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Integrative Physiology

Opioid-Induced Cardioprotection Occurs via Glycogen Synthase Kinase ß Inhibition During Reperfusion in Intact Rat Hearts

Eric R. Gross, Anna K. Hsu, Garrett J. Gross

From the Medical College of Wisconsin, Department of Pharmacology and Toxicology, Milwaukee, Wis.

Correspondence to Garrett J. Gross, PhD, Medical College of Wisconsin, Department of Pharmacology and Toxicology, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail ggross{at}mcw.edu

	Abstract

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Glycogen synthase kinase (GSK) inhibition produced by ischemic preconditioning has been previously shown to be regulated through phosphatidylinositol-3 kinase (PI3K). Therefore, we determined whether opioid-induced cardioprotection (OIC) occurs during reperfusion by altering GSK phosphorylation through PI3K and target of rapamycin (TOR). Furthermore, we determined if selective GSK inhibitors, SB216763(SB21) or SB415286(SB41), emulate OIC. Rats were treated with the nonselective opioid agonist, morphine (MOR, 0.3 mg/kg), the -selective opioid agonist BW373U86 (BW, 1 mg/kg), or the GSK inhibitors, SB21 (0.6 mg/kg) or SB41(1.0 mg/kg), either 10 minutes before ischemia or 5 minutes before reperfusion. Five minutes before opioid or SB21 treatment, some rats received either the PI3K inhibitor wortmannin (15 µg/kg) or LY294002 (0.3 mg/kg) or the TOR inhibitor rapamycin (3 µg/kg). After 30 minutes of ischemia followed by 2 hours of reperfusion, infarct size was assessed. MOR, BW, SB41, and SB21 reduced infarct size compared with vehicle when administered before ischemia (42.9±2.6, 40.3±2.3, 46.6±1.6, 42.2±1.8 versus 60.0±1.1%, respectively; P<0.001) and showed similar protection when administered 5 minutes before reperfusion (43.6±2.3, 40.2±2.6, 44.8±2.8, 39.4±0.8%, respectively; P<0.001). Wortmannin, LY294002, and rapamycin were found to inhibit OIC; however, they did not abrogate SB21-induced infarct size reduction. At 5 minutes of reperfusion, both MOR and BW increased P-GSKß at Ser⁹ in the ischemic zone compared with vehicle (181±20, 178±15 versus 75±17 DU, respectively; P<0.05), and this effect was abrogated by prior administration of wortmannin or rapamycin in MOR-treated rats. Furthermore, no differences were seen in phosphorylation of GSK (Ser²¹ or Tyr²⁷⁹) or phosphorylation of GSKß (Tyr²¹⁶). These data indicate that OIC occurs via the phosphorylation of GSKß at Ser⁹ during reperfusion.

Key Words: reperfusion • glycogen synthase kinase • phosphatidylinositol-3 kinase • target of rapamycin • opioids

	Introduction

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Glycogen synthase kinase (GSK) contributes to many cellular processes such as transcription, metabolism, cell division, adhesion, and apoptosis.¹ Abnormal GSK function has been linked to pathophysiological states including Alzheimer’s, diabetes, and cardiac hypertrophy.^2–4 Moreover, selective inhibition of GSK is similar to the effects of ischemic preconditioning (IPC) in isolated rat hearts, with IPC shown to reduce GSKß activity by phosphorylation at the N-terminal serine residue, Ser⁹.⁵

There are two known isoforms of GSK, GSK, and GSKß, with GSK differing from GSKß by an N-terminal glycine-rich tail, which contributes to a difference in molecular weight between GSK (51 kDa) and GSKß (47 kDa). The GSK isoforms are regulated by phosphorylation at Ser²¹ and Tyr²⁷⁹ for GSK and Ser⁹ and Tyr²¹⁶ for GSKß. Both the Ser^21/9 and Tyr^279/216 sites on GSK modulate function in opposing directions, with phosphorylation of Ser^21/9 decreasing GSK activity and phosphorylation of Tyr^279/216 increasing GSK activity.^5,6 Both GSK isoforms have high basal activity and are constitutively activated due to basal phosphorylation of their respective Tyr^279/216 residue, which is different than most other protein kinases that usually have low basal activity. Tyr²¹⁶ phosphorylation also increases in neuronal cells 3 hours after cerebral damage and has been suggested to contribute to a proapoptotic environment.⁷ In contrast, growth factors and insulin have been found to phosphorylate Ser^21/9 in nonmyocardial cells, which results in a decrease in GSK activity and occurs independently of Tyr^279/216 phosphorylation.^8,9

