Atorvastatin Upregulates The Expression Of PPARs And Inhibits The Hypertrophy Of Cardiac Myocytes In Vitro

Objective: Cardiac hypertrophy is fundamental response of cardiac myocytes to various stimuli, which is associated with significantly increased risk of heart failure and malignant arrhythmia. Statins, HMG-CoA reductase inhibitors, are widely prescribed cholesterol-lowering drugs that also appear to play beneficial roles in the prevention of cardiac hypertrophy and inflammation, however, little is known about the mechanism. Recently, it has been proved that peroxisome peroliferator activated-receptors (PPARs) are involved in prevention of cardiac hypertrophy, and statins exert their effect on upregulation of PPARs in rat hepatocytes and rabbit adipocytes, respectively. The purposes of this study were: 1. to investigate the effects of atorvastatin on Ang II -induced cardiomyocytes hypertrophy and cytokines;2. to characterize the role of PPARs in regulating hypertrophic gene expressions in response to atorvastatin;3. to study the role of GW0742, a PPAR β/δ agonist, on Ang II-induced hypertrophic cardiomyocyte growth and gene expression of cytokines.Methods: 1 .Primary cultures of cardiac myocytes (MC) were prepared from the ventricles of Wistar rats (1-2d). Hypertrophy in neonatal rat cardiac MC was established with angiotensin II (Ang II) and treated with atorvastatin (10, 1, 0.1 μmol·L^-1) or GW0742 (10μmol·L^-1). 2. The characteristic parameters of hypertrophic myocytes include the surface area of MC which was analyzed by the aid of NIH Image J software, the synthetic rate of protein in MC that was detected by ³H-leucine incorporation, and the mRNA expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was measured by reverse transcription-polymerase chain reaction (RT-PCR). 3. At mRNA level, the changes of MMP9,MMP2, IL-ip and Corin in all groups were measured by RT-PCR. 4. PPAR a, p/5, y subtypes were measured by RT-PCR at mRNA level, and detected by immuno-fluorescence staining at protein level, activation and intracellular localization. 5. A reporter gene assay was applied to evaluate the effect of atorvastatin on the transcriptional activities of PPARs.Results: 1. Atorvatatin inhibited Ang II -induced neonatal cardiacmyocytes hypertrophy in a dose-dependent manner, through decreasing surface area (PO.01), mRNA expression of ANP, BNP, Corin (PO.01), and 3H-leucine incorporation (PO.01) in MC. 2. Atorvastatin decreased the mRNA level of MMP9, MMP2 and IL-ip (PO.01) in hypertrophic myocytes. 3. The expression of PPARs was downregulated by Ang II at both mRNA and protein levels, otherwise, atorvastatin administration could reverse the changes above and improve activities of PPARs, but no effect was investaged on the cells exposed to DM SO, the solvent of atorvastatin. 4. To confirm PPARs-dependent pathway is involved in the inhibition of cardiac hypertrophy, a plasmid carrying PPRE derived from the promoter of rat acyl-CoA oxidase (ACO) gene was constructed. With it, HepG2 cells were transiently transfected with or without atorvastatin. Luciferase activities of transfected cells were measured using dual-luciferase reporter assay kit. The results indicated that the luciferase expression markedly increased in the atorvastatin-treated group compared with the vehicle group. 5. At lOumol'L"', GW0742, a highly selective PPAR p/5 activator, inhibited Ang II -induced protein synthesis (3H-leucine incorporation) (P<0.05), induction of the fetal-type gene ANP (PO.05), BNP (P<0.05) and cardiac myocyte size (P<0.05). Meanwhile, GW0742 has no such effect on normal myocytes (P > 0.05). 6. Induction of cardiac hypertrophy with Ang II also led to an augment at the transcription levels of MMP9, MMP2, IL-lp, and these changes were reversed by GW0742. 7. The agonist upregulated PPAR p/5 both at mRNA level (PO.05) and protein level (P<0.05), and increased PPAR p/5 activity, promote its translocation into cellularnucli.Conclusion: 1. Atorvastatin inhibits cardiac hypertrophy and has a potential role in the prevention of cytokine expressions induced by Angll in vitro;2. Atorvastatin upregulates and activates PPARs in hypertrophic myocytes;3. PPAR p/5 agonist GW0742 exerts anti-hypertophy and anti-inflammatory effect on myocytes stimulated by Angll. Furthermore, it increases the expression of PPAR p75 at mRNA and protein levels.In summary, the present investigation indicates that atorvastatin inhibits the Ang II -induced cardiomyocyte hypertrophy through activation of PPARs pathway partly. Our data may provide a novel insight into the development mechanism for the pathological process of cardiac hypertrophy and be helpful to further explore the novel pharmacyological effect of statins.