VDAC phosphorylation Author Manuscript NIH Public Access ... · phosphorylation of downstream targets to physiologic effects on mitochondrial function. We propose that cardioprotection

Glycogen Synthase Kinase 3 (GSK-3) Inhibition SlowsMitochondrial Adenine Nucleotide Transport and RegulatesVDAC phosphorylation

Samarjit Das1, Renee Wong2, Nishadi Rajapakse3, Elizabeth Murphy2, and CharlesSteenbergen11 Department of Pathology, Johns Hopkins University, Baltimore, MD 212052 Cardiac Physiology Section, Vascular Medicine Branch, NHLBI, NIH, Bethesda, MD 208923 Laboratory of Signal Transduction, NIEHS, NIH, RTP, NC 27712

AbstractInhibition of GSK-3 reduces ischemia-reperfusion injury by mechanisms that involve themitochondria. The goal of this study was to explore possible molecular targets and mechanisticbasis of this cardioprotective effect. In perfused rat hearts, treatment with GSK inhibitors prior toischemia significantly improved recovery of function. To assess the effect of GSK inhibitors onmitochondrial function under ischemic conditions, mitochondria were isolated from rat heartsperfused with GSK inhibitors and treated with uncoupler or cyanide, or made anoxic. GSKinhibition slowed ATP consumption under these conditions, which could be due to inhibition ofATP entry into the mitochondria through VDAC and/or ANT or to inhibition of the F1F0-ATPase.To determine the site of the inhibitory effect on ATP consumption, we measured the conversion ofADP to AMP by adenylate kinase located in the intermembrane space. This assay requires adeninenucleotide transport across the outer but not the inner mitochondrial membrane, and we found thatGSK inhibitors slow AMP production similar to their effect on ATP consumption. This suggeststhat GSK inhibitors are acting on outer mitochondrial membrane transport. In sonicatedmitochondria, GSK inhibition had no effect on ATP consumption or AMP production. In intactmitochondria, cyclosporin A had no effect, indicating that ATP consumption is not due to openingof the mitochondrial permeability transition pore. Since GSK is a kinase, we assessed whetherprotein phosphorylation might be involved. Therefore, we performed western blot and 1D/2D gelphosphorylation site analysis using phos-tag staining to indicate proteins that had decreasedphosphorylation in hearts treated with GSK inhibitors. LC/MS analysis revealed one of theseproteins to be VDAC2. Taken together, we found that GSK mediated signaling modulatestransport through the outer membrane of the mitochondria. Both proteomics and adeninenucleotide transport data suggest that GSK regulates VDAC and suggest that VDAC may be animportant regulatory site in ischemia-reperfusion injury.

KeywordsMitochondria; GSK-3; VDAC; adenine nucleotide transport

Correspondence: Charles Steenbergen, M.D., Ph.D., Johns Hopkins University, Baltimore, MD 21205, Tel: (410) 502-5982, Fax:(410) 502-5862, Email: [email protected] OF INTEREST: None

NIH Public AccessAuthor ManuscriptCirc Res. Author manuscript; available in PMC 2009 October 24.

Published in final edited form as:Circ Res. 2008 October 24; 103(9): 983–991. doi:10.1161/CIRCRESAHA.108.178970.

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INTRODUCTIONIschemic heart disease is a major cause of morbidity and mortality worldwide. This hasmotivated a search for new approaches to reduce ischemic injury, propelled by the discoveryof ischemic preconditioning (IPC) (1). Considerable data suggest that IPC leads to therelease of adenosine, opioids, or bradykinin (2,3), which bind to G-protein coupled receptorsand initiate a signaling cascade that involves activation of phosphatidylinositol-3 kinase(PI-3 Kinase) and downstream kinases such as Akt, eNOS, PKC, mTOR, p70S6-Kinase,ERK, and GSK-3β (4–6).

Glycogen Synthase Kinase-3 (GSK-3), first identified as a regulator of glycogenmetabolism, is also an important regulator of cell function, including gene expression, cellcycle, survival and apoptosis (7). GSK-3 belongs to the serine/threonine kinase family ofproteins and the two known isoforms are GSK-3α (51 kDa) and GSK-3β (47 kDa), whichshare 98% homology and similar substrate specificities.

The activity of GSK-3β is regulated by phosphorylation (7). GSK-3β is active under normalresting conditions when it is not phosphorylated, and phosphorylation at serine 9 leads to itsinactivation. The activity of GSK-3β is regulated by several signaling pathways as a numberof kinases (Akt, PKC, PKA, and others) can phosphorylate GSK-3β on serine 9 andinactivate it.

In addition to its role in cell survival, proliferation and differentiation, GSK-3β has beenproposed to be involved in the cardioprotective effects of IPC (8,9) and various forms ofpharmacologic cardioprotection (10–14). Our laboratory (8) previously demonstrated thatthe cardioprotection elicited by IPC is mediated by the inhibition of GSK via PI3 Kinase. Ithas also been reported that opioid-induced cardioprotection occurs via inactivation ofGSK-3β by phosphorylation at serine 9 through PI3 Kinase and TOR-dependent pathways(11). Protective effects have been observed when GSK-3β inhibitors were administeredimmediately prior to ischemia (8), 24 hours prior to ischemia (13), or immediately uponreperfusion (11,14). GSK inhibition is also protective during heart failure (15).