Negative upstream regulators of GSK in nonmyocardial cells include phosphatidylinositol-3 kinase (PI3K), protein kinase C (PKC), target of rapamycin (TOR), and mitogen-activated protein kinase (MAPK).^1,10 PI3K is a pivotal mediator of lipid signaling, which generates phosphatidylinositols, second messengers that activate numerous downstream pathways by interacting with pleckstrin homology domains.¹¹ TOR has been found to regulate p70s6 kinase and eIF-4E BP1 in parallel, which enables TOR to control protein translation.¹²

Previously, our laboratory demonstrated that pretreatment with several opioid agonists produced a cardioprotective effect that occurs via PKC- and MAPK-dependent pathways.^13,14 However, it is unknown whether opioids produce beneficial effects when administered at reperfusion and if opioids require the activation of PI3K and TOR to reduce infarct size. It is also unknown if opioid-induced infarct size reduction occurs by modulation of Ser^21/9 and/or Tyr^279/216 phosphorylation of GSK.

Therefore, we determined whether opioid-induced infarct size reduction requires the PI3K and TOR pathways and if opioids reduce infarct size equally when given before ischemia or before reperfusion. Furthermore, we quantified whether opioids modulate either GSK and GSKß at their respective Ser^21/9 and Tyr^279/216 phosphorylation sites during reperfusion and if this effect could be blocked by PI3K or TOR inhibitors. We also examined whether GSK inhibitors reduce infarct size when administered either before ischemia or before reperfusion and determined if a GSK inhibitor could reduce infarct size in the presence of PI3K or TOR inhibitors.

	Materials and Methods

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The experimental procedures and protocols used in this study were reviewed and approved by the Animal Care and Use Committee of the Medical College of Wisconsin and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Pharmacological Agents
The opioid agonists used for this study were morphine sulfate (Research Biochemicals International, RBI) and BW373U86 (Ardent Pharmaceuticals). Specific inhibitors used included wortmannin (PI3K, Sigma), LY294002 (PI3K, Calbiochem), rapamycin (TOR, NCI), SB216763 (GSK, Tocris), and SB415286 (GSK, Tocris). Morphine sulfate was dissolved in water, whereas all other agents were dissolved in DMSO. All agents and vehicle were administered intravenously via the right jugular vein.

Experimental Protocols
Male Sprague-Dawley rats (250 to 300 g) were obtained from Charles River Laboratories, Wilmington, Mass. An in vivo anesthetized intact rat model was used for these experiments. The general surgical protocol and determination of infarct size have been described previously in detail.¹⁵ After surgical intervention and stabilization, rats were separated into groups.

Infarct Size Studies With Opioids
Rats (n=6 to 8 per group) were subjected to treatment with either the nonselective opioid agonist morphine (0.3 mg/kg), the selective agonist BW373U86 (BW, 1 mg/kg) or vehicle (DMSO) 10 minutes before ischemia. Other groups were pretreated with selective blockers 5 minutes before opioid administration. These included the selective irreversible PI3K inhibitor wortmannin (15 µg/kg), the reversible PI3K inhibitor LY294002 (0.3 mg/kg), or the selective TOR inhibitor rapamycin (3 µg/kg). Morphine (0.3 mg/kg) or BW (1 mg/kg) were also administered 5 minutes before reperfusion in two additional groups.

