Phosphorylation of Glycogen Synthase Kinase-3β During Preconditioning Through a Phosphatidylinositol-3-Kinase–Dependent Pathway Is Cardioprotective

Abstract

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-3␤ (GSK-3␤), a downstream target of PI3-kinase and protein kinase B (PKB), an effect that was blocked by wortmannin. Because phosphorylation inactivates GSK-3␤, we examined whether PC-induced phosphorylation and inhibition of GSK-3␤ is important in PC by using two inhibitors of GSK-3␤, 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-3␤ 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-3␤. 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-3␤ in PC. Our results suggest that phosphorylation and subsequent inactivation of GSK-3␤ play an important role in the protection afforded by PC in the heart. Materials and Methods Isolated Rat Heart PreparationAll 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 Experimental ProtocolsThe 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, nϭ19) were perfused with Krebs-Henseleit (KH) buffer. Group II hearts (PC, nϭ11) were preconditioned with four cycles of 5 minutes of ischemia (I) and 5 minutes of reflow (R). Group III hearts (SB, nϭ14) were treated with 3 mol/L SB 216763 for 10 minutes. Group IV hearts (Li, nϭ6) were treated with 3 mmol/L LiCl for 10 minutes. For studies assessing infarct size, the groups (nϭ4 for all) and protocol...

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3β during reperfusion injury

“…As previously demonstrated by our group, 6,7 mice treated with SB21 at reperfusion (IR+SB21, 70 μg/kg) exhibit a significant reduction of infarct size after prolonged IR injury when compared with control (IR), averaging 31.0 ± 0.6% versus 44.6 ± 1.7%, respectively (Figure 5a). SB21 administered at reperfusion significantly decreased the phosphorylation of both GSK3β (Tyr 216 ) and IP 3 R (Ser 1756 ) induced by reperfusion injury (Figures 5b and c (Figure 5d).…”

“…It has been previously demonstrated that inhibition of GSK3β at reperfusion is cardioprotective by preventing PTP opening. 6,7 However, the relative cardioprotective mechanism still remains unknown. We here hypothesized that this beneficial effect might be linked to the limitation of both cytosolic and mitochondrial Ca 2+ overload (and subsequent Ca 2+ -dependent PTP opening) by reducing Ca 2+ release from IP 3 R at reperfusion.…”

“…4 In the heart, GSK3β has several important roles in cardiac hypertrophy 5 and ischemia-reperfusion (IR) injury. 6 Accumulating evidence indicates that phospho-Ser9-GSK3β-mediated cytoprotection is achieved by an increased threshold for permeability transition pore (PTP) opening. [6][7][8][9] The mechanism by which GSK3β delays PTP opening still remains unclear.…”

“…6 Accumulating evidence indicates that phospho-Ser9-GSK3β-mediated cytoprotection is achieved by an increased threshold for permeability transition pore (PTP) opening. [6][7][8][9] The mechanism by which GSK3β delays PTP opening still remains unclear. It has been reported that GSK3β could interact with ANT at the inner mitochondrial membrane in the heart 9 and/or to phosphorylate voltage-dependent anion channel (VDAC) and cyclophilin D (CypD) in cancer cells.…”

Glycogen synthase kinase-3β plays a pro-apoptotic role in β-adrenergic receptor-stimulated apoptosis in adult rat ventricular myocytes: Role of β1 integrins

“…Furthermore, direct inhibition of GSK-3β reduces infarct size and improves post-ischemic heart function [27]. Collectively these data suggest activation of GSK-3β may play an important role in cardiac myocyte apoptosis and heart function.…”