Recently GSK-3β has been described in mitochondria, and GSK inhibition is thought toblock opening of the mitochondrial permeability transition pore in cardiomyocytes (10).However, the molecular targets of GSK-3β in mitochondria and the precise physiologiceffects of GSK-3β inhibition on mitochondrial function have not been fully elucidated.

The present study was designed to identify downstream targets of GSK-3 and to link thephosphorylation of downstream targets to physiologic effects on mitochondrial function. Wepropose that cardioprotection resulting from inhibition of GSK-3 is partly mediated bymodulation of mitochondrial bioenergetics.

MATERIALS AND METHODSAnimals

Male Sprague-Dawley rats were used in this study. All experimental procedures wereapproved by the Institutional Animal Care and Use Committee of Johns Hopkins University.

Langendorff Rat Heart PreparationHearts were perfused as described previously (16). Details are provided in the supplement.

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Isolated Mitochondria ProtocolsMitochondria were isolated as described previously (16). The supplement provides details ofthe assays.

ProteomicsDetails of the Western Blots, 1D/2D gel electrophoresis, and phospho-protein detection areprovided in the supplement.

RESULTSGSK-3 inhibitors increase recovery of contractile function after ischemia

We previously reported that GSK-3 inhibitors improved recovery of function and reducednecrosis following ischemia. In this study, we examined the mechanisms involved, afterconfirming the protective effects of two GSK-3 inhibitors, SB216763 and SB415286. Dose-response experiments were performed to establish effective concentrations. As shown inFigure 1, either 3 μM SB216763 or 10 μM SB415286 significantly enhanced recovery ofleft ventricular developed pressure (LVDP) during reperfusion, to 48.9 ± 6.2% and 56.8 ±8.1% of baseline compared with 34.7 ± 7.5% in control hearts (p< 0.05). Previous workfrom our laboratory (8) and others (11,13–14) has shown that GSK-3 inhibitors also reducenecrosis.

GSK-3 inhibitors reduce the rate of ATP consumption in isolated mitochondriaWe previously found that addition of recombinant Bcl-2 to mitochondria resulted in reducedbreakdown of ATP under de-energized conditions when the mitochondrial F1F0-ATPaseruns in reverse (16). This reduced rate of ATP consumption was attributed to altered ATPtransport into the mitochondria. To determine whether GSK-3 inhibitors also decreased ATPconsumption under de-energized conditions, we studied mitochondria rapidly de-energizedusing the uncoupler, dinitrophenol. Under these conditions, mitochondria from hearts treatedwith GSK inhibitors consumed less ATP than control mitochondria (Figure 2A). Similarly,GSK inhibitors significantly reduced ATP consumption when sodium cyanide was used tostop mitochondrial respiration (Figure 2B). In the physiologically relevant model ofmitochondrial de-energization, mitochondria were allowed to consume all of the oxygen inan oxygraph chamber, and then the consumption of ATP during the ensuing anoxic periodwas measured. ATP consumption was linear in both control and GSK inhibitor treatedmitochondria, and after 60 minutes of anoxia, the amount of ATP consumed wassignificantly less in the GSK inhibitor treated mitochondria (Figure 2C). Mitochondria fromischemically preconditioned myocardium showed a similar slowing of ATP consumption(Supplemental Figure 1). This slowing of ATP consumption could be accomplished byattenuating ATP entry into the mitochondria through VDAC and/or the adenine nucleotidetransporter (ANT) or by inhibition of the mitochondrial F1F0-ATPase.

To determine whether this effect is due to inhibition of the mitochondrial F1F0-ATPase,mitochondria were sonicated to disrupt the mitochondria and expose the F1F0-ATPase. Wefound (figure 3A) that ATPase activity in the sonicated mitochondrial fragments was similarin the presence or absence of uncoupler, as expected if we had fully disrupted themitochondria, and GSK inhibitors had no effect on ATP consumption by the sonicatedmitochondrial fragments. Only in the intact mitochondria was there an inhibitory effect ofGSK inhibitors. Thus the effect of GSK inhibitors does not appear to be due to directinhibition of F1F0-ATPase activity.

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The effect of GSK-3 inhibitors on mitochondrial adenine nucleotide metabolism is not dueto a direct effect on the mitochondrial permeability transition pore

To examine whether the effect of GSK inhibitors on adenine nucleotide metabolism is dueto a direct effect on the mitochondrial permeability transition pore (mPTP), we examined theeffect of cyclosporin A (CsA) on ATP consumption under de-energized conditions. Toestablish that an effective dose of CsA was being used, we measured mitochondrial swellinginduced by calcium, and we found that 200 μM CsA markedly reduced mitochondrialswelling in response to calcium compared to control. However, this concentration of CsAhad no effect on adenine nucleotide transport (figure 3B). Thus GSK inhibitors areinhibiting transport through physiologic transport mechanisms, and there is no evidence thatadenine nucleotide entry into the mitochondria during early anoxia is related to mPTPopening. However, inhibiting physiologic transport could indirectly protect the mitochondriaand reduce the probability of mPTP opening as injury progresses.