Western Analysis of GSK
Additional experiments (n=3 to 4 per group) were performed to quantify the extent of GSK phosphorylation at Ser^21/9 and Tyr^279/216 in the presence of opioids or vehicle. Opioids, BW or morphine, were administered 10 minutes before ischemia, and when applicable, pharmacological inhibitors of PI3K and TOR were administered 5 minutes before opioids. Five minutes after reperfusion, hearts were excised, separated into normal and ischemic zones, homogenized in lysis buffer (50 mmol/L HEPES, 150 mmol/L NaCl, 1.5 mmol/L MgCl₂, 1 mmol/L EGTA, 1% Triton X, 10% Glycerol plus 8.6 µmol/L leupeptin, 5.8 µmol/L pepstatin A, 4 mmol/L phenylmethylsulfonyl fluoride (PMSF), 0.6 µmol/L aprotinin, 4 mmol/L sodium fluoride, and 0.8 mmol/L sodium orthovanadate), and centrifuged to remove nuclei and debris at 10 000g for 15 minutes. The supernatant was collected and centrifuged at 100 000g to enrich for the cytosolic fraction. Protein concentration in the cytosolic fraction was determined by the Pierce assay. Protein (35 µg) was loaded onto 10% Tris-HCl gels and after electrophoresis (150 V, 1.5 hours), transferred to a PVDF membrane (50 V, 2 hours). Equal loading of samples was confirmed by Ponceau S staining. Membranes were blocked with 3% bovine serum albumin solution followed by probing overnight with an antibody for either P-Ser or P-Tyr for both GSK and ß (P-Ser^21/9 GSK/ß, 1:1000, Cell Signaling, P-Tyr^279/216 GSK/ß, 1:1000, Upstate), followed by secondary antibody application (1:2500 for P-Ser^21/9 GSK/ß, 1:10 000 for P-Tyr^279/216 GSK/ß) and ECL (Amersham). Protein was detected by X-ray film and densitometry assessed by NIH image 1.62.

Infarct Size Studies With GSK Inhibitors
Rats (n=6 to 8 per group) were subjected to treatment with either the selective GSK inhibitors SB21673 (S21, 0.6 mg/kg), SB415286 (S41, 1.0 mg/kg), or vehicle (DMSO) 10 minutes before ischemia or 5 minutes before reperfusion. One of the two GSK inhibitors, SB21, was also given in the presence of the selective blockers wortmannin (15 µg/kg), LY294002 (0.3 mg/kg), or rapamycin (3 µg/kg), administered 5 minutes before GSK inhibitor administration 10 minutes before ischemia.

Statistical Measurements
All values were denoted as mean±SEM. Statistical significance was determined by performing a one-way ANOVA with Bonferroni’s correction for multiplicity. Values were significantly different from vehicle or groups treated with opioids alone when P<0.001 or P<0.05.

	Results

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Two hundred and seven rats were used to obtain 191 successful experiments. Sixteen rats were excluded due to ventricular fibrillation during reperfusion (8), acidosis (2), the suture breaking during negative staining (3), and anesthetic overdose (3).

Infarct Size Studies With Opioids
Rats treated with morphine or BW 10 minutes before ischemia produced significant and similar reductions in infarct size when compared with vehicle (Figure 1; 42.9±2.6% and 40.3±2.3% versus 60.0±1.1%, respectively; P<0.001). Wortmannin administration before either morphine or BW abolished the reduction in infarct size (Figure 1, top; 54.7±2.3% and 57.7±2.6%, respectively) with similar results obtained for LY294002 administered before morphine or BW (Figure 1, middle; 60.8±1.6% and 61.4±1.4%, respectively). Furthermore, rapamycin administration before morphine or BW also abolished the reduction in infarct size (Figure 1, bottom; 56.6±3.1% and 61.0±1.1%, respectively). Wortmannin, LY294002, or rapamycin had no effect on infarct size when administered alone as compared with vehicle (Figure 1; 57.0±2.1%, 60.5±1.0%, and 58.1±1.7%, respectively). Either morphine or BW administered 5 minutes before reperfusion also reduced infarct size when compared with vehicle (Figure 2; 43.6±2.3% and 40.2±2.6% compared with 60.0±1.1%, respectively; P<0.001), an effect that is similar to that observed when morphine or BW were administered before ischemia (Figure 2: 42.9±2.6% and 40.3±2.3%, respectively; P<0.001).

