Atorvastatin Alleviates Experimental Diabetic Cardiomyopathy by Regulating the GSK-3β-PP2Ac-NF-κB Signaling Axis

Abstract

Recent studies reported that atorvastatin (ATOR) alleviated progression of experimental diabetic cardiomyopathy (DCM), possibly by protecting against apoptosis. However, the underlying mechanisms of this protective effect remain unclear. Therefore, our study investigated the role of the glycogen synthase kinase (GSK)-3β-protein phosphatase 2A(PP2A)-NF-κB signaling pathway in the anti-apoptotic and cardioprotective effects of ATOR on cardiomyocytes cultured in high glucose (HG) and in DCM. Our results showed that, in HG-cultured cardiomyocytes, phosphorylation of GSK-3β was decreased, while that of the PP2A catalytic subunit C (PP2Ac) and IKK/IкBα was increased, followed by NF-кB nuclear translocation and apoptosis. IKK/IкBα phosphorylation and NF-кB nuclear translocation were also increased by treatment of cells with okadaic acid (OA), a selective PP2A inhibitor, or by silencing PP2Ac expression. The opposite results were obtained by silencing GSK-3β expression, which resulted in PP2Ac activation. Furthermore, IKK/IкBα phosphorylation and NF-кB nuclear translocation were markedly inhibited and apoptosis attenuated in cells treated with ATOR. These effects occurred through inactivation of GSK-3β and subsequent activation of PP2Ac. They were abolished by treatment of cells with OA or PP2Ac siRNA. In mice with type 1 diabetes mellitus, treatment with ATOR, at 10 mg-kg-1-d-1, significantly suppressed GSK-3β activation, IKK/IкBα phosphorylation, NF-кB nuclear translocation and caspase-3 activation, while also activating PP2Ac. Finally, improvements in histological abnormalities, fibrosis, apoptosis and cardiac dysfunction were observed in diabetic mice treated with ATOR. These findings demonstrated that ATOR protected against HG-induced apoptosis in cardiomyocytes and alleviated experimental DCM by regulating the GSK-3β-PP2A-NF-κB signaling pathway.

Fig 1. HG treatment induced apoptosis in cardiomyocytes by activation of the IKK-IκBα-NF-κB pathway.

(A). Primary cultures of neonatal rat cardiomyocytes were exposed to HG for different time periods (0, 1, 4, 12, 24 or 36 h), then cytoplasmic and nuclear proteins were extracted and nuclear translocation of NF-κB detected by western blotting. (B). H9C2 cardiomyocytes were exposed to HG for 36 h, in the presence of OA, PP2Ac-siRNA or ATOR, and NF-κB luciferase reporter assays were performed. (C). Neonatal rat cardiomyocytes were pretreated with okadaic acid (20 nM) for 1 h, treated with ATOR (20 μM) for 30 min, then exposed to HG for an additional 36 h. The immunofluorescence assay was performed to determine nuclear translocation of NF-κB. (D). Neonatal rat cardiomyocytes were exposed to HG for different time periods (0, 1, 2, 4, 12, 24 or 36 h) and total proteins were extracted. Phospho-IKK (p-IKK), p-IκBα, total IKK (t-IKK) and t-IκBα were detected by western blotting.

Fig 2. HG-induced apoptosis in neonatal rat cardiomyocytes was mediated by activation of the NF-κB pathway.

(A-B). Neonatal rat cardiomyocytes were pretreated with 10–20 μM JSH-23 and exposed to HG for 36 h. Levels of caspase-3 and cleaved caspase-3 and Bax/Bcl-2 ratios were determined by western blotting. (C). Apoptotic cells stained with Annexin V-FITC and PI were detected by flow cytometry. In the 2D coordinates, the X-axis shows intensity of the FITC signal and the Y-axis shows that of the PI signal.

Fig 3. Effects of PP2Ac on HG-induced sustained phosphorylation of IKK/IκBα in H9C2cells.

