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Activation of the endocannabinoid system mediates cardiac hypertrophy induced by rosiglitazone - PubMed

Activation of the endocannabinoid system mediates cardiac hypertrophy induced by rosiglitazone

Ya-Han Liu et al. Acta Pharmacol Sin. 2022 Sep.

Abstract

Rosiglitazone (RSG) is a synthetic agonist of peroxisome proliferator-activated receptor-γ (PPARγ), which plays a central role in the regulation of metabolism. Meta-analyses have suggested that RSG is associated with increased cardiovascular risk. However, the mechanisms underlying such adverse cardiac effects are still poorly understood. Here, we found that activation of PPARγ by RSG stimulated the endocannabinoid system (ECS), a membrane lipid signaling system, which induced cardiac hypertrophy. In neonatal rat cardiomyocytes, RSG increased the level of anandamide (AEA); upregulated the expression of N-acyl phosphatidylethanolamine phospholipase D (NapePLD), a key enzyme for AEA synthesis; and downregulated the expression of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of AEA. Importantly, PPARγ activation increased the expression of cannabinoid receptor type 1 (CB1) through an identified binding site for PPARγ in the CB1 promoter region. Moreover, both the in vitro and in vivo results showed that inhibition of the ECS by rimonabant, an antagonist of CB1, attenuated RSG-induced cardiac hypertrophy, as indicated by decreased expression of cardiac hypertrophy markers (ANP and BNP), deactivation of the mTOR pathway, and decreased cardiomyocyte size. Thus, these results demonstrated that the ECS functions as a novel target of PPARγ and that the AEA/CB1/mTOR axis mediates RSG-induced cardiac remodeling.

Keywords: cardiac hypertrophy; endocannabinoids; mammalian target of rapamycin; peroxisome proliferator-activated receptor-γ.

© 2022. The Author(s), under exclusive licence to Shanghai Institute of Materia Medica, Chinese Academy of Sciences and Chinese Pharmacological Society.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. TZDs regulated the levels of eCBs in cardiomyocytes.

The levels of eCBs in per 106 NCMCs after RSG (20 μmol · L−1) and PIO (20 μmol · L−1) treatment for 24 h. a, b AEA and 2-AG were assayed by LC–MS with a standard curve, and c, d the relative intensity of the ions (arbitrary units) of OEA and PEA were acquired by comparing peak area. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. Ctrl from one-way ANOVA followed by Sidak’s post hoc tests. n = 4.

Fig. 2
Fig. 2. RSG activated ECS in cardiomyocytes.

NCMCs were treated with RSG at 1 or 20 μmol · L−1. mRNA was harvested after indicated time periods. a–c The relative mRNA expression levels of NapePLD, FAAH, and CB1 were measured. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. Ctrl from two-way ANOVA followed by Sidak’s post hoc tests. n = 3–4. RSG1, RSG 1 μmol · L−1; RSG20, RSG 20 μmol · L−1. d The protein level of CB1 was measured in NCMCs with RSG treatment for 48 h. Data are mean ± SEM. *P < 0.05 vs. Ctrl from one-way ANOVA followed by Sidak’s post hoc tests. n = 4. e NCMCs were treated with PIO (20 μmol · L−1) for 24 h, the relative mRNA expression of NapePLD, FAAH, CB1, and FABP4 was measured by qPCR. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. Ctrl from t-test. n = 3–6. For in vivo study, rats were treated with RSG (15 mg · kg−1 · d−1) by gavage for 2 weeks. f The mRNA expression of genes related to ECS and g the protein level of CB1 were measured in cardiac tissue from the left ventricle. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. Ctrl from t-test. n = 3–4.

Fig. 3
Fig. 3. RSG activated ECS via PPARγ activation.

NCMCs were pretreated with GW9662 (40 μmol · L−1) for 2 h and subjected to RSG (20 μmol · L−1) for 24 or 48 h. a After treated with RSG for 24 h, the mRNA relative expression levels of CB1, NapePLD, FAAH, and FABP4 were measured and normalized to Ctrl. b Protein levels of CB1 were analyzed after 48 h of treatment. NCMCs lysates were immunoblotted with the antibody of CB1 and shown as relative fold changes in the bar graph. c After treated with RSG for 24 h, the level of AEA was measured by LC–MS and normalized in per 106 cardiomyocytes. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. Ctrl, #P < 0.05, ##P < 0.01 vs. RSG from one-way ANOVA followed by Sidak’s post hoc tests. n = 3–8. d NCMCs were co-infected with Ad-CA-PPARγ and Ad-tTA with or without tetracycline (Tc, 1 µg · mL−1) for 36 h, and the mRNA expression level of related genes was measured. Data are mean ± SEM. *P < 0.05 vs. Mock from t-test. n = 3. e NCMCs were treated with RSG (20 μmol · L−1) for 24 h, then cross-linked and immunoprecipitated with the antibody of PPARγ or IgG. The immunoprecipitates or supernatants before immunoprecipitation (Input) were PCR amplified using the primers flanking the motifs in the rat CB1 gene.