View larger version (27K): [in this window] [in a new window]   Figure 1. Top, Infarct size expressed as a percent of the area at risk in rats (n=8 per group) treated with vehicle (V) or the opioids morphine (MOR) or BW373U86 (BW), with or without the irreversible PI3K antagonist wortmannin (W). Middle, Infarct size expressed as a percent of the area at risk in rats (n=8 per group) treated with vehicle (V) or the opioids morphine (MOR) or BW373U86 (BW), with or without the reversible PI3K antagonist LY294002 (LY). Bottom, Infarct size expressed as a percent of the area at risk in rats (n=8 per group) treated with vehicle (V) or the opioids morphine (MOR) or BW373U86 (BW), with or without the TOR antagonist rapamycin (R). *Significant differences vs vehicle (P<0.001); +Significant differences vs when opioids administered alone (P<0.001). Values represent mean±SEM.

View larger version (33K): [in this window] [in a new window]   Figure 2. Infarct size expressed as a percent of the area at risk in rats treated with opioids morphine (MOR) or BW373U86 (BW) either 10 minutes before ischemia (n=8 per group, white bars) or 5 minutes before reperfusion (n=6 per group, gray bars). *Significant differences vs vehicle (P<0.001). Values represent mean±SEM.

Western Analysis of GSK With Opioids
Tyr^279/216 GSK Phosphorylation
Western blotting for Tyr^279/216 phosphorylation for GSK and GSKß in tissue obtained from ischemic and normal zones of vehicle-, BW-, or morphine-treated groups at 5 minutes of reperfusion were performed (Figure 3, top). No significant changes were found for P-Tyr²⁷⁹ for GSK in BW- or morphine-treated rats when compared with vehicle (Figure 3, middle; ischemic zone, 113±23 and 108±26 versus 117±25 DU, respectively; normal zone, 105±25 and 99±23 versus 127±26 DU). No significant differences were found for P-Tyr²¹⁶ GSKß expression in tissue obtained from the ischemic or normal zone in BW- or morphine-treated rats when compared with vehicle (Figure 3, bottom; ischemic zone, 143±12 and 154±10 versus 158±14 DU, respectively; normal zone, 145±12 and 127±7 versus 157±10 DU, respectively).

View larger version (35K): [in this window] [in a new window]   Figure 3. Top, Representative Western analysis of tissue samples acquired from the ischemic zone (I) and normal zone (N) 5 minutes after reperfusion in rats treated with vehicle (V, n=3), BW (B, n=4), or morphine (M, n=4) and probed for Tyr279 and Tyr216 phosphorylation of GSK (, Tyr279) and GSKß (, Tyr216), respectively. Middle, Relative densitometries of P-GSK at Tyr279 for ischemic and normal zone tissue at 5 minutes of reperfusion. Bottom, Relative densitometries of P-GSKß at Tyr216 for ischemic and normal zone tissue at 5 minutes of reperfusion. Values represent mean±SEM.

Ser^21/9 GSK Phosphorylation
Western blotting for Ser^21/9 phosphorylation for GSK and GSKß in tissue obtained from ischemic and normal zones of vehicle-, BW-, or morphine-treated groups at 5 minutes of reperfusion was determined (Figure 4, top). No differences were seen in P-Ser²¹ expression for GSK in either the ischemic or normal zone at 5 minutes of reperfusion in morphine- or BW-treated rats when compared with vehicle (Figure 4, middle; ischemic zone, 53±6 and 38±7 versus 47±8 DU, respectively; normal zone, 63±6 and 43±10 versus 50±16 DU, respectively). In contrast, tissue samples obtained from the ischemic zone at 5 minutes of reperfusion in rats treated with morphine or BW showed a significant elevation of P-Ser⁹ for GSKß compared with vehicle (Figure 4, bottom; ischemic zone, 181±20 and 178±15 versus 75±17 DU, respectively; P<0.05). No significant differences were found for P-Ser⁹ GSKß expression in tissue obtained from the normal zone for morphine or BW compared with vehicle (Figure 4, bottom; normal zone, 123±9 and 107±30 versus 88±15 DU, respectively). Prior administration of either wortmannin or rapamycin abrogated morphine-induced phosphorylation of GSKß at Ser⁹ in ischemic zone tissue [Figure 5; 78±25 and 77±37 DU compared to morphine alone 178±15 DU (P<0.05), respectively].

