Phosphorylation of Glycogen Synthase Kinase-3β During Preconditioning Through a Phosphatidylinositol-3-Kinase–Dependent Pathway Is Cardioprotective
We previously reported that activation of phosphatidylinositol-3-kinase (PI3-kinase) is involved in ischemic preconditioning (PC). Our goal was to determine downstream targets of PI3-kinase. In perfused rat hearts, PC (4 cycles of 5 minutes of ischemia and 5 minutes of reflow) increased phosphorylation of glycogen synthase kinase-3β (GSK-3β), a downstream target of PI3-kinase and protein kinase B (PKB), an effect that was blocked by wortmannin. Because phosphorylation inactivates GSK-3β, we examined whether PC-induced phosphorylation and inhibition of GSK-3β is important in PC by using two inhibitors of GSK-3β, lithium and SB 216763. Pretreatment of perfused rat hearts with lithium or SB 216763, before ischemia, mimicked the protective effects of PC; hearts treated with either lithium or SB 216763 had improved postischemic function and reduced infarct size. These findings indicate that inhibition of GSK-3β is protective and that this PI3-kinase–dependent signaling pathway may play an important role in ischemic preconditioning.
Brief intermittent periods of ischemia and reflow, termed ischemic preconditioning (PC), have been shown to protect the myocardium against injury produced by a subsequent sustained period of ischemia.1 PC has been shown to significantly reduce infarct size, arrhythmias, and postischemic contractile dysfunction. We previously reported that activation of phosphatidylinositol-3-kinase (PI3-kinase) is important in PC.2 PI3-kinase has been reported to enhance cell survival via phosphorylation of GSK-3β.3 Expression of a mutant GSK that cannot be phosphorylated blocked the antiapoptotic action of PI3-kinase.3 We therefore examined the role of GSK-3β in PC. Our results suggest that phosphorylation and subsequent inactivation of GSK-3β play an important role in the protection afforded by PC in the heart.
Materials and Methods
Isolated Rat Heart Preparation
All rats received humane care in accordance with the NIH guidelines (NIH publication No. 8523, revised 1985). Hearts from male Sprague-Dawley rats (200 to 300 g; Taconic, Germantown, NY) were perfused in Langendorff mode under constant pressure (67 mm Hg) as described previously.2
The protocol is illustrated in Figure 1. For studies assessing recovery of postischemic function, the protocol consisted of a 30-minute control period, a treatment period, 20 minutes of global normothermic ischemia, and 30 minutes of reperfusion. Recovery of left ventricular developed pressure (LVDP) was measured after 30 minutes of reflow and expressed as a percentage of the initial LVDP, before PC or drug administration. Group I hearts (control, n=19) were perfused with Krebs-Henseleit (KH) buffer. Group II hearts (PC, n=11) were preconditioned with four cycles of 5 minutes of ischemia (I) and 5 minutes of reflow (R). Group III hearts (SB, n=14) were treated with 3 μmol/L SB 216763 for 10 minutes. Group IV hearts (Li, n=6) were treated with 3 mmol/L LiCl for 10 minutes. For studies assessing infarct size, the groups (n=4 for all) and protocol were the same except that the period of ischemia was extended to 25 minutes and reperfusion was extended to 2 hours.
Western Blot Analysis
For Western blot analysis, four groups of rat hearts (n=4 each group) were snap-frozen at the end of the treatment period. Frozen hearts were homogenized in ice-cold lysis buffer and subjected to SDS-polyacrylamide gel electrophoresis as described previously.2 The membranes were probed with anti–phospho-GSK3β (Cell Signaling Technology) (1:1000 dilution).
GSK-3β Kinase Assay
GSK-3β activity (n=3 to 5/group) was measured in control and PC hearts snap-frozen at the end of the treatment period and after 20 minutes of global ischemia. Frozen hearts were homogenized in ice-cold lysis buffer as described previously.2 Immunoprecipitated GSK-3β (using GSK-3β antibody [Transduction Laboratory]) activity was assessed by a kinase assay using 32P-[ATP] and glycogen synthase peptide-2 (Upstate Biotechnology) as substrate, as described by Haq et al.4
At the end of 2 hours of reperfusion, hearts were perfused with 40 mL of 0.8% solution of 2,3,5-triphenyltetrazolium chloride (TTC) dissolved in KH buffer and then incubated in 0.8% TTC at 37°C for 15 minutes, followed by fixation in 10% formaldehyde. Cross-sectional slices through the ventricles were then photographed using a digital camera. Infarct size is expressed as the percent of total heart (LV+RV) that does not stain with TTC.
