Glycogen Synthase Kinase 3 Inhibition Slows Mitochondrial Adenine Nucleotide Transport and Regulates Voltage-Dependent Anion Channel Phosphorylation

Abstract

Abstract-Inhibition of glycogen synthase kinase (GSK)-3 reduces ischemia/reperfusion injury by mechanisms that involve the mitochondria. The goal of this study was to explore possible molecular targets and mechanistic basis of this cardioprotective effect. In perfused rat hearts, treatment with GSK inhibitors before ischemia significantly improved recovery of function. To assess the effect of GSK inhibitors on mitochondrial function under ischemic conditions, mitochondria were isolated from rat hearts perfused with GSK inhibitors and were treated with uncoupler or cyanide or were made anoxic. GSK inhibition slowed ATP consumption under these conditions, which could be attributable to inhibition of ATP entry into the mitochondria through the voltage-dependent anion channel (VDAC) and/or adenine nucleotide transporter (ANT) or to inhibition of the F 1 F 0 -ATPase. To determine the site of the inhibitory effect on ATP consumption, we measured the conversion of ADP to AMP by adenylate kinase located in the intermembrane space. This assay requires adenine nucleotide transport across the outer but not the inner mitochondrial membrane, and we found that GSK inhibitors slow AMP production similar to their effect on ATP consumption. This suggests that GSK inhibitors are acting on outer mitochondrial membrane transport. In sonicated mitochondria, GSK inhibition had no effect on ATP consumption or AMP production. In intact mitochondria, cyclosporin A had no effect, indicating that ATP consumption is not caused by opening of the mitochondrial permeability transition pore. Because GSK is a kinase, we assessed whether protein phosphorylation might be involved. Therefore, we performed Western blot and 1D/2D gel phosphorylation site analysis using phos-tag staining to indicate proteins that had decreased phosphorylation in hearts treated with GSK inhibitors. Liquid chromatographic-mass spectrometric analysis revealed 1 of these proteins to be VDAC2. Taken together, we found that GSK-mediated signaling modulates transport through the outer membrane of the mitochondria. Both proteomics and adenine nucleotide transport data suggest that GSK regulates VDAC and that VDAC may be an important regulatory site in ischemia/reperfusion injury. (Circ Res. 2008;103:983-991.)

GSK-3α directly regulates β-adrenergic signaling and the response of the heart to hemodynamic stress in mice

“…To confirm that the dysregulation of cAMP production in the KO mouse was due to a direct effect of deletion of Gsk3a, we used SB415286, a relatively selective, small molecule inhibitor of GSK-3 (25,26). Pretreatment of WT cardiomyocytes for 30 minutes with SB415286 led to a significant reduction in the contractile response to isoproterenol, mimicking the findings in the KO cells ( Figure 8C).…”

Intracellular zinc release-activated ERK-dependent GSK-3β–p53 and Noxa–Mcl-1 signaling are both involved in cardiac ischemic-reperfusion injury

“…GSK-3b inhibition), which protects the adult-rat heart from I/R injury for 2-4 h. 4,[13][14][15][16] This study showed a link between the Zn 2 þ i release/increase and the activation of GSK-3b, resulting in myocyte apoptosis. We observed an early increase in p-Ser9 GSK-3b levels after Zn 2 þ Figure S2b), we conclude that Zn 2 þ i supplementation-induced early phosphorylation at Ser9 is not necessarily cardioprotective and may induce cytotoxicity after return to normal medium for 4 or 24 h. In support of the above conclusions, Tyr216 phosphorylation is required for nuclear GSK-3b accumulation in neurons and fibroblasts, 17,18 as mutation of Tyr216 to phenylalanine or its dephosphorylation results in less GSK-3b being found in nuclei.…”

“…When the reperfusion injury salvage kinase (RISK) family members PKA, PKB/Akt, PKC, and MEK-ERK are activated by pre-or post-conditioning, a common downstream protein, GSK-3b, is phosphorylated at Ser9, resulting in GSK-3b inhibition (both events occurring within 20 min of the end of conditioning), leading to the inhibition of mPTPs and the salvaging of 20-50% live myocardium. 4,5,[13][14][15][16] A potent GSK-3 inhibitor, SB216763 (SB21), 4,13 had a relatively weak protective effect against H 2 O 2 -, ONOO À -induced myocyte death (Supplementary Figure S2c), but gave better protection against 10 mM Zn 2 þ -induced cell toxicity at 4 h (Figure 2d (TUNEL test) were performed as follows: after ischemia for 40 min, followed by reperfusion for 4 h, myocytes were returned to normal medium without inhibitors for 24 h, then were examined for apoptotic markers. Compared with I/R alone ('I/R', Figure 4b), the presence of 15 mM TPEN at the start of reperfusion for only B20 min ('TPEN (20 min)') markedly inhibited I/R injury at 24 h after TPEN removal, suggesting that the reperfusion-induced initial Zn 2 þ i release (Figure 3b) is important in I/R injury.…”

“…In contrast, when Zn 2 þ /pyrithione was added for only B20 min at the beginning of reperfusion, it had little protective effect for both cardiomyocytes and H9c2 cells (Figures 4a and b; Supplementary Figures S2a and b). Inclusion of any of three potent flavonoids or U0126 for 4 h in the reperfusion solution again had a marked cardioprotective effect for at least 24 h (Figures 4a and b; Supplementary Figures S2a and b), while Ca 2 þ -free medium (inhibition of I/R-induced Ca 2 þ i overload), 5 CsA (an mPTP inhibitor), 13 diazoxide (an mK ATP opener), 7 or MK886 (a selective 5-LOX inhibitor 8 ) were less protective (Figures 4a and b).…”

Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse, a Model of Leigh Syndrome

“…Indeed, the impact of charge modification on VDAC channel function has been shown previously as VDAC treatment with succinic anhydride, which increases the negative charge of the channel, eliminates VDAC voltage-dependence and changes its selectivity from anion to cation (79). Further, VDAC phosphorylation leading to increased negative charge was shown to decrease the conductance of the channel, though the modified residue(s) were not identified (80,81). It is also possible that the association of VDAC with modulatory proteins is altered when VDAC is succinated; an increase in the negative charge of the protein could e.g.…”