View larger version (35K): [in this window] [in a new window]   Figure 4. Top, Representative Western analysis of tissue samples acquired from the ischemic zone (I) and normal zone (N) 5 minutes after reperfusion in rats treated with vehicle (V, n=3), BW (B, n=4), or morphine (M, n=4) and probed for Ser21 and Ser9 phosphorylation of GSK (, Ser21) and GSKß (ß, Ser9), respectively. Middle, Relative densitometries of P-GSK at Ser21 for ischemic and normal zone tissue at 5 minutes of reperfusion. Bottom, Relative densitometries of P-GSKß at Ser9 for ischemic and normal zone tissue at 5 minutes of reperfusion. #Significant differences vs vehicle (P<0.05). Values represent mean±SEM.

View larger version (33K): [in this window] [in a new window]   Figure 5. Top, Representative Western analysis of tissue samples acquired from the ischemic zone 5 minutes after reperfusion in rats treated with vehicle (V, n=3), morphine (MOR, n=4), wortmannin+morphine (W+MOR, n=3), and rapamycin+morphine (R+MOR, n=3) and probed for Ser9 phosphorylation of GSKß (ß, Ser9). Bottom, Relative densitometries of P-GSKß at Ser9 for ischemic zone tissue at 5 minutes of reperfusion. #Significant differences vs vehicle (P<0.05); &Significant differences vs when opioids are administered alone (P<0.05). Values represent mean±SEM.

Infarct Size Studies With GSK Inhibitors
The GSK inhibitors, SB41 or SB21, reduced infarct size when administered before ischemia when compared with vehicle (Figure 6, 46.6±1.6% and 42.2±1.8% versus 60.0±1.1%, respectively; P<0.001) and SB41 and SB21 also produced a similar reduction in infarct size when administered 5 minutes before reperfusion (Figure 6; 44.8±2.8% and 39.4±0.9%, respectively; P<0.001). Wortmannin, LY294002, or rapamycin given before SB21 had no effect on SB21-induced infarct size reduction (Figure 7; 42.9±2.6%, 42.3±0.8%, and 42.8±2.6%, respectively; P<0.001), whereas wortmannin, LY294002, or rapamycin had no effect on infarct size when given alone as compared with vehicle (Figure 7; 57.0±2.1%, 60.5±1.0%, and 58.1±1.7%, respectively).

View larger version (33K): [in this window] [in a new window]   Figure 6. Infarct size expressed as a percent of the area at risk in rats treated with the GSK inhibitors SB216763 (SB21) or SB415286 (SB41), either 10 minutes before ischemia (n=8 per group, white bars) or 5 minutes before reperfusion (n=6 per group, gray bars). *Significant differences vs vehicle (P<0.001). Values represent mean±SEM.

View larger version (21K): [in this window] [in a new window]   Figure 7. Top, Infarct size expressed as a percent of the area at risk in rats (n=8 per group) treated with vehicle (V) or the GSK inhibitor SB216763 (SB21) with or without the irreversible PI3K antagonist wortmannin (W). Middle, Infarct size expressed as a percent of the area at risk in rats (n=8 per group) treated with vehicle (V) or the GSK inhibitor SB216763 (SB21), with or without the reversible PI3K antagonist LY294002 (LY). Bottom, Infarct size expressed as a percent of the area at risk in rats (n=8 per group) treated with vehicle (V) or the GSK inhibitor SB216763 (SB21), with or without the TOR antagonist rapamycin (R). *Significant differences vs vehicle (P<0.001). Values represent mean±SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
These data are the first to show that both opioids and GSK inhibitors reduce infarct size when administered just before reperfusion. We have also demonstrated for the first time that opioid-induced infarct size reduction occurs through PI3K- and TOR-dependent and ischemia-induced GSKß phosphorylation at Ser⁹. Furthermore, the GSK inhibitor, SB21, reduced infarct size in the presence of PI3K or TOR inhibitors, suggestive that PI3K and TOR are upstream negative regulators of GSK in the heart.