GSK-3 inhibitors reduce adenine nucleotide transport through the outer mitochondrialmembrane

To further explore the mechanism whereby GSK inhibitors slow ATP consumption underde-energized conditions, an assay was developed that isolated effects of outer mitochondrialmembrane transport from effects on ANT or the F1F0-ATPase. This assay took advantage ofadenylate kinase, which resides in the intermembrane space. Under de-energized conditions,comparable to the conditions used to measure mitochondrial ATP consumption but in thepresence of oligomycin to block matrix adenine nucleotide metabolism, ADP was added toisolated mitochondria, and AMP generation was measured. This assay is independent ofANT or F1F0-ATPase activity; it relies solely on VDAC to transport ADP into themitochondria, adenylate kinase to convert ADP into AMP and ATP, and VDAC to transportAMP out of the mitochondria. If the effect of GSK inhibitors is on adenine nucleotidetransport across the outer mitochondrial membrane, then there should be less ADPconsumed and less AMP produced in the mitochondria treated with GSK inhibitorscompared to control. There is significantly less ADP consumed (Figure 4A) and less AMPproduced (Figure 4B) in the GSK inhibitor treated mitochondria, as compared to theuntreated mitochondria. In a separate series of experiments, we assessed the effect of GSKinhibitors on adenylate kinase activity in sonicated mitochondria where the outer membranewould be disrupted, and GSK inhibitors had no effect on ADP consumption or AMPproduction (right side, Figure 4A 4B). The data suggest that GSK inhibitors do not inhibitadenylate kinase and that the ATP-sparing effect of GSK inhibitors is related to decreasedadenine nucleotide transport across the outer mitochondrial membrane.

GSK-3 inhibitors reduce phosphorylation of VDACSince the functional data suggest that GSK inhibitors are slowing adenine nucleotidetransport through the outer mitochondrial membrane, and VDAC is the channel that isresponsible for this transport, we explored the effect of GSK inhibitors on VDACphosphorylation. We used several approaches. First, we used an antibody that recognizesproteins that are phosphorylated at serine/threonine residues of Akt substrates, and we didwestern blotting to determine if GSK inhibition affects protein phosphorylation. Weobserved a dramatic change in a protein band at ~32 kD. As shown in figure 5A (top), thereis much less phosphorylation of this band when the hearts were treated with a GSK inhibitorthan in untreated hearts. The molecular weight of VDAC is 32kD. The gel was stripped andre-probed with antibody to VDAC, and the phosphorylated protein overlaps with VDAC,and as shown in figure 5A (bottom), there is the same amount of VDAC in each lane. Nextwe performed 2D gel electrophoresis to verify this finding when proteins are betterseparated. As shown in figure 5B, the location of VDAC in the 2D gel was established withVDAC antibody (upper panels), and phosphorylation was assessed using phospho-Akt

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substrate antibodies (lower panels). The lower panels show VDAC phosphorylation in thecontrol extracts on the left but not in the GSK inhibitor treated extracts on the right. Toexamine this further, we used Phos-tag to identify phosphorylated proteins, and weperformed 1D as well as 2D gel electrophoresis. In the 1D gels, the GSK inhibitorssignificantly reduced protein phosphorylation in the 32 kD region. To validate this finding,2D gel electrophoresis was performed with narrow pH range strips that focused the VDACregion more clearly, and then stained with Phos-tag. Figure 6 shows several spots that hadhigh levels of phosphorylation in untreated heart extracts but very little phosphorylation inGSK inhibitor treated heart extracts. Mass spectrometry demonstrated that VDAC2 ispresent in all 3 spots in the 2D gel.

VDAC can be phosphorylated by either Akt or GSK-3βTo determine whether Akt or GSK-3β can phosphorylate VDAC in vitro, we partiallypurified VDAC and performed an in vitro kinase assay using recombinant active Akt andrecombinant GSK-3β. VDAC was partially purified using a hydroxyapatite/celite column asused by others (17). We then performed an in vitro kinase assay, and measured the extent ofphosphorylation using Pro-Q Diamond staining. Although there was some endogenousphosphorylation, this was further increased by either Akt or GSK-3β (figure 7A). We alsoexamined the ability of recombinant active Akt to phosphorylate VDAC using isolatedmitochondria. Recombinant Akt added to the medium, in the presence of ATP, increasedphosphorylation of the ~32 kD protein band (figure 7B). Thus external Akt canphosphorylate the protein, indicating that the phosphorylation site is on the outside of themitochondria, consistent with the location of VDAC.

