Distinct roles of GSK-3alpha and GSK-3beta phosphorylation in the heart under pressure overload

Abstract

Glycogen synthase kinase-3 (GSK-3) is a master regulator of growth and death in cardiac myocytes. GSK-3 is inactivated by hypertrophic stimuli through phosphorylation-dependent and -independent mechanisms. Inactivation of GSK-3 removes the negative constraint of GSK-3 on hypertrophy, thereby stimulating cardiac hypertrophy. N-terminal phosphorylation of the GSK-3 isoforms GSK-3alpha and GSK-3beta by upstream kinases (e.g., Akt) is a major mechanism of GSK-3 inhibition. Nonetheless, its role in mediating cardiac hypertrophy and failure remains to be established. Here we evaluated the role of Serine(S)21 and S9 phosphorylation of GSK-3alpha and GSK-3beta in the regulation of cardiac hypertrophy and function during pressure overload (PO), using GSK-3alpha S21A knock-in (alphaKI) and GSK-3beta S9A knock-in (betaKI) mice. Although inhibition of S9 phosphorylation during PO in the betaKI mice attenuated hypertrophy and heart failure (HF), inhibition of S21 phosphorylation in the alphaKI mice unexpectedly promoted hypertrophy and HF. Inhibition of S21 phosphorylation in GSK-3alpha, but not of S9 phosphorylation in GSK-3beta, caused phosphorylation and down-regulation of G1-cyclins, due to preferential localization of GSK-3alpha in the nucleus, and suppressed E2F and markers of cell proliferation, including phosphorylated histone H3, under PO, thereby contributing to decreases in the total number of myocytes in the heart. Restoration of the E2F activity by injection of adenovirus harboring cyclin D1 with a nuclear localization signal attenuated HF under PO in the alphaKI mice. Collectively, our results reveal that whereas S9 phosphorylation of GSK-3beta mediates pathological hypertrophy, S21 phosphorylation of GSK-3alpha plays a compensatory role during PO, in part by alleviating the negative constraint on the cell cycle machinery in cardiac myocytes.

Fig. 1.

The effect of TAC in DKI mice. (A) Heart homogenates from WT mice 1 week after TAC and sham were subjected to immunoblotting analyses with isoform-specific antibodies for phosphorylated and total GSK-3α and GSK-3β. (B) HW/TL in DKI mice and WT mice 4 weeks after TAC. (C) Measurement of LVEF in DKI and WT mice 4 weeks after TAC. (D) Fibrosis in DKI and WT mice 4 weeks after TAC. Representative pictures of Masson's trichrome staining are shown. In (B) and (C), data are reported as mean ± SEM. *P < .05; NS, not significant.

Fig. 2.

The effect of 4 weeks of TAC in αKI and βKI mice. (A) HW/TL was measured in αKI, βKI, and WT mice 4 weeks after TAC. (B) LVEF was measured in αKI, βKI, and WT mice 4 weeks after TAC. (C) LW/TL was measured in αKI, βKI, and WT mice 4 weeks after TAC. (D) Fibrosis in the αKI, βKI, and WT mice 4 weeks after TAC. Representative images of Masson's trichrome staining are shown. (E) TUNEL staining in the αKI, βKI, and WT mice 4 weeks after TAC. In (A), (B), (C), and (E), data are reported as mean ± SEM. *P < .05 versus respective sham; ^#P < .05.

Fig. 3.

Activation of GSK-3α, but not GSK-3β, inhibits cell proliferation. (A and B) αKI, βKI, and WT mice were subjected to TAC for 1 week. Representative confocal microscopic images of staining with Ki67 (A) or phosphorylated histone H3 (B) (green), DAPI (blue), and Troponin I (red), a cardiac-specific marker, are shown. Ki67- or phosphorylated histone H3-positive myocytes are indicated by arrows. In (A), the frequency of Ki67-positive cells after TAC also is shown. Data are reported as mean ± SEM. *P < .05; **P < .01 versus respective sham; ^#P < .05. (C) Total myocyte cell numbers per heart were measured 1 week after TAC in the αKI, βKI, and WT mice, as described in Methods. Data are reported as mean ± SEM. **P < .01 versus respective sham; ^#P < .05; ^##P < .01; n = 4–5.

Fig. 4.

Regulation of the cyclin D/E2F pathway by GSK-3α and GSK-3β. (A) Myocytes were transfected with the E2F-Luc reporter and expression plasmids encoding GSK-3α (S21A) or GSK-3β (S9A). Data are reported as mean ± SEM. *P < .05. Experiments were performed three times in triplicate. (B) Staining of endogenous GSK-3α and -3β in cardiac myocytes with and without PE treatment. (C) Subcellular localization of endogenous GSK-3α and -3β in cultured cardiac myocytes was determined by immunoblotting analyses. (D) Localization of endogenous GSK-3α and -3β in control mouse hearts subjected to TAC for 1 week or sham was determined by immunostaining with anti-GSK-3α or -3β antibody (green), either troponin T or I antibody (red), and DAPI (blue). Arrows indicate positive staining (GSK-3α) and negative staining (GSK-3β) of GSK-3 in the nucleus. Magnifications of nuclei indicated by asterisks are shown in the insets. Representative confocal microscopic images are shown. In (B–D), the results are representative of three or four experiments.

Fig. 5.

The effect of cyclin D1-NLS on the cardiac phenotype after TAC in αKI mice. Ad-CycD1-NLS or Ad-LacZ was injected into αKI hearts 2 days before TAC. After 2 weeks of TAC, the hearts were harvested. (A) LVEF after TAC in Ad-CycD1-NLS- or Ad-LacZ-injected αKI hearts. (D) Data are reported as mean ± SEM. **P < .01. (B) (Left) Representative confocal microscopic images of Ki67 staining after TAC in αKI hearts injected with Ad-LacZ (Left) or Ad-CycD1-NLS (Right). Triple-staining with anti-Ki67 (green) and anti-troponin T (red) antibodies and DAPI (blue) is shown. (Right) The frequency of Ki67-positive cells after TAC in αKI mice with or without Ad-CycD1-NLS injection. Data are reported as mean ± SEM. **P < .01. NS, not significant. (C) Representative pictures of Masson's trichrome staining (Top) and quantitative analysis of the fibrotic area (Bottom). (D) The extent of apoptosis was determined by the number of TUNEL-positive myocytes divided by the number of DAPI-positive nuclei in 20 separate fields. In (C) and (D), data are reported as mean ± SEM. *P < .05; **P < .01.