The results of our study demonstrate that GSK inhibition reduces infarct size as previously found in the preconditioned isolated rat heart, and further extends these findings to show that cardioprotection afforded through GSK inhibition occurs during reperfusion.⁵ Interestingly, bradykinin, insulin, and urocortin administered at reperfusion also acutely mediate cardioprotection via the PI3K pathway.^16–19 The importance of PI3K for pharmacological agents to induce cardioprotection likely stems from PI3K-generating phosphatidylinositols (PIPs), which are found to bind to protein pleckstrin homology domains and allow for cellular translocation and protein-protein interactions and/or lipid-protein interactions.²⁰ Additionally, PI3K and TOR have also recently been found to be involved as a trigger of delayed IPC-induced cardioprotection, suggesting the overall importance of these proteins for infarct size reduction.²¹

TOR has been previously found to regulate p70s6 kinase and eIF-4E BP1, two proteins downstream of TOR that are activated in parallel.¹² TOR likely regulated GSK through p70s6, because addition of p70s6 kinase in vitro was found to diminish GSK activity for both GSK isoforms.²² Furthermore, TOR and p70s6 kinase have been suggested to be downstream mediators of PI3K for IPC-induced delayed cardioprotection.²¹ Although both PI3K and TOR pathways are required for opioid-induced infarct size reduction, it was not discerned in this study whether opioids activate TOR dependently or independently of PI3K. PI3K-independent TOR activation has been shown in human gastrocnemius muscle myoblasts by addition of amino acids, which suggests that these two proteins may be activated independently.²³ Further studies will need to be conducted to determine whether PI3K and TOR activation are dependent or independent events that converge at GSK and whether p70s6 kinase is the protein that directly interacts with GSK to result in GSK phosphorylation.

This is also the first study in hearts that has examined opioid-induced modulation of Ser^21/9 and Tyr^279/216 phosphorylation sites of both GSK isoforms. Our findings that GSKß Ser⁹ phosphorylation produced by opioids was localized to the ischemic zone is similar to the regional differences of GSKß phosphorylation found during persistent myocardial stunning.²⁴ Furthermore, no differences were found in P-Tyr^279/216 for GSK and GSKß, respectively, when comparing opioid to vehicle-treated animals for the ischemic zones and normal zones within experiments. This would suggest that opioids, similar to insulin-like growth factors or insulin, selectively modulate GSKß only at Ser⁹.^8,9 Our results also differ from those obtained in neuronal cells after cerebral damage that were found to have an increased amount of P-Tyr²¹⁶ at 3 hours after reperfusion in a middle cerebral artery ischemia/reperfusion model.⁷ Modulation of Tyr²¹⁶ may be more important in signaling proapoptotic pathways once salvage of tissue cannot be obtained. The difference between these results and our study is likely the time point selected for investigation of GSK phosphorylation.

Another intriguing finding in this study is the selectivity of opioids to phosphorylate GSKß at Ser⁹ and not GSK at Ser²¹. The selective phosphorylation of GSKß could perhaps involve PKC, which has been previously found to be important in opioid-induced cardioprotection.¹⁴ In human neuroblastoma cells, PKC directly phosphorylates GSKß, which is dependent on cleavage of PKC by caspase-3.²⁵ In contrast, purified PKC isoforms ₁, ß₁, ß₂, and , but not , from sf9 cells can inactivate GSKß, without affecting GSK.¹⁰ However, PKC may indirectly regulate GSK through PI3K, because induction of a dominant-negative PKC has previously been found to block phorbol ester and insulin induced activity of PI3K.²⁶ Therefore, the selective PKC isoform responsible for modulating GSK and GSKß remains unknown and is an interesting area for future studies.

The two selective GSK inhibitors used in this study, SB21 and SB41, are ATP-dependent and have selectivity and preference for GSK inhibition, without inhibition of Akt, MAPK, p70s6 kinase, or PDK1.²⁷ Inhibition of GSK by either selective inhibitor reduced infarct size to a similar extent as opioids, which further supports our hypothesis that opioids reduce infarct size through GSK inhibition. The selectivity of the GSK inhibitor SB21 was further strengthened by the ability of SB21 to reduce infarct size in the presence of either PI3K or TOR inhibitors and suggests that GSK inhibition is a common mediator of infarct size reduction downstream of both PI3K and TOR.

