Resveratrol attenuates angiotensin II-induced cellular hypertrophy through the inhibition of CYP1B1 and the cardiotoxic mid-chain HETE metabolites

Abstract

Several reports demonstrated the direct contribution of cytochrome P450 1B1 (CYP1B1) enzyme and its associated cardiotoxic mid-chain, hydroxyeicosatetraenoic acid (HETEs) metabolites in the development of cardiac hypertrophy. Resveratrol is commercially available polyphenol that exerts beneficial effects in wide array of cardiovascular diseases including cardiac hypertrophy, myocardial infarction and heart failure. Nevertheless, the underlying mechanisms responsible for these effects are not fully elucidated. Since resveratrol is a well-known CYP1B1 inhibitor, the purpose of this study is to test whether resveratrol attenuates angiotensin II (Ang II)-induced cellular hypertrophy through inhibition of CYP1B1/mid-chain HETEs mechanism. RL-14 and H9c2 cells were treated with vehicle or 10 μM Ang II in the absence and presence of 2, 10 or 50 μM resveratrol for 24 h. Thereafter, the level of mid-chain HETEs was determined using liquid chromatography-mass spectrometry (LC/MS). Hypertrophic markers and CYP1B1 gene expression and protein levels were measured using real-time PCR and Western blot analysis, respectively. Our results demonstrated that resveratrol, at concentrations of 10 and 50 μM, was able to attenuate Ang-II-induced cellular hypertrophy as evidenced by substantial inhibition of hypertrophic markers, β-myosin heavy chain (MHC)/α-MHC and atrial natriuretic peptide. Ang II significantly induced the protein expression of CYP1B1 and increased the metabolite formation rate of its associated mid-chain HETEs. Interestingly, the protective effect of resveratrol was associated with a significant decrease of CYP1B1 protein expression and mid-chain HETEs. Our results provided the first evidence that resveratrol protects against Ang II-induced cellular hypertrophy, at least in part, through CYP1B1/mid-chain HETEs-dependent mechanism.

Keywords: CYP1B1; Cardiac hypertrophy; Mid-chain HETEs; Resveratrol.

Fig. 1

Effect of resveratrol on cell viability in RL-14 and H9c2 cells. RL-14 (a) and H9c2 (b) cells were treated for 24 h with 1–100 μM of resveratrol. Cell cytotoxicity was assessed using the MTT assay. Data are presented as the percentage of control (set at 100%) ± SEM (n = 4). Data were analyzed using one-way ANOVA followed by Student–Newman–Keuls as post hoc test

Fig. 2

Effect of resveratrol on Ang II-mediated induction of hypertrophic markers in RL-14 and H9c2 cells. RL-14 (a) and H9c2 (b) cells were treated for 24 h with vehicle, 10 μM Ang II, 10 μM Ang II in combination with (2, 10 or 50 μM resveratrol) or (2, 10 or 50 μM resveratrol alone) in SFM. Total RNA was isolated using TRIzol, the mRNA expression levels of β-MHC/α-MHC and ANP were assessed using real-time PCR and their levels were normalized to β-actin housekeeping gene. The results are presented as the mean and SEM based on at least 3 individual experiments. Data were analyzed using one-way ANOVA followed by Student–Newman–Keuls as post hoc test. ⁺p < 0.05 significantly different from control group. *p < 0.05 significantly different from Ang II-treated group

Fig. 3

Effect of resveratrol on mRNA expression and protein expression levels of CYP1B1 in RL-14 cells. RL-14 cells were treated for 24 h with vehicle, 10 μM Ang II, 10 μM Ang II in combination with (2, 10 or 50 μM resveratrol) or (2, 10 or 50 μM resveratrol alone) in SFM. CYP1B1 mRNA expression (a) and protein expression levels (b) were determined using real-time PCR and Western blot analysis, respectively. For real-time PCR, total RNA was isolated using TRIzol reagent, the mRNA level was quantified and its level was normalized to β-actin housekeeping gene. For Western blot analysis, protein levels were detected using the enhanced chemiluminescence method. The intensity of protein band was normalized to the signals obtained for β-actin or GAPDH protein and quantified using ImageJ®. The results are presented as the mean and SEM based on at least 3 individual experiments. Data were analyzed using one-way ANOVA followed by Student–Newman–Keuls as post hoc test. ⁺p < 0.05 significantly different from control group. *p < 0.05 significantly different from Ang II-treated group

Fig. 4

Effect of resveratrol on mRNA expression and protein expression levels of CYP1B1 in H9c2 cells. H9c2 cells were treated for 24 h with vehicle, 10 μM Ang II, 10 μM Ang II in combination with (2, 10 or 50 μM resveratrol) or (2, 10 or 50 μM resveratrol alone) in SFM. CYP1B1 mRNA expression (a) and protein expression levels (b) were determined using real-time PCR and Western blot analysis, respectively. For real-time PCR, total RNA was isolated using TRIzol reagent, the mRNA level was quantified and its level was normalized to β-actin housekeeping gene. For Western blot analysis, protein levels were detected using the enhanced chemiluminescence method. The intensity of protein band was normalized to the signals obtained for β-actin or GAPDH protein and quantified using ImageJ®. The results are presented as the mean and SEM based on at least 3 individual experiments. Data were analyzed using one-way ANOVA followed by Student–Newman–Keuls as post hoc test. ⁺p < 0.05 significantly different from control group. *p < 0.05 significantly different from Ang II-treated group

Fig. 5

Effect of resveratrol on mid-chain HETE metabolite formation rate in RL-14 cells. RL-14 cells were treated for 24 h with vehicle, 10 μM Ang II and 10 μM Ang II in combination with (2, 10 or 50 μM resveratrol). 5-, 8-, 9-HETE (a) and 11-, 12-, 15-HETE (b) metabolites were measured using LC–ESI–MS. The results are presented as the mean and SEM (n = 3). Data were analyzed using one-way ANOVA followed by Student–Newman–Keuls as post hoc test. ⁺p < 0.05 significantly different from control group. *p < 0.05 significantly different from Ang II-treated group

Fig. 6

Effect of resveratrol on mid-chain HETE metabolite formation rate in H9c2 cells. H9c2 cells were treated for 24 h with vehicle, 10 μM Ang II and 10 μM Ang II in combination with (2, 10 or 50 μM resveratrol). 5-, 8-, 9-HETE (a) and 11-, 12-, 15-HETE (b) metabolites were measured using LC–ESI–MS. The results are presented as the mean and SEM (n = 3). Data were analyzed using one-way ANOVA followed by Student–Newman–Keuls as post hoc test. ⁺p < 0.05 significantly different from control group. *p < 0.05 significantly different from Ang II-treated group