Fig. 4
Fig. 4. eCBs exerted hypertrophic effects on NCMCs via CB1.

a NCMCs were treated with AEA (10 μmol · L−1) or OEA (50 μmol · L−1) for 24 h. The relative mRNA expression levels of ANP and BNP were measured and normalized to Ctrl. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. Ctrl from one-way ANOVA followed by Sidak’s post hoc tests. n = 4. b The F-actin of NCMCs was stained with rhodamine-conjugated phalloidin. The cardiomyocyte area was digitized and the areas (mm2) of 100 cells from each treatment were determined using Image J. Data are mean ± SEM. *P < 0.05 vs. Ctrl from t-test. n = 6. c NCMCs were pretreated with Rimo (3 μmol · L−1) for 2 h and then subjected to AEA (10 μmol · L−1) for 24 h. The relative mRNA expression levels of ANP and BNP were determined by qPCR. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. Ctrl, #P < 0.05, ##P < 0.01 vs. AEA from one-way ANOVA followed by Sidak’s post hoc tests. n = 3–7. d After 48 h treatment as indicated, NCMCs were collected for determining protein level by Western blotting. Data are mean ± SEM. **P < 0.01 vs. Ctrl, ##P < 0.01 vs. AEA from one-way ANOVA followed by Sidak’s post hoc tests. n = 4.

Fig. 5
Fig. 5. CB1 antagonist attenuated RSG-induced cardiac hypertrophy in NCMCs.

a, b NCMCs were treated with the indicated dose of RSG for 24 or 48 h. The relative mRNA expression levels of ANP and BNP were measured and normalized to Ctrl. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. RSG (0 μmol · L−1) from one-way ANOVA followed by Sidak’s post hoc tests. n = 3–4. c The lysates from NCMCs treated with RSG for 48 h were immunoblotted with the indicated antibody for mTOR pathway. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. Ctrl from one-way ANOVA followed by Sidak’s post hoc tests. n = 4. RSG1, RSG 1 μmol · L−1; RSG20, RSG 20 μmol · L−1. d NCMCs were pretreated with Rimo (3 μmol · L−1) or AM251 (5 μmo · L−1) and then subjected to RSG (20 μmol · L−1) for 24 h. The relative mRNA expression levels of ANP and BNP were measured and normalized to Ctrl. Data are mean ± SEM. **P < 0.01 vs. Ctrl, ##P < 0.01 vs. RSG from one-way ANOVA followed by Sidak’s post hoc tests. n = 4. e NCMCs were pretreated with Rimo (3 μmol · L−1) for 2 h and then subjected to RSG (20 μmol · L−1) for 48 h. The cardiomyocyte size was measured using rhodamine-phalloidin staining. A total of 100 cells were counted for statistical analysis. Scale bar = 50 µm. Data are mean ± SEM. **P < 0.01 vs. Ctrl, ##P < 0.01 vs. RSG from one-way ANOVA followed by Sidak’s post hoc tests. n = 3. f After 48 h treatment as indicated, the cell lysate was immunoblotted with the indicated antibody for the mTOR pathway. Data are mean ± SEM. **P < 0.01 vs. Ctrl, #P < 0.05, ##P < 0.01 vs. RSG from one-way ANOVA followed by Sidak’s post hoc tests. n = 3.

Fig. 6
Fig. 6. CB1 antagonist attenuated RSG-induced cardiac hypertrophy in rats.

SD rats were treated with RSG (15 mg · kg−1 ·  d−1) alone or combined with Rimo (10 mg · kg−1 · d−1) by gavage for 4 weeks. a Representative photograph of hearts from each group. b, c The hearts and left ventricles were weighed and normalized to body weight. d, e Histological analysis by HE staining in the cross-section of cardiac tissue. Scale bar = 50 µm. The relative cross-sectional areas of cardiomyocytes were calculated by Image J. f The relative mRNA expression levels of ANP and BNP in left ventricles were measured and normalized to Ctrl. g The lysate from the left ventricular was immunoblotted with the indicated antibody for the mTOR pathway. Data are mean ± SEM. *P < 0.05, **P < 0.01 vs. Ctrl, #P < 0.05, ##P < 0.01 vs. RSG from one-way ANOVA followed by Sidak’s post hoc tests. n = 3–8.

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