GSK-3 inhibitors increase Bcl-2 binding to mitochondriaPrevious work (16) had demonstrated that cardiac overexpression of Bcl-2 protects the heartfrom ischemia-reperfusion injury, and this protection is associated with inhibitedmitochondrial ATP consumption under de-energized conditions and with binding of Bcl-2 toVDAC. To determine if GSK inhibitors are protective, at least in part, by enhancing Bcl-2binding to VDAC, cell fractionation experiments were performed, and the amount of Bcl-2in the mitochondrial and cytosolic fractions were determined by western blotting. GSKinhibition causes a significant loss of Bcl-2 from the cytosol and a significant increase in themitochondrial fraction (figure 8A). This indicates that binding of Bcl-2 to mitochondrialtargets increases in the presence of GSK inhibitors. To test whether this increased binding ofBcl-2 to mitochondria is specific binding to VDAC, we added equal amounts ofrecombinant Bcl-2 to GSK inhibitor treated mitochondria and untreated mitochondria,immunoprecipitated VDAC, and measured the amount of Bcl-2 that was bound to VDAC.As shown in figure 8B, there was significantly more Bcl-2 bound to VDAC in the GSKinhibitor treated mitochondria. Thus phosphorylation of VDAC may affect the bindingaffinity for Bcl-2, which may regulate outer mitochondrial membrane transport.

DISCUSSIONPreviously, our group has demonstrated that ischemic preconditioning results inphosphorylation and inactivation of GSK-3β and that this is mediated by the PI3-kinasepathway (8). Furthermore, pretreatment with GSK-3 inhibitors is approximately asprotective as ischemic preconditioning. Others (11–14) have shown that GSK-3β is involvedin a variety of forms of pharmacologic preconditioning, and the GSK-3β inhibitors areprotective when added at the start of reperfusion (11,14). This suggests that GSK-3β mayplay a central role in a final pathway of cardioprotection, as suggested by Sollott (10).Furthermore, previous work has suggested that the effects of GSK-3β inhibition are

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primarily focused on the mitochondria, although the molecular target has not beendetermined.

The present study suggests a possible molecular target for GSK-3β in the mitochondria thatmay be involved in cardioprotection, and a possible mechanism whereby protection isachieved. The molecular target appears to be VDAC in the outer mitochondrial membraneand phosphorylation may be a mechanism for regulating VDAC activity, either directly orby altering the binding of Bcl-2. There are 3 VDAC isoforms in mammalian cells (18–20),which are primarily responsible for transport of molecules up to 5000 kD across the outermitochondrial membrane. Transport of anions is highly voltage sensitive with highconductance at low voltages and low conductance when voltage is increased, eitherpositively or negatively. In contrast, cation conductance is relatively voltage insensitive.Nucleotides such as ATP are readily permeable when VDAC is in the open state, and arevirtually impermeant when VDAC is closed. Ablation of the mouse VDAC1 gene results inaltered mitochondrial sensitivity for ADP in muscle, but since VDAC2 and VDAC3 are alsopresent at high levels in the heart, the mice are viable (21). Mice deficient for VDAC3 areviable, but deficiency of VDAC2 is embryologically lethal. Our proteomic studies suggestthat it is VDAC2 that is a primary GSK target in the outer mitochondrial membrane.

Several investigators have suggested that VDAC can be an important regulator ofmitochondrial function. Under conditions of anoxia, there is evidence of inhibition ofmitochondrial anion exchange (22). Others (23–24) have found evidence for VDACregulation of mitochondrial function under conditions that stimulate apoptosis. In theapoptosis studies, it has been suggested that early in the apoptotic program, the outermitochondrial membrane becomes impermeable to small molecules such as ATP andcreatine phosphate, and that this leads to the eventual loss of outer mitochondrial membraneintegrity and cytochrome c release, which can be prevented by anti-apoptotic Bcl-2 familymembers (23–24). Although the conditions and mechanisms are different in our study, thefindings support the concept that VDAC transport can be regulated, and plays a role in cellinjury. Under conditions leading to apoptosis, there is some controversy concerning Bcl-2regulation of VDAC. Some reports suggest that during apoptosis, VDAC closes and thatBcl-2 is protective by maintaining VDAC in an open configuration (23–24), whereas otherssuggest that Bcl-2 promotes VDAC closure (25). This could be related to the specificexperimental conditions or the timing of the measurements. Regardless of the controversy,our data as well as the apoptosis literature support the concept that altered VDACconductance can be involved in the evolution of cell injury.

Although our data suggest that VDAC phosphorylation under the control of GSK-3 is animportant mechanism that alters mitochondrial function under energy deficient conditions,others (26) suggest that mitochondrial protein phosphorylation is not involved in theprotective effect of ischemic preconditioning. One might expect that ischemicpreconditioning and GSK inhibition would involve many of the same mechanisms sinceischemic preconditioning inhibits GSK through phosphorylation. However, this thoroughstudy by Clarke et al (26) was performed on extracts following mitochondrial purification,and we found that we had to use whole-tissue extracts, immediately placed in detergent, tosee the phosphorylation clearly. If we purified the mitochondria and then prepared extracts,the phosphorylation was barely detectable. This suggests that the phosphorylation is highlylabile, presumably because the phosphatases must be separated from the substrate rapidly topreserve the phosphorylated state. To minimize this problem, we used a rapid isolationprocedure for preparing mitochondria and analyzing mitochondrial function. It is possiblethat even under these conditions, our mitochondrial functional assays are underestimatingthe magnitude of the effect that is present in situ.