This study is not without potential limitations, because many of the pharmacological agents used may cause nonspecific effects when applied in vivo. However, the use of two different opioids and GSK inhibitors would suggest our results are valid. Furthermore, wortmannin, LY294002, and rapamycin have been found to be selective inhibitors for PI3K and TOR.²⁸ Although the kinase activity of GSK was not measured in this study, a previous report has demonstrated that changes in phosphorylation at Ser⁹ for GSK produces a decreased activity in isolated rat hearts, with only a 25% reduction of GSKß needed for IPC-induced infarct size reduction to occur, due to the high basal activity of GSKß.⁵

GSK requires a S/T-X-X-X-S/T-P sequence motif, where either the serine or threonine site is phosphorylated (P), or primed, to allow for GSK to interact with proteins on the other S/T amino acid.²⁹ The pleiotropic nature of GSK suggests multiple downstream targets that could induce cardioprotection by modulating the putative GSK substrates eIF2,³⁰ cyclin D1,³¹ glycogen synthase,²⁹ heat shock transcription factor (HSF-1),³² and NFB³³ or yet to be discovered proteins with the GSK sequence motif. These alterations could lead to a change in the cellular state that is more conducive to cardioprotection and are potential future directions of study.

Interestingly, GSKß contributes an essential role for NFB in the regulation of TNF-–induced apoptosis, because GSKß-null mice are embryonic lethal between E13.5 and E14.5.³⁴ This study also displays the isoform selectivity of GSK to regulate NF-B function, because liver-induced apoptosis could not be rescued by GSK. However, in rat primary astrocytes, presence of a constitutively active or phosphorylation-deficient form of GSKß at Ser⁹ results in astrocyte cell death, abrogates NF-B activation, and decreases NF-B–mediated COX-2 expression.³³ Hence, the role of GSKß in regulating NF-B differs between organ systems. Therefore, the cardiovascular role for GSKß regulating NF-B will need to be investigated; however, the essential role for NF-B activation in cardiac cells to reduce infarct size in delayed cardioprotection models may suggest that GSKß negatively regulates NF-B activation as found for astrocytes.³³

Immunohistochemical analysis in cerebral cells has shown that both GSK isoforms associate with the rough endoplasmic reticulum, ribosomes, and microtubules, with GSKß, but not GSK, also associating with the mitochondria.³⁵ The mitochondrial localization of GSKß may suggest that the GSKß pathway may regulate mitochondrial-dependent apoptosis in the heart. Whether the mitochondrial K_ATP channel (mK_ATP) interacts with GSKß either directly or indirectly, due to the mitochondrial localization of GSKß, is also a future area of interest. Oxygen-derived free radicals have previously been implicated as a downstream trigger in the protection afforded by the mK_ATP channel, because the free radical scavenger, N-2-mecaptopropionyl-glycine, abrogates the infarct size reduction afforded by K_ATP channel openers P-1075, diazoxide, or BMS-191095.¹⁵ Generation of oxygen-derived free radicals have been found to activate PI3K and in turn trigger activation of Akt,³⁶ a known upstream modulator of GSK. This suggests a possible link may exist between mitochondrial K_ATP channel opening, free radical generation, and GSKß modulation through a PI3K-dependent pathway.

In summary, our results show that opioid-induced cardioprotection occurs by GSKß inactivation at Ser⁹ through PI3K- and TOR-dependent pathways (Figure 8). The ability for SB21 to reduce infarct size in the presence of either PI3K or TOR inhibitors also suggests that GSK is a downstream target of PI3K and TOR. Our results also demonstrate that opioid-induced infarct size reduction is similar to the infarct size reduction produced by two GSK inhibitors when administered just before ischemia or reperfusion.

View larger version (35K): [in this window] [in a new window]   Figure 8. Proposed mechanism of opioid-induced infarct size reduction. Opioids activate both PI3K and TOR pathways to inactivate GSKß at Ser9. Inactivation may lead to many cellular downstream signaling events that create a cardioprotective phenotype.

	Acknowledgments

 
This work was supported by NIH grant HL08311 (G.J.G.) and an American Heart Predoctoral Fellowship (Northland, E.R.G.). Rapamycin was generously supplied by the National Cancer Institute. The authors would also like to thank Lynda Ludwig for her technical assistance.

	Footnotes

 
Original received September 9, 2003; resubmission received December 23, 2003; revised resubmission received February 4, 2004; accepted February 5, 2004.

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