Values are expressed as mean±SEM. Significance was determined using ANOVA (StatView). The level of statistical significance was taken as P≤0.05.
PC Increases the Phosphorylation of GSK-3β
In this study, we examined whether GSK-3β, a downstream target of protein kinase B (PKB), is involved in PC. As shown in Figure 2A, PC caused an increase in GSK-3β phosphorylation, and this increase was blocked by wortmannin (P<0.05 compared with PC). The addition of wortmannin alone had no effect on GSK-3β phosphorylation.
To confirm that PC-induced phosphorylation of GSK-3β altered activity, we performed an immune complex kinase assay. As shown in Figure 2B, at the end of the PC protocol (time=0), PC hearts had significantly lower GSK-3β activity compared with non-PC control hearts. During sustained ischemia, there was a similar decline in GSK-3β in non-PC hearts and in PC hearts.
SB 216763 and Lithium Mimic the Protective Effect of PC on Recovery of Function
To investigate whether GSK-3β might be involved in the cardioprotective effect of PC, we examined whether the inhibitors of GSK-3β, lithium and SB 216763, would mimic the cardioprotective effect of PC. Treatment with the GSK-3β inhibitors SB 216763 or lithium had no effect on preischemic LVDP, heart rate, or coronary flow rate. As shown in Figure 3A, PC resulted in a significant improvement in recovery of postischemic LVDP compared with control (50.3±4.4% versus 31.7±2.7% P<0.05). Similar to PC, lithium treatment significantly improved postischemic LVDP (59.2±6.3%, P<0.01 versus control). Similar to the results obtained with LiCl, we found that SB 216763 treatment significantly improved postischemic LVDP compared with control (41.7±3.0%, P<0.05 versus control).
As it is well established that PC reduces infarct size, we also examined whether lithium or SB 216763 treatment would reduce infarct size. As shown in Figure 3B, after 25 minutes of ischemia and 2 hours of reperfusion, PC hearts have significantly smaller infarcts compared with control non-PC hearts (37% infarct in control, non-PC hearts versus 6% in PC hearts). Similar to the protection afforded by PC, hearts treated with lithium or SB 216763 had infarcts that were significantly smaller than control. Taken together, these data suggest that inhibition of GSK-3β, as occurs with PC, is cardioprotective.
PI3-kinase has been shown to be involved in the signaling pathway of PC.2 The downstream targets of PI3-kinase and their role in preconditioning have not been identified. The data presented here provide the first evidence linking GSK-3β to PC and cardioprotection. We found that PC results in phosphorylation and inactivation of GSK-3β, consistent with a role for GSK-3β in PC. We also found that the specific PI3-kinase inhibitor wortmannin blocks the PC-induced phosphorylation of GSK-3β. Because phosphorylation leads to inhibition of GSK-3β, we examined whether inhibitors of GSK-3β, SB 216763 or lithium, would mimic the protective effects of PC. We used a specific inhibitor of GSK-3β, SB 216763, which is a potent, cell-permeant competitive inhibitor of the ATP binding site of GSK-3β.5 SB 216763 does not inhibit other related protein kinases, including PKB and PDK-1.5 Another inhibitor, lithium, has no known effects on other protein kinases, but does have effects on other enzymes, including the inositol monophosphatase and adenylyl cyclase.6 Lithium has been used extensively4,6⇓ to study the effect of GSK-3β. We found that treating hearts with lithium or SB 216763 before ischemia and reperfusion improved recovery of postischemic function and reduced infarct size, suggesting that inhibition of GSK-3β is cardioprotective.