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Functional Consequences of GSK Signaling in the MitochondriaOne of the downstream effects of GSK inhibition is delayed opening of the mitochondrialpermeability transition pore (mPTP) in response to oxygen radicals (10), and opening of themPTP has been suggested to be a causative event in cell death during myocardial ischemia-reperfusion injury (27–28). This effect on mPTP opening could be a direct or indirect effectof GSK inhibition. Since VDAC does not appear to be necessary for mPTP formation (29),it seems unlikely that altered phosphorylation of VDAC would necessarily prevent mPTPformation. Also the studies of Sollott’s group show that mPTP opening occurs even in thepresence of GSK inhibitors, just more slowly (10). In accordance with these observations,our present study has also revealed that cyclosporin A treatment does not change the rate ofATP hydrolysis following de-energization, indicating an effect independent of mPTPopening. Alternatively, GSK inhibitors could alter mitochondrial function in a way thatmakes the mPTP less likely to form or open, possibly by limiting the factors that are knownto activate the mPTP, oxidant stress and high calcium concentrations (30). If GSK inhibitorseither reduced mitochondrial calcium uptake, reduced endogenous oxygen radicalproduction, or both, this could account for the ability of GSK inhibitors to slow mPTPopening. The importance of calcium loading in mPTP opening in situ has been questioned(31) and ROS may be a more important factor in intact tissue.

One mechanism whereby GSK inhibitors could reduce mitochondrial calcium loading wouldbe by inhibiting ATP influx through VDAC during ischemia. During ischemia, ATP isproduced by anaerobic glycolysis, and this ATP can enter the mitochondria and behydrolyzed by the matrix F1F0-ATPase. This is a significant ATP consuming process; whenischemic myocardium is treated with oligomycin, ATP consumption slows (16,32), by asmuch as 50%. Although inhibition of this process may be protective by preserving ATP, thisis not likely to be its major effect. Rather, mitochondrial ATP hydrolysis is used to preservethe mitochondrial membrane potential (ΔΨ), and direct measurement of ΔΨ in isolatedmyocytes subjected to simulated ischemia (33) shows that ΔΨ falls much more rapidly inthe presence of oligomycin than in its absence, indicating that the matrix F1F0-ATPase isusing cytosolic ATP to slow the decay of ΔΨ. Since ΔΨ is the primary driving force forcalcium uptake into the mitochondria, it is not surprising that there is virtually completeelimination of mitochondrial calcium uptake in isolated myocytes subjected to simulatedischemia in the presence of oligomycin, whereas mitochondrial calcium uptake is observedunder similar conditions in the absence of oligomycin (33). Although this calcium uptakemay be transient and may not directly open the mPTP, it could facilitate mPTP opening inresponse to other stimuli.

Another possible mechanism whereby inhibition of VDAC transport by GSK inhibitors maybe protective under anoxic conditions, is by reducing oxygen radical production during earlyreperfusion (34). Agents that reduce ΔΨ by a small amount are cardioprotective, such asmitochondrial K-ATP channel openers, presumably because production of oxygen radicalsis enhanced when the electrochemical proton gradient is high (35). However, there is noconsensus that a decrease in ΔΨ is critical for the protective effect of K-ATP channelopeners. A decrease in ΔΨ during ischemia could result in less oxygen radical productionduring early reperfusion, which would reduce the probability of mPTP opening. Therefore,GSK inhibitors could be protective by allowing ΔΨ to decrease during ischemia, reducingmitochondrial calcium loading during ischemia and reducing oxygen radical productionduring reperfusion, both of which would tend to reduce the probability of mPTP openingand thereby be protective. Others have suggested that the protective effect of ischemicpreconditioning involves suppression of oxidant stress (26). These effects on calcium andoxidant stress would not be direct effects on the mPTP but rather effects on mPTPactivators.

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One interesting aspect of this study is that we found that GSK inhibitors reduced VDACphosphorylation using an antibody that detects proteins phosphorylated at Akt sites. Thiscould be because the antibody is not entirely specific for Akt phosphorylation sites and alsorecognizes other serine/threonine phosphorylations. However, it is also possible that VDACis phosphorylated at multiple sites. GSK-3β is known to prefer substrates that have alreadybeen phosphorylated by other kinases (9,36–37). Thus it is possible that physiologicregulation of VDAC is achieved through a dual phosphorylation mechanism involving Aktas the priming kinase and GSK-3β as the secondary kinase that is required for regulation ofVDAC function. An alternative possibility is that VDAC is phosphorylated by Akt and thatGSK-3β regulates the phosphatase that dephosphorylates VDAC. If GSK-3β inactivates thephosphatase that dephosphorylates VDAC, then GSK inhibitors could decrease VDACphosphorylation by increasing the activity of the phosphatase. Further characterization of thephosphorylation sites of VDAC will be necessary to resolve this issue.

