Abstract Body: Introduction: Glycogen synthase kinase-3β (GSK3β) is inactivated by phosphorylation at serine-9 (S9). Pressure overload (PO) induces phosphorylation of GSK3β at S9, whereas cardiac dysfunction and hypertrophy are attenuated in GSK3β-S9A knock-in (KI) mice. Thus, S9 phosphorylation of GSK3β mediates pathological hypertrophy. Forkhead box O1 (FoxO1) promotes cardiomyocyte survival by upregulating antioxidants in a context-dependent manner. We hypothesize that GSK3β phosphorylates FoxO1, thereby protecting the heart during PO.
Methods and results: Co-immunoprecipitation assays demonstrated that GSK3β interacts with FoxO1. Mass spectrometry analysis showed that GSK-3b phosphorylates FoxO1 at serine-284 (S284). To examine the role of FoxO1 phosphorylation at S284, FoxO1 (S284A)-KI mice were generated. Baseline left ventricular ejection fraction (LVEF) was lower in FoxO1 (S284A)-KI mice than in wild-type (WT) mice (47 ± 5.2% vs. 52 ± 1.6%, p = 0.077, N = 5) at 6 weeks of age. The heart weight, normalized to tibial length, was significantly greater in FoxO1 (S284A)-KI mice than in WT mice (131 ± 15 mg/mm vs. 101 ± 7.8 mg/mm, p = 0.0048, N = 5). In addition, the gene expression level of catalase was significantly reduced in FoxO1 (S284A)-KI mice compared to in WT mice (p = 0.04). These results suggest that phosphorylation of FoxO1 at S284 promotes pathological hypertrophy through oxidative stress. Four weeks of transverse aortic constriction (TAC) significantly reduced LVEF in both WT and S284A-KI mice compared to that in WT-sham (53 ± 1.2%, both p < 0.01, N > 5). However, LVEF in WT mice after TAC was not significantly different from that in S284A-KI mice after sham and TAC, suggesting that TAC and FoxO1 (S284)-KI do not have additive effects upon cardiac dysfunction (LVEF in S284A-sham, WT-TAC and S284A-TAC: 42 ± 3.6% vs. 38 ± 8.9% vs. 40 ± 5.0, p = 0.70, N > 5). These results indicate that TAC-induced cardiac dysfunction is mediated through FoxO1 phosphorylation at S284.
Conclusions: GSK-3b-induced phosphorylation of FoxO1 at S284 is protective in the heart, whereas TAC induces pathological hypertrophy by inhibiting S284 phosphorylation of FoxO1.