(A). H9C2 cells were exposed to HG for different time periods (0, 5, 15 or 30 min; or 1, 2, 4, 6, 12 or 24 h), total proteins were extracted and PP2Ac phosphorylation detected by western blotting. (B). After transfection with PP2Ac-siRNA for 48 h, cells were exposed to HG for an additional 4 h and IKK/IκBα was detected by western blotting. (C). After transfection with PP2Ac-siRNA for 24 h, cells were exposed to HG for 36 h. Then, cytoplasmic and nuclear proteins were extracted and NF-κB detected by western blotting. (D). Total protein was extracted after transfecting cells with PP2Ac-siRNA and exposing them to HG for 36 h. Apoptotic proteins were detected by western blotting. (E). After H9C2 cells were transfected with or without PP2Ac-siRNA, then exposed to HG for different time periods (4, 24 or 48 h), IKK/IκBα expression and phosphorylation and nuclear translocation of NF-κB were (F) detected by western blotting. (G). H9C2 cells were exposed to HG for 4, 24 or 48 h, after adding OA, and IKK/IκBα expression and phosphorylation were determined by western blotting.

Fig 4. Role of GSK-3β in HG-induced sustained phosphorylation of PP2Ac-IKK-IκBα in H9C2 cells.

(A). H9C2 cardiomyocytes were exposed to HG for 0, 5, 15 or 30 min; or 1, 2, 4, 6, 12, 24 or 48 h, then total protein was extracted and GSK-3β phosphorylation detected by western blotting. (B). After transfection with GSK-3β-siRNA for 48 h, cells were exposed to HG for an additional 4 h and phosphorylation of PP2Ac and IKK/IκBα detected by western blotting. (C). After transfection with GSK-3β-siRNA for 24 h, cells were exposed to HG for another 36 h. Then, cytoplasmic and nuclear proteins were extracted and NF-κB detected by western blotting. (D). Total protein was extracted after transfecting cells with GSK-3β-siRNA and exposing them to HG for 36 h. Apoptotic proteins were detected by western blotting.

Fig 5. Effects of ATOR on HG-induced activation of the GSK-3β-PP2Ac-IKK-IκBα-NF-κB pathway in H9C2 cells.

(A). H9C2 cells were pretreated with ATOR at different concentrations (1, 5, 10 or 20 μM) then exposed to HG for 4 h and proteins extracted. GSK-3β phosphorylation was detected by western blotting. (B). H9C2 cells were pretreated with ATOR at different concentrations then exposed to HG for 24 h and phosphorylation of PP2Ac detected by western blotting. (C). H9C2 cells were pretreated with ATOR at different concentrations then exposed to HG for 4 h and proteins extracted. IKK/IκBα phosphorylation was detected by western blotting. (D). H9C2 cardiomyocytes were pretreated with ATOR at different concentrations and exposed to HG for 36 h, then cytoplasmic and nuclear proteins extracted. Nuclear translocation of NF-κB was determined by western blotting. (E). Cells were treated as described for Fig 5D, total proteins were extracted and apoptosis-associated proteins detected by western blotting. (F). H9C2 cells were pretreated with OA, OA+ATOR, PP2Ac-siRNA+ATOR or PP2Ac-siRNA before exposure to HG for 4 h. IKK and IκBα expression was detected by western blotting.

Fig 6. Effects of ATOR on cardiomyocyte apoptosis and the NF-κB pathway in diabetic hearts.

(A). Total protein extracted from heart tissues was subjected to western blot analysis for caspase-3 and cleaved caspase-3. (B). Heart tissues were sectioned at 3–4 μm and the slides were processed for TUNEL staining to detect apoptotic cells (arrows represent TUNEL-positive cells). (C). Total protein was extracted from heart tissues and IKK/IκBα expression detected by western blotting. (D). Cytoplasmic and nuclear proteins were extracted from heart tissues and nuclear translocation of NF-κB determined. (E). GSK-3β and PP2Ac phosphorylation was detected in total protein extracts from heart tissues. TUNEL assay images were obtained by microscopy with original magnification 400×.

Fig 7. Effects of ATOR on diabetes-induced histological alterations and cardiac dysfunction.

(A). H-E staining for heart tissues in each group. (B). Masson's trichrome staining for heart tissues in each group. Data are means ± SD from three independent experiments. *, p<0.05 vs control; #, p<0.05 vs HG. H-E staining and Masson's trichrome staining images were obtained by microscopy with original magnification 400×.

Fig 8. Effects of ATOR on diabetes-induced alterations in cardiac function.

Doppler echocardiographic images obtained from hearts of normal mice (A), normal mice treated with ATOR (B), diabetic mice (C) and diabetic mice treated with ATOR (D) for 12 wk. E, peak velocity of the early ventricular filling (E wave); A, peak velocity of the late ventricular filling (A wave).