Although dephosphorylation of VDAC by GSK inhibition may be protective by alteringchannel conductance directly, an alternative mechanism would involve differential proteinbinding to the phosphorylated versus dephosphorylated VDAC. In a previous study (16), wefound that Bcl-2 overexpression inhibited mitochondrial ATP consumption under de-energized conditions, similar to what we report here with GSK inhibitors. One possibleexplanation for the similar effect of Bcl-2 overexpression and GSK inhibition is that animportant mechanism of VDAC regulation involves Bcl-2 binding, and this binding can beincreased either by overexpression or by altering affinity, and one mechanism for alteringaffinity may be through phosphorylation. Data presented here suggests that Bcl-2 affinity formitochondria and for VDAC specifically, is increased by GSK inhibitors, consistent with aregulatory role for VDAC phosphorylation. Others have provided convincing evidence thatBcl-2 binding to a component of the mPTP is critical for cardioprotection, and that GSK-3 isa critical regulator of this interaction (31). An alternate possibility is that binding ofhexokinase to VDAC could have important regulatory properties (38). The binding ofhexokinase to VDAC appears to require activated Akt (39), although it is not clear ifphosphorylation plays a role.

CONCLUSIONSThe data suggest that VDAC is an important site of regulation of mitochondrial metabolismand function under de-energized conditions. GSK inhibition decreases adenine nucleotideentry through the outer mitochondrial membrane, most likely through VDAC, under anoxicor similar conditions. This reduction in adenine nucleotide transport is likely to result in adecrease in ΔΦ, less mitochondrial calcium loading during anoxic conditions, and lessendogenous oxygen radical production during reoxygenation/reperfusion. Furthermore, GSKinhibitors alter the phosphorylation status of VDAC and alter the affinity for Bcl-2, whichmay also contribute to the decrease in transport through VDAC. All of these factors, lesscalcium loading, less oxidant stress, and more Bcl-2 binding, likely contribute to thecardioprotective effect of GSK inhibitors.

AcknowledgmentsSupported in part by NIH grant HL39752. RW, NR, and EM were supported by the NIH intramural program.

References1. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in

ischemic myocardium. Circulation 1986;74:1124–1136. [PubMed: 3769170]

Das et al. Page 8


NIH


NIH


NIH


2. Gross ER, Gross GJ. Ligand triggers of classical preconditioning and postconditioning. CardiovascRes 2006;70:212–221. [PubMed: 16448635]

3. Downey JM, Davis AM, Cohen MV. Signaling pathways in ischemic preconditioning. Heart FailRev 2007;12:181–188. [PubMed: 17516169]

4. Tong H, Rockman HA, Koch WJ, Steenbergen C, Murphy E. G protein-coupled receptorinternalization signaling is required for cardioprotection in ischemic preconditioning. Circ Res2004;94:1133–1141. [PubMed: 15031261]

5. Hausenloy DJ, Yellon DM. Reperfusion injury salvage kinase signaling: taking a RISK forcardioprotection. Heart Fail Rev 2007;12:217–234. [PubMed: 17541822]

6. Liem DA, Honda MH, Zhang J, Woo D, Ping P. Past and present course of cardioprotection againstischemia-reperfusion injury. J Appl Physiol 2007;103:2129–2136. [PubMed: 17673563]

7. Kockeritz L, Doble B, Patel S, Woodgett JR. Glycogen Synthase Kinase-3 – An overview of anover-achieving protein kinase. Curr Drug Targets 2006;7:1377–1388. [PubMed: 17100578]

8. Tong H, Imahashi K, Steenbergen C, Murphy E. Phosphorylation of glycogen synthase kinase-3βduring preconditioning through a phosphatidylinositol-3-kinase-dependent pathway iscardioprotective. Circ Res 2002;90:377–379. [PubMed: 11884365]

9. Murphy E, Steenbergen C. Inhibition of GSK-3β as a target for cardioprotection: the importance oftiming, location, duration, and degree of inhibition. Expert Opin Ther Targets 2005;9:447–456.[PubMed: 15948666]

10. Juhaszova M, Zorov DB, Kim SH, Pepe S, Fu Q, Fishbein KW, Ziman BD, Wang S, Ytrehus K,Antos CL, Olson EN, Sollott SJ. Glycogen synthase kinase-3β mediates convergence of protectionsignaling to inhibit the mitochondrial permeability transition pore. J Clin Invest 2004;113:1535–1549. [PubMed: 15173880]

11. Gross ER, Hsu AK, Gross GJ. Opioid-induced cardioprotection occurs via glycogen synthasekinase β inhibition during reperfusion in intact rat hearts. Circ Res 2004;94:960–966. [PubMed:14976126]

12. Nishihara M, Miura T, Miki T, Sakamoto J, Tanno M, Kobayashi H, Ikeda Y, Ohori K, TakahashiA, Shimamoto K. Erythropoietin affords additional cardioprotection to preconditioned hearts byenhanced phosphorylation of glycogen synthase kinase-3β. Am J Physiol Heart Circ Physiol2006;291:H748–H755. [PubMed: 16565311]

13. Gross ER, Hsu AK, Gross GJ. Delayed cardioprotection afforded by the glycogen synthase kinase3 inhibitor SB-216763 occurs via a KATP- and MPTP-dependent mechanism at reperfusion. Am JPhysiol 2008;294:H1497–H1500.

14. Gross ER, Hsu AK, Gross GJ. GSK3 inhibition and KATP channel opening mediate acute opioid-induced cardioprotection at reperfusion. Bas Res Cardiol 2007;102:341–349.

