Eric R. Gross

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

Abstract— 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 δ-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-GSKβ 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&agr; (Ser21 or Tyr279) or phosphorylation of GSKβ (Tyr216). These data indicate that OIC occurs via the phosphorylation of GSKβ at Ser9 during reperfusion.

Heat Shock Protein 90 Mediates the Balance of Nitric Oxide and Superoxide Anion from Endothelial Nitric-oxide Synthase

The balance of nitric oxide (·NO) and superoxide anion (O⨪2) plays an important role in vascular biology. The association of heat shock protein 90 (Hsp90) with endothelial nitric-oxide synthase (eNOS) is a critical step in the mechanisms by which eNOS generates ·NO. As eNOS is capable of generating both ·NO and O⨪2, we hypothesized that Hsp90 might also mediate eNOS-dependent O⨪2 production. To test this hypothesis, bovine coronary endothelial cells (BCEC) were pretreated with geldanamycin (GA, 10 μg/ml; 17.8 μm) and then stimulated with the calcium ionophore,A23187 (5 μm). GA significantly decreasedA23187-stimulated eNOS-dependent nitrite production (p < 0.001, n = 4) and significantly increased A23187-stimulated eNOS-dependent O⨪2production (p < 0.001, n = 8).A23187 increased phospho-eNOS(Ser-1179) levels by >1.6-fold over vehicle (V)-treated levels. Pretreatment with GA by itself or with A23187 increased phospho-eNOS levels. In unstimulated V-treated BCEC cultures low amounts of Hsp90 were found to associate with eNOS. Pretreatment with GA and/or A23187 increased the association of Hsp90 with eNOS. These data show that Hsp90 is essential for eNOS-dependent ·NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eNOS activity and increases eNOS-dependent O⨪2production.

Sarcolemmal and mitochondrial adenosine triphosphate- dependent potassium channels: mechanism of desflurane-induced cardioprotection.

Background Volatile anesthetic–induced preconditioning is mediated by adenosine triphosphate–dependent potassium (KATP) channels; however, the subcellular location of these channels is unknown. The authors tested the hypothesis that desflurane reduces experimental myocardial infarct size by activation of specific sarcolemmal and mitochondrial KATP channels. Methods Barbiturate-anesthetized dogs (n = 88) were acutely instrumented for measurement of aortic and left ventricular pressures. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3-h reperfusion. In four separate groups, dogs received vehicle (0.9% saline) or the nonselective KATP channel antagonist glyburide (0.1 mg/kg intravenously) in the presence or absence of 1 minimum alveolar concentration desflurane. In four additional groups, dogs received 45-min intracoronary infusions of the selective sarcolemmal (HMR 1098; 1 &mgr;g · kg−1 · min−1) or mitochondrial (5-hydroxydecanoate [5-HD]; 150 &mgr;g · kg−1 · min−1) KATP channel antagonists in the presence or absence of desflurane. Myocardial perfusion and infarct size were measured with radioactive microspheres and triphenyltetrazolium staining, respectively. Results Desflurane significantly (P < 0.05) decreased infarct size to 10 ± 2% (mean ± SEM) of the area at risk as compared with control experiments (25 ± 3% of area at risk). This beneficial effect of desflurane was abolished by glyburide (25 ± 2% of area at risk). Glyburide (24 ± 2%), HMR 1098 (21 ± 4%), and 5-HD (24 ± 2% of area at risk) alone had no effects on myocardial infarct size. HMR 1098 and 5-HD abolished the protective effects of desflurane (19 ± 3% and 22 ± 2% of area at risk, respectively). Conclusion Desflurane reduces myocardial infarct size in vivo, and the results further suggest that both sarcolemmal and mitochondrial KATP channels could be involved.

Targeting Aldehyde Dehydrogenase 2: New Therapeutic Opportunities

A family of detoxifying enzymes called aldehyde dehydrogenases (ALDHs) has been a subject of recent interest, as its role in detoxifying aldehydes that accumulate through metabolism and to which we are exposed from the environment has been elucidated. Although the human genome has 19 ALDH genes, one ALDH emerges as a particularly important enzyme in a variety of human pathologies. This ALDH, ALDH2, is located in the mitochondrial matrix with much known about its role in ethanol metabolism. Less known is a new body of research to be discussed in this review, suggesting that ALDH2 dysfunction may contribute to a variety of human diseases including cardiovascular diseases, diabetes, neurodegenerative diseases, stroke, and cancer. Recent studies suggest that ALDH2 dysfunction is also associated with Fanconi anemia, pain, osteoporosis, and the process of aging. Furthermore, an ALDH2 inactivating mutation (termed ALDH2*2) is the most common single point mutation in humans, and epidemiological studies suggest a correlation between this inactivating mutation and increased propensity for common human pathologies. These data together with studies in animal models and the use of new pharmacological tools that activate ALDH2 depict a new picture related to ALDH2 as a critical health-promoting enzyme.

