GSK3β Serine 389 Phosphorylation Modulates Cardiomyocyte Hypertrophy and Ischemic Injury

Figures

• 

  Figure 1. Analysis for phosphorylation of GSK3β in diseased human and mouse hearts. (A) Western blot analysis of phosphorylated glycogen synthase kinase 3β (GSK3β) at Ser9 and Ser389 and total GSK3β in left ventricular samples from human dilated cardiomyopathy (DCM) and ischemic cardiomyopathy (ICM) hearts. *** p < 0.001 compared to control. (B) Western blot analysis of phosphorylated GSK3β and total GSKβ in left ventricular samples of mice subjected to transverse aortic constriction (TAC) for 6 weeks. ** p < 0.01 compared to sham. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control; n = 4–6 for each group.

• 

  Figure 2. Validation of adenoviruses encoding for GSK3β S9A, GSK3β S389A, and GSK3β S9A/S389A. Neonatal rat cardiomyocytes were infected with LacZ, wild-type (WT) GSK3β, or GSK3β carrying Ser→Ala mutation at Ser9 and Ser389. Phenylephrine (PE) was used to stimulate GSK3β phosphorylation where indicated. Shown is Western blot analysis of phosphorylated GSK3β at Ser9, Ser389, and for total GSK3β. (A) Overexpression of GSK3β S9A and PE stimulus. (B) Overexpression of GSK3β S389A and PE stimulus. (C) Overexpression of GSK3β S9A/S389A and PE stimulus. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control; n = 4 for each group.

• 

  Figure 3. Overexpression of GSK3β S389A reduces cardiomyocyte viability. Cultured adult rat cardiomyocytes were infected with adenoviruses indicated in the figure. (A) Twenty-four hours later, cells were subjected to hypoxia–reoxygenation and cell viability was measured with adenylate kinase release. Cells cultivated in normoxia were used as a control. *** p < 0.001 compared to control LacZ, ## p < 0.01, ### p < 0.001 compared to GSK3β WT, $$$ p < 0.001 compared to GSK3β S9A and S389A. (B) Analysis for mitochondrial membrane potential by JC1 assay in normoxia and following hypoxia–reoxygenation. * p < 0.05. (C) Cardiomyocytes were infected with adenoviruses encoding for LacZ, p38α, p38β GSK3β WT, and S389A as indicated, and cell viability was measured with adenylate kinase release. * p < 0.05, ** p < 0.01, *** p < 0.001; n = 5–6 for each group.

• 

  Figure 4. Overexpression of GSK3β S389A enhances protein synthesis in cardiomyocytes. Cultured rat cardiomyocytes were infected with adenoviruses as depicted in the figure, and 24 h later treated with FGF (20 ng/mL) or insulin (20 µg/mL) for 24 h. (A) Shown is analysis for [3H]-leucine incorporation in neonatal cardiomyocytes. Left panel, *** p < 0.001 compared to LacZ, ### p < 0.001 compared to GSKβ WT and GSK3β S9A. Right panel, ** p < 0.01, *** p < 0.001 compared to LacZ, ### p < 0.001 compared to LacZ + FGF, $$$ p < 0.001 compared to GSK3β WT + FGF, GSK3β S9A + FGF. (B) Shown is analysis for [3H]-leucine incorporation in adult cardiomyocytes. * p < 0.05, *** p < 0.001 compared to LacZ, # p < 0.05, ### p < 0.001 compared to LacZ + insulin, $$$ p < 0.001 compared to GSK3β WT + insulin and GSK3β S9A + insulin; n = 4 for each group.

• 

  Figure 5. GSK3β S389 regulates p70S6 kinase. Cultured neonatal rat cardiomyocytes were infected with adenoviruses depicted in the figure, and 24 h later treated with FGF (20 ng/mL) for 24 h where indicated. (A) Western blot analysis of β-catenin in cytosolic fractions. Vinculin was used as loading control. (B) Western blot analysis for β-catenin and total GSK3β in nuclear fractions. Lamin B was used as a loading control. (C) Western blot analysis of phosphorylated p70S6 kinase (Thr389) and phosphorylated Akt (Ser473) in cytosol. * p < 0.05 compared to LacZ. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control; n = 4 for each group.