15. Hirotani S, Zhai P, Tomita H, Galeotti J, Marquez JP, Go S, Hong C, Yatani A, Avila J, SadoshimaJ. Inhibition of glycogen synthase kinase 3β during heart failure is protective. Circ Res2007;101:1164–1174. [PubMed: 17901358]

16. Imahashi K, Schneider MD, Steenbergen C, Murphy E. Transgenic expression of Bcl-2 modulatesenergy metabolism, prevents cytosolic acidification during ischemia, and reduces ischemia/reperfusion injury. Circ Res 2004;95:734–741. [PubMed: 15345651]

17. Lai JC, Tan W, Benimetskaya L, Miller P, Colombini M, Stein CA. A pharmacologic target ofG3139 in melanoma cells may be the mitochondrial VDAC. Proc Natl Acad Sci U S A2006;103:7494–7499. [PubMed: 16648253]

18. Columbini M. VDAC: The channel at the interface between mitochondria and the cytosol. MolCell Biochem 2004;256/257:107–115.

19. Blachly-Dyson E, Zambronicz EB, Yu WH, Adams V, McCabe ERB, Adleman J, Columbini M,Forte M. Cloning and functional expression in yeast of two human isoforms of the outermitochondrial membrane channel, the voltage-dependent anion channel. J Biol Chem1993;268:1835–1841. [PubMed: 8420959]

20. Sampson MJ, Lovell RS, Davison DB, Craigen WJ. A novel mouse mitochondrial voltage-dependent anion channel gene localizes to chromosome 8. Genomics 1996;36:192–196. [PubMed:8812436]

Das et al. Page 9


NIH


NIH


NIH


21. Anflous K, Armstrong DD, Craigen WJ. Altered mitochondrial sensitivity for ADP andmaintenance of creatine-stimulated respiration in oxidative striated muscles from VDAC1-deficient mice. J Biol Chem 2001;276:1954–1960. [PubMed: 11044447]

22. Lemasters JJ, Holmuhamedov E. Voltage-dependent anion channel (VDAC) as mitochondrialgovernator--thinking outside the box. Biochim Biophys Acta 2006;1762:181–190. [PubMed:16307870]

23. Vander Heiden MG, Li XX, Gottleib E, Hill RB, Thompson CB, Colombini M. Bcl-xL promotesthe open configuration of the voltage-dependent anion channel and metabolite passage through theouter mitochondrial membrane. J Biol Chem 2001;276:19414–19419. [PubMed: 11259441]

24. Vander Heiden MG, Chandel NS, Li XX, Schumacker PT, Colombini M, Thompson CB. Outermitochondrial membrane permeability can regulate coupled respiration and cell survival. Proc NatAcad Sci USA 2000;97:4666–4671. [PubMed: 10781072]

25. Shimizu S, Narita M, Tsujimoto Y. Bcl-2 family proteins regulate the release of apoptogeniccytochrome c by the mitochondrial channel VDAC. Nature 1999;399:483–487. [PubMed:10365962]

26. Clarke SJ, Khaliulin I, Das M, Parker JE, Heesom KJ, Halestrap AP. Inhibition of mitochondrialpermeability transition pore opening by ischemic preconditioning is probably mediated byreduction of oxidative stress rather than mitochondrial protein phosphorylation. Circ Res 2008;102in press.

27. Di Lisa F, Menabò R, Canton M, Barile M, Bernardi P. Opening of the mitochondrial permeabilitytransition port causes depletion of mitochondrial and cytosolic NAD+ and is a causative event inthe death of myocytes in postischemic reperfusion of the heart. J Biol Chem 2001;276:2571–2575.[PubMed: 11073947]

28. Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore opening duringmyocardial reperfusion – a target for cardioprotection. Cardiovasc Res 2004;61:372–385.[PubMed: 14962470]

29. Baines CP, Kaiser RA, Sheiko T, Craigen WJ, Molkentin JD. Voltage-dependent anion channelsare dispensable for mitochondrial-dependent cell death. Nat Cell Biol 2007;9:550–555. [PubMed:17417626]

30. Griffiths EJ, Halestrap AP. Mitochondrial non-specific pores remain closed during cardiacischaemia, but open upon reperfusion. Biochem J 1995;307:93–98. [PubMed: 7717999]

31. Juhaszova M, Wang S, Zorov DB, Nuss HB, Gleichmann M, Mattson MP, Sollott SJ. The identityand regulation of the mitochondrial permeability transition pore. Where the known meets theunknown. Ann N Y Acad Sci 2008;1123:197–212. [PubMed: 18375592]

32. Jennings RB, Reimer KA, Steenbergen C. Effect of inhibition of the mitochondrial ATPase on netmyocardial ATP in total ischemia. J Mol Cell Cardiol 1991;23:1383–1395. [PubMed: 1839801]

33. Ruiz-Meana M, Garcia-Dorado D, Miró-Casas E, Abellán A, Soler-Soler J. Mitochondrial Ca2+uptake during simulated ischemia does not affect permeability transition pore opening uponsimulated reperfusion. Cardiovasc Res 2006;71:715–724. [PubMed: 16860295]

34. Holmuhamedov EL, Jahangir A, Oberlin A, Komarov A, Colombini M, Terzic A. Potassiumchannel openers are uncoupling protonophores: implication in cardioprotection. FEBS Lett2004;568:167–170. [PubMed: 15196941]