Diabetes Abolishes Morphine-Induced Cardioprotection via Multiple Pathways Upstream of Glycogen Synthase Kinase-3β

The cardioprotective effect of opioids or glycogen synthase kinase (GSK) inhibitors given at reperfusion has not been investigated in diabetes models. Therefore, nondiabetic (NDBR) or streptozotocin-induced diabetic (DBR) rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Groups of NDBR or DBR were administered either vehicle, morphine (0.3 mg/kg), or the GSK inhibitor SB216763 (0.6 mg/kg) 5 min before reperfusion. SB216763 (but not morphine) reduced infarct size in DBRs (44 ± 1* and 55 ± 2%, respectively), while both agents reduced infarct size in NDBRs versus untreated NDBRs or DBRs (44 ± 3*, 42 ± 3*, 60 ± 2, and 56 ± 2%, respectively, *P < 0.001). Morphine-induced phospho- (P-)GSK3β was reduced 5 min after reperfusion in DBRs compared with NDBRs (0.83 ± 0.29 and 1.94 ± 0.12 [P < 0.05] pg/μg tissue, respectively). The GSK3β mediators, P-Akt, P–extracellular signal–related kinase (ERK)1, and P–signal transducer and activator of transcription (STAT)3, were also significantly reduced in untreated DBR compared with NDBR rats. Morphine-induced elevations of P-Akt, P-ERK1, P-p70s6, P–janus-activated kinase-2, and P-STAT3 in NDBRs were also blunted in DBRs. H9C2 cells raised in 25 mmol/l compared with 5.56 mmol/l glucose media also demonstrated reduced morphine-induced P-GSK3β, P-Akt, P-STAT3, and P-ERK1 after 15 min. Hence, acute GSK inhibition may provide a novel therapeutic strategy for diabetic patients during an acute myocardial infarction, whereas morphine is less effective due to signaling events that adversely affect GSK3β.

Inhibition of Cytochrome P450ω-Hydroxylase A Novel Endogenous Cardioprotective Pathway

Cytochrome P450s (CYP) and their arachidonic acid (AA) metabolites have important roles in regulating vascular tone, but their function and specific pathways involved in modulating myocardial ischemia–reperfusion injury have not been clearly established. Thus, we characterized the effects of several selective CYP&ohgr;-hydroxylase inhibitors and a CYP&ohgr;-hydroxylase metabolite of AA, 20-hydroxyeicosatetraenoic acid (20-HETE), on the extent of ischemia–reperfusion injury in canine hearts. During 60 minutes of ischemia and particularly after 3 hours of reperfusion, 20-HETE was produced at high concentrations. A nonspecific CYP inhibitor, miconazole, and 2 specific CYP&ohgr;-hydroxylase inhibitors, 17-octadecanoic acid (17-ODYA) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly inhibited 20-HETE production during ischemia–reperfusion and produced a profound reduction in myocardial infarct size (expressed as a percent of the area at risk) (19.6±1.7% [control], 8.4±2.5% [0.96 mg/kg miconazole], 5.9±2.2% [0.28 mg/kg 17-ODYA], and 10.8±1.8% [0.40 mg/kg DDMS], P<0.05, respectively). Conversely, exogenous 20-HETE administration significantly increased infarct size (26.9±1.9%, P<0.05). Several CYP&ohgr;-hydroxylase isoforms, which are known to produce 20-HETE such as CYP4A1, CYP4A2, and CYP4F, were demonstrated to be present in canine heart tissue and their activity was markedly inhibited by incubation with 17-ODYA. These results indicate an important endogenous role for CYP&ohgr;-hydroxylases and in particular their product, 20-HETE, in exacerbating myocardial injury in canine myocardium. The full text of this article is available online at http://circres.ahajournals.org.

Isoflurane Preconditions Myocardium Against Infarction via Activation of Inhibitory Guanine Nucleotide Binding Proteins

Background Isoflurane-induced myocardial protection during ischemia is mediated by adenosine triphosphate–regulated potassium (KATP) channels; however, the intracellular signal transduction cascade responsible for this process has been incompletely evaluated. The authors tested the hypothesis that isoflurane reduces myocardial infarct size through a Gi protein–mediated process. Methods Forty-eight hours after pretreatment with vehicle (0.9% saline) or the Gi protein inhibitor pertussis toxin (10 &mgr;g/kg intravenously), barbiturate-anesthetized dogs (n = 43) were instrumented for measurement of aortic and left ventricular pressures and maximum rate of increase of left ventricular pressure. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3-h reperfusion. In four separate groups, vehicle- or pertussis toxin–pretreated dogs were studied with or without administration of 1 minimum alveolar concentration isoflurane. In two additional groups, dogs received the direct KATP channel agonist nicorandil (100 &mgr;g/kg bolus and 10 &mgr;g · kg−1 · min−1 intravenous infusion) in the presence or absence of pertussis toxin pretreatment. Myocardial perfusion and infarct size were measured with radioactive microspheres and triphenyltetrazolium staