Aldose reductase modulates cardiac glycogen synthase kinase-3β phosphorylation during ischemia-reperfusion

Abstract

Earlier studies have demonstrated that aldose reductase (AR) plays a key role in mediating ischemia-reperfusion (I/R) injury. Our objective was to investigate if AR mediates I/R injury by influencing phosphorylation of glycogen synthase kinase-3β (p-GSK3β). To investigate this issue, we used three separate models to study the effects of stress injury on the heart. Hearts isolated from wild-type (WT), human expressing AR transgenic (ARTg), and AR knockout (ARKO) mice were perfused with/without GSK3β inhibitors (SB-216763 and LiCl) and subjected to I/R. Ad-human AR (Ad-hAR)-expressing HL-1 cardiac cells were exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)) conditions. I/R in a murine model of transient occlusion and reperfusion of the left anterior descending coronary artery (LAD) was used to study if p-GSK3β was affected through increased AR flux. Lactate dehydrogenase (LDH) release and left ventricular developed pressure (LVDP) were measured. LVDP was decreased in hearts from ARTg mice compared with WT and ARKO after I/R, whereas LDH release and apoptotic markers were increased (P < 0.05). p-GSK3β was decreased in ARTg hearts compared with WT and ARKO (P < 0.05). In ARKO, p-GSK3β and apoptotic markers were decreased compared with WT (P < 0.05). WT and ARTg hearts perfused with GSK3β inhibitors improved p-GSK3β expression and LVDP and exhibited decreased LDH release, apoptosis, and mitochondrial pore opening (P < 0.05). Ad-hAR-expressing HL-1 cardiac cells, exposed to hypoxia (0.5% O(2)) and reoxygenation (20.9% O(2)), had greater LDH release compared with control HL-1 cells (P < 0.05). p-GSK3β was decreased and correlated with increased apoptotic markers in Ad-hAR HL-1 cells (P < 0.05). Treatment with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor increased injury demonstrated by increased LDH release in ARTg, WT, and ARKO hearts and in Ad-hAR-expressing HL-1 cells. Cells treated with protein kinase C (PKC) α/β inhibitor displayed significant increases in p-Akt and p-GSK3β expression, and resulted in decreased LDH release. In summary, AR mediates changes in p-GSK3β, in part, via PKCα/β and Akt during I/R.

Fig. 1.

Glycogen synthase kinase-3β (GSK3β) phosphorylation expression in wild-type (WT), aldose reductase transgenic (ARTg), and aldose reductase knockout (ARKO) mice. Western blot analysis for phospho (p)-GSK3β-Ser⁹ [isolated heart (A) and LAD ligation (C)], p-GSK3β-Tyr²¹⁶ [isolated heart (B) and LAD ligation (D)], cytochrome c (E), and aldose reductase (AR) expression (F) in untreated WT, ARTg, and ARKO mice hearts subjected to ischemia-reperfusion (I/R). ARTg hearts showed decreased p-GSK3β inhibition compared with WT hearts. ARKO hearts had significant increases in p-GSK3β (Ser⁹) (P < 0.05). ARTg hearts displayed a more than threefold increase in apoptosis compared with WT and ARKO hearts as was measured by cytochrome c expression (P < 0.05). ARKO hearts displayed significant decreases in apoptotic levels compared with WT (P < 0.05). ARTg hearts displayed a twofold increase in AR expression compared with WT (n = 4–16 mice/group).

Fig. 2.

Pharmacological inhibition of GSK3β protects mice hearts upon I/R. Determination of myocardial ischemic injury and function with and without GSK3β inhibitor treatment as shown by left ventricular developed pressure (LVDP) recovery (A) and lactate dehydrogenase (LDH) release (B). Western blot analysis for p-GSK3β-Ser⁹ (C), p-GSK3β-Tyr²¹⁶ (D), and cytochrome c (E) in WT and ARTg hearts perfused with and without GSK3β inhibitors SB-216763 and LiCl. Treatment with SB-216763 and LiCl protected WT and ARTg mice hearts, significantly increased p-GSK3β inhibition (P < 0.05), and decreased apoptosis (n = 4–16 mice/group).

Fig. 3.

Mitochondrial permeability in WT and ARTg hearts treated with and without GSK3β inhibitors SB-216763 and LiCl and subjected to I/R. Mitochondrial permeability transition pore opening (mPTP) was determined by monitoring swelling of mitochondria by measuring light scattering at 540 nm in the presence and absence of 50 μM (A) and 100 μM (B) Ca²⁺. Pore opening was reduced in WT and ARTg hearts treated with SB-216763 and LiCl in response to increasing calcium concentrations (P < 0.05) (n = 4–16/group).