35. Korshunov SS, Skulachev VP, Starkov AA. High protonic potential actuates a mechanism ofproduction of reactive oxygen species in mitochondria. FEBS Lett 1997;416:15–18. [PubMed:9369223]

36. Fiol CJ, Mahrenholz AM, Wang Y, Roeske RW, Roach PJ. Formation of protein kinaserecognition sites by covalent modification of the substrate. Molecular mechanism for thesynergistic action of casein kinase II and glycogen synthase kinase 3. J Biol Chem1987;262:14042–14048. [PubMed: 2820993]

37. Fiol CJ, Williams JS, Chou CH, Wang QM, Roach PJ, Andrisani OM. A secondaryphosphorylation of CREB341 at Ser129 is required for the cAMP-mediated control of geneexpression. A role for glycogen synthase kinase-3 in the control of gene expression. J Biol Chem1994;269:32187–32193. [PubMed: 7798217]

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38. Pastorino JG, Shulga N, Hoek JB. Mitochondrial binding of hexokinase II inhibits Bax-inducedcytochrome C release and apoptosis. J Biol Chem 2002;277:7610–7618. [PubMed: 11751859]

39. Majewski N, Nogueira V, Bhaskar P, Coy PE, Skeen JE, Gottlob K, Chandel NS, Thompson CB,Robey RB, Hay N. Hexokinase-mitochondria interaction mediated by Akt is required to inhibitapoptosis in the presence or absence of Bax and Bak. Mol Cell 2004;16:819–830. [PubMed:15574336]

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Figure 1.Hearts were perfused for 15 minutes with control buffer, then 15 minutes with GSKinhibitor or vehicle, 20 minutes global ischemia, followed by 40 minutes of reperfusion.Results are Means SEM (n=6). *p<0.05 vs. control.

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Figure 2.GSK inhibitors slow ATP consumption. Panel A, mitochondria were de-energized withdinitrophenol for 5 minutes, in Panel B, mitochondria were treated with cyanide for 20minutes, and in Panel C, mitochondria were anoxic for 60 minutes. Both GSK inhibitorssignificantly decrease the rate of ATP hydrolysis. Results are Means ± SEM (n=6). *p<0.05vs. control.

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Figure 3.Effect of GSK inhibition on mitochondrial adenine nucleotide metabolism. Panel A,mitochondria were de-energized using dinitrophenol, and ATP remaining after 2.5 minutesof incubation was measured. GSK inhibitors have no effect on ATPase activity in sonicatedmitochondria. Panel B, cyclosporin A (200 μM) was added to de-energized mitochondria;this had no effect on ATP hydrolysis, in control and GSK inhibitor groups. Results areMeans ± SEM (n=6). *p<0.05 vs. intact control. #p<0.05 vs. intact SB216763 treated.ζp<0.05 vs. intact+DNP, SB 216763 treated.

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Figure 4.GSK inhibitors slow the rate of ADP consumption (Panel A) and the rate of AMPproduction (Panel B) by cyanide-treated mitochondria. Results are Means ± SEM (n=6).*p<0.05 vs. control.

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Figure 5.Effect of GSK inhibition on phosphorylation of a 32 kD protein. Panel A-top is arepresentative 1D gel showing the amount of 32 kD phosphorylated Akt substrate protein incontrol and GSK inhibitor treated heart extracts. Panel A-middle shows the gel re-probedwith VDAC antibody, showing no significant difference in total VDAC expression. Geldensitometry was performed for quantitation, and the ratio of 32 kD phospho-protein to totalVDAC is plotted. *p<0.05 vs control. In Panel B, whole heart homogenates were furtherseparated using 2D gel electrophoresis. In the upper panels, control homogenate (left) andGSK inhibitor treated homogenate (right) is probed with VDAC antibody. Then themembranes were stripped and reprobed for phosphorylated Akt substate protein (lower

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images). There is phosphorylated protein in the VDAC region in control but not in GSKinhibitor treated homogenate. The region of interest is circled and shown at highermagnification at the bottom.

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Figure 6.Whole heart homogenate protein was separated by 2D gel electrophoresis and stained withphos-tag to detect phosphorylated proteins. Three spots showing marked differences inphosphorylation level, in the 32 kD region (shown in higher magnification), were analyzedby LC-MS, and VDAC-2 was identified by at least 4 peptide fragments, in all three spots.

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Figure 7.Panel A illustrates in vitro phosphorylation of semi-purified VDAC, by Akt and GSK-3β.Panel B shows increased 32 kD Akt substrate phosphorylation in isolated mitochondriafollowing addition of recombinant Akt (rAkt). *p<0.05 vs control.

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Figure 8.Panel A shows the effect of GSK inhibition on Bcl-2 levels in cytosolic and mitochondrialfractions. Panel B shows the effect of GSK inhibition on the amount of Bcl-2 that isimmunoprecipitated by VDAC antibodies. *p<0.05 vs control.

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VDAC phosphorylation Author Manuscript NIH Public Access ... · phosphorylation of downstream targets to physiologic effects on mitochondrial function. We propose that cardioprotection