expression of CYP2J2 or treatment with 11,12-EET attenuate Ang II-induced Gα_12/13 activity via NO/cGMP activation. A: Quantitation of NO and cGMP level in cardiac tissue. NO concentration (picomoles per milligram protein): Saline-WT, 2.725 ± 0.50; Saline-Ang II, 5.025 ± 0.75; Ang II-WT, 2.225 ± 0.50; Ang II-CYP2J2, 4.400 ± 0.25. cGMP concentration (picomoles per milligram protein): Saline-WT, 0.1650 ± 0.045; Saline-CYP2J2, 0.2835 ± 0.030; Ang II-WT, 0.1350 ± 0.015; Ang II-CYP2J2, 0.2340 ± 0.030. *P < 0.05 versus WT-saline (n = 5 for each group). B: Quantitation of NO and cGMP level in cardiac fibroblasts treated with various reagents for 5 min. NO concentration (picomoles per milligram protein): DMSO, 1.68 ± 0.210; EET, 3.78 ± 0.420; Ang II, 2.10 ± 0.315; Ang II + EET, 3.57 ± 0.420; Ang II + EET + EEZE, 2.31 ± 0.294. cGMP concentration (picomoles per milligram protein): DMSO, 0.117 ± 0.0208; EET, 0.246 ± 0.0299; Ang II, 0.143 ± 0.0260; Ang II + EET, 0.234 ± 0.0130; Ang II + EET + EEZE, 0.156 ± 0.0247. *P < 0.05 versus DMSO; ^#P < 0.05 versus Ang-II + 11,12-EET (n = 5 for each experiment). C: Representative immunoblots of G12 (TPR), G12 (Total), G13 (TPR), and G13 (Total) in cardiac fibroblasts treated with various reagents for 5 min. D: Quantitation of Gα₁₂ activity and Gα₁₃ activity by determining the ratio of G12 (TPR)/G12 (Total) and G13 (TPR)/G13 (Total), respectively. **P < 0.05 versus DMSO; ^#P < 0.05 versus Ang II; ^&P < 0.05 versus Ang II + 11,12-EET (n = 5 for each experiment).

Fig. 7.

Schematic of mechanisms on EET-mediated signaling in response to cardiac hypertrophy and fibrosis. EETs inhibit cardiac hypertrophy by acting directly on cardiomyocytes through the PPAR-γ/oxidative stress/SERCA2a/ER stress pathway and interaction of Akt1 and AMPKα2β2γ1 (8, 13, 14). Moreover, EETs attenuate pro-fibrotic and pro-inflammatory responses of cardiomyocytes transmitted to cardiac fibroblasts and macrophages, respectively (13, 15). This study indicates that EETs inhibit cardiac fibrosis by inhibiting fibrotic response of cardiac fibroblasts, including inhibition of cardiac fibroblast activation, proliferation, migration, and collagen secretion. Mechanistically, EETs stimulate the NO/cGMP signaling pathway, which reduces Gα_12/13 and subsequently inhibits RhoA/ROCK activation (Dashed lines represent previous findings and solid lines represent present findings).