Fig. 4.

p-Protein kinase B (Akt). Western blot analysis for p-Akt in LAD ligation of WT, ARTg, and ARKO hearts (A), determination of myocardial ischemic injury and function, as shown by LVDP (B) and LDH release (C) in WT, ARTg, and ARKO perfused with and without phosphatidylinositol 3-kinase (PI3K)/AKT inhibitor LY-294002. Western blot analysis for p-Akt (D), p-GSK3β-Ser⁹ (E), and cytochrome c (F) in WT, ARTg, and ARKO hearts perfused with and without PI3K/Akt inhibitor LY-294002. Treatment with LY-294002 decreased p-Akt and p-GSK3β levels in all 3 groups (P < 0.05). Cytochrome c levels were increased in WT and ARKO hearts (P < 0.05), but not ARTg upon perfusion with LY-294002 (n = 4–16 mice/group).

Fig. 5.

GSK3β small-interfering RNA (siRNA) knock down in HL-1 cells. Western blot analysis for total GSK3β dose-dependent siRNA knockdown (A) and AR expression (B). Determination of cardiac injury as determined by LDH release in control and human AR (hAR) siRNA knock down of GSK3β (C). D: Western blot analysis of cytochrome c expression in control and hAR-overexpressing cells transfected with GSK3β siRNA. No change in LDH release was observed in control and hAR-overexpressing cells exposed to 30 min of normoxia alone. Thirty minutes of hypoxia followed by 60 min of reoxygenation increased LDH release (P < 0.05), whereas inhibiting GSK3β with siRNA significantly reduced LDH release and injury (P < 0.05). hAR-transfected cells, as expected, had greater AR expression than control cells. There was no difference in AR expression between HL-1 control cells subjected to normoxic and H/R conditions. GSK3β siRNA knock down significantly reduced GSK3β expression in both control and hAR-overexpressing cells. Inhibition of GSK3β significantly reduced apoptotic levels in both control and hAR-overexpressing cells. NS, not significant. Error bars not visible. Control normoxia: SE ± 0.003; control hAR: SE ± 0.006; control GSK3β siRNA: ±0.005.

Fig. 6.

HL-1 cardiomyocyte studies. Western blot analysis for p-Akt (A), p-GSK3β-Ser⁹ (B), p-GSK3β-Tyr²¹⁶ (C), and cytochrome c (D) in control and hAR-overexpressing HL-1 cells subjected to H/R alone. E: determination of cardiomyocyte H/R injury as shown by LDH release in control and hAR-overexpressing HL-1 cell supernatants. F: LDH release in control and hAR-overexpressing HL-1 cells treated with and without PI3K/Akt inhibitor LY-294002. Western blot analysis for p-GSK3β-Ser⁹ (G) and cytochrome c (H) in control and hAR-overexpressing HL-1 cells treated with and without PI3K/Akt inhibitor LY-294002. Cells were incubated with LY-294002 (10 μM) or its vehicle control dimethyl sulfoxide (DMSO) for 1 h, followed by 30 min of hypoxia and 1 h reoxygenation (H/R). Treatment with LY-294002 decreased p-Akt levels, decreased inhibition of GSK3β, and increased apoptosis in both control and hAR-overexpressing cells. LDH release in supernatants in both control and hAR-overexpressing cells was increased with PI3K/Akt inhibitor LY-294002 treatment. Error bars not visible. Control: SE ± 0.006; and hAR: SE ± 0.011.

Fig. 7.

Protein kinase C (PKC) α/β inhibition. Western blot analysis for p-Akt (A) and p-GSK3β (B). C: determination of cardiomyocyte injury as shown by LDH release. Treatment with Gö-6976 resulted in decreased cellular injury and an increase in p-GSK3β. Cells were incubated with Gö-6976 (1 μM) or its vehicle control DMSO for 1 h, followed by 30 min of hypoxia and 1 h reoxygenation (H/R).

Fig. 8.

Schematic diagram. Increased flux via AR after I/R resulted in generation of diacylglycerol (DAG) and activation of PKCα and -β isoforms, leading to decreased phosphorylation of Akt and GSK3β kinases. Decreased GSK3β ultimately decreased cardioprotection via increased apoptotic mechanisms and mitochondrial permeability opening.