Background Inhibition of Glycogen synthase kinase-3 (GSK-3) protects the heart during ischemia/reperfusion (I/R). However, the underlying mechanisms of cardioprotection afforded by beta isoform-specific inhibition of GSK-3 remain to be elucidated. Methods and Results We studied the molecular mechanism mediating the effect of GSK-3β activation/inhibition upon myocardial injury during prolonged ischemia and I/R. Beta isoform-specific inhibition of GSK-3 by dominant negative GSK-3β in transgenic mice (Tg-DnGSK-3β) or in heterozygous GSK-3β knock-out mice (GSK-3β +/−) significantly increased, whereas activation of GSK-3β in constitutively active GSK-3β knock-in mice (βKI) significantly decreased, myocardial ischemic injury after prolonged ischemia. In contrast, inhibition of GSK-3β in Tg-DnGSK-3β or GSK-3β +/− significantly reduced, while activation of GSK-3β in βKI significantly enhanced, myocardial I/R injury. Inhibition of GSK-3β stimulated mTOR signaling and inhibited autophagy through a rapamycin-sensitive (mTOR-dependent) mechanism. Rapamycin enhanced autophagy and, at the same time, abolished the effects of GSK-3β inhibition on both prolonged ischemic injury and I/R injury. Importantly, the influence of rapamycin over the effects of GSK-3β inhibition on myocardial injury was reversed by inhibition of autophagy. Conclusions Our results suggest that beta isoform-specific inhibition of GSK-3 exacerbates ischemic injury but protects against I/R injury by modulating mTOR and autophagy.

Abstract-Glycogen synthase kinase (GSK)-3, a negative regulator of cardiac hypertrophy, is inactivated in failing hearts. To examine the histopathological and functional consequence of the persistent inhibition of GSK-3␤ in the heart in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative GSK-3␤ (Tg-GSK-3␤-DN) and tetracycline-regulatable wild-type GSK-3␤. GSK-3␤-DN significantly reduced the kinase activity of endogenous GSK-3␤, inhibited phosphorylation of eukaryotic translation initiation factor 2B, and induced accumulation of ␤-catenin and myeloid cell leukemia-1, confirming that GSK-3␤-DN acts as a dominant negative in vivo. Tg-GSK-3␤-DN exhibited concentric hypertrophy at baseline, accompanied by upregulation of the ␣-myosin heavy chain gene and increases in cardiac function, as evidenced by a significantly greater E max after dobutamine infusion and percentage of contraction in isolated cardiac myocytes, indicating that inhibition of GSK-3␤ induces well-compensated hypertrophy. Although transverse aortic constriction induced a similar increase in hypertrophy in both Tg-GSK-3␤-DN and nontransgenic mice, Tg-GSK-3␤-DN exhibited better left ventricular function and less fibrosis and apoptosis than nontransgenic mice. Induction of the GSK-3␤ transgene in tetracycline-regulatable wild-type GSK-3␤ mice induced left ventricular dysfunction and premature death, accompanied by increases in apoptosis and fibrosis. Overexpression of GSK-3␤-DN in cardiac myocytes inhibited tumor necrosis factor-␣-induced apoptosis, and the antiapoptotic effect of GSK-3␤-DN was abrogated in the absence of myeloid cell leukemia-1. These results suggest that persistent inhibition of GSK-3␤ induces compensatory hypertrophy, inhibits apoptosis and fibrosis, and increases cardiac contractility and that the antiapoptotic effect of GSK-3␤ inhibition is mediated by myeloid cell leukemia-1. Thus, downregulation of GSK-3␤ during heart failure could be compensatory. Key Words: GSK-3␤ Ⅲ heart failure Ⅲ cardiac hypertrophy Ⅲ apoptosis G SK-3␤ is a ubiquitously expressed serine/threonine kinase that has versatile biological functions in cells, including regulation of metabolism, cell growth/death, and protein translation and transcription. 1,2 Unlike most protein kinases, GSK-3␤ remains active in the resting state and is inactivated when cells are stimulated by mitogens, by other protein kinases, such as Akt, or by the Wnt pathway. In cardiac myocytes, GSK-3␤ phosphorylates ␤-catenin, 3 eukaryotic translation initiation factor (eIF)2B, 4 NFAT, 5 GATA4, 6 myocardin, 7 and other proteins, thereby negatively regulating protein synthesis and gene expression. GSK-3␤ downregulates SERCA2a 8 and enhances mitochondrial permeability transition, 9 thereby leading to an inability to normalize cytosolic Ca 2ϩ in diastole and reduced cell survival, respectively.GSK-3␤ is an important negative regulator of cardiac hypertrophy. 10 GSK-3␤ negatively regulates ␤-adrenergic and endothelin-induced cardiac hypertrophy in cultured ...

Abstract-We have shown previously that tyrosine 319 in a conserved YIPP motif in the C terminus of angiotensin II (Ang II) type 1 receptors (AT 1 Rs) is essential for transactivation of epidermal growth factor receptor (EGFR) in vitro. We hypothesized that the signaling mechanism mediated through the specific amino acid sequence in the G protein-coupled receptor plays an important role in mediating cardiac hypertrophy in vivo. Transgenic mice with cardiac-specific overexpression of wild-type AT 1 R (Tg-WT) and an AT 1 R with a mutation in the YIPP motif (Tg-Y319F) were studied. Tg-Y319F mice developed no significant cardiac hypertrophy, in contrast to the significant development of hypertrophy in Tg-WT mice. Expression of fetal-type genes, such as atrial natriuretic factor, was also significantly lower in Tg-Y319F than in Tg-WT mice. Infusion of Ang II caused an enhancement of hypertrophy in Tg-WT mice but failed to induce hypertrophy in Tg-Y319F mice. Left ventricular myocardium in Tg-Y319F mice developed significantly less apoptosis and fibrosis than that in Tg-WT mice. EGFR phosphorylation was significantly inhibited in Tg-Y319F mice, confirming that EGFR was not activated in Tg-Y319F mouse hearts. In contrast, activation/phosphorylation of protein kinase C, STAT3, extracellular signal-regulated kinase, and Akt and translocation of G␣q/11 to the cytosolic fraction were maintained in Tg-Y319F hearts. Furthermore, a genetic cross between Tg-WT and transgenic mice with cardiac-specific overexpression of dominant negative EGFR mimicked the phenotype of Tg-Y319F mice.

Activation of the endocytosis machinery is required for activation of Akt, which, in turn, critically mediates beta1-AR-induced cardiac hypertrophy.