In contrast to many protein kinases, GSK-3 is active in resting cells and is inactivated by phosphorylation.7 On stimulation, GSK-3 is phosphorylated at serine 21 in GSK-3α or serine 9 in GSK-3β, resulting in inhibition of GSK-3 kinase activity.7–9⇓⇓ PKB phosphorylates GSK-3 at both of these sites in vitro and in vivo.7,8⇓ Inhibition of GSK-3β has been shown to reduce apoptosis and enhance cell survival,3 providing a plausible mechanism by which phosphorylation and inhibition of GSK-3β might mediate cardioprotection. Several groups have reported that a dominant-negative GSK-3β or inhibitors of GSK-3β prevent apoptosis.3,10⇓ However, the precise mechanism by which GSK-3β inhibits apoptosis is not clear. GSK-3 was originally identified as an enzyme that regulates glycogen synthesis in response to insulin.10 Recent studies have demonstrated a growing list of substrates for GSK-3β, such as β-catenin, tau, NF-AT, cyclin D1, c-myc, E-cadherin, and eIF2B,10 some of which may be involved in GSK inhibition of apoptosis. Data in Figure 2B show that at the start of sustained ischemia, GSK-3β activity is significantly lower in PC versus non-PC hearts. During ischemia, GSK-3β is inhibited in non-PC as well as PC hearts, but there is a trend toward lower activity in PC hearts. These data suggest that the lower GSK-3β activity at the start of the sustained ischemia is protective.
Recent studies suggested that inhibition of GSK-3β leads to cardiomyocyte hypertrophy.4,11⇓ Myocytes that were transfected with a mutant GSK-3β, which could not be phosphorylated, did not undergo hypertrophy.4,11⇓ Hypertrophy is initially a compensatory mechanism, and although prolonged hypertrophy is associated with heart failure, the exact relationship between signals that activate hypertrophy and those that activate failure are not clearly delineated. Thus, the protective effects of GSK-3β inhibition are not necessarily inconsistent with stimulation of hypertrophy. It is also possible that short-term activation of GSK-3β is protective whereas long-term activation may have other detrimental effects.
In summary, the present study demonstrates that PC increases phosphorylation of GSK-3β through a PI3-kinase–PKB-dependent pathway, since wortmannin blocks the PC-induced phosphorylation of GSK-3β. The GSK-3β inhibitors, SB 216763 and lithium, mimic the cardioprotective effect of PC, suggesting that inhibition of GSK-3β is protective in classic PC. We conclude that GSK-3β is involved in the cardioprotective effect of PC through a PI3-kinase–dependent pathway. These data indicate that pharmacological modulation of GSK-3β activity could have utility for protecting the heart against ischemic injury.
H.T., K.I., and E.M. were supported by the National Institute of Environmental Health Sciences Intramural Program. C.S. was supported by NIH Grant HL-39752.
Original received August 9, 2001; resubmission received January 11, 2002; revised resubmission received January 28, 2002; accepted January 28, 2002.
- ↵Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986; 74: 1124–1136.
- ↵Tong H, Chen W, Steenbergen C, Murphy E. Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C. Circ Res. 2000; 87: 309–315.
- ↵Pap M, Cooper GM. Role of glycogen synthase kinase-3 in the phosphatidylinositol-3-kinase/Akt cell survival pathway. J Biol Chem. 1998; 273: 19929–19932.
- ↵Haq S, Choukroun G, Kang ZB, Ranu H, Matsui T, Rosenzweig A, Molkentin JD, Alessandrini A, Woodgett J, Hajjar R, Michael A, Force T. Glycogen synthase kinase-3β is a negative regulator of cardiomyocyte hypertrophy. J Cell Biol. 2000; 151: 117–130.
- ↵Coghlan MP, Culbert AA, Cross DA, Corcoran SL, Yates JW, Pearce NJ, Rausch OL, Murphy GJ, Carter PS, Roxbee-Cox L, Mills D, Brown MJ, Haigh D, Ward WR, Smith DG, Muray KJ, Reith AD, Holder JC. Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription. Chem Biol. 2000; 7: 793–803.
- ↵Klein PS, Melton DA. A molecular mechanism for the effect of lithium on development. Proc Natl Acad Sci U S A. 1996; 93: 8455–8459.
- ↵Stambolic V, Woodgett JR. Mitogen inactivation of glycogen synthase kinase-3β in intact cells via serine 9 phosphorylation. Biochem J. 1994; 303: 701–704.
- ↵Morisco C, Zebrowski D, Condorelli G, Tsichlis P, Vatner SF, Sadoshima J. The Akt-glycogen synthase kinase 3β pathway regulates transcription of atrial natriuretic factor induced by β-adrenergic receptor stimulation in cardiac myocytes. J Biol Chem. 2000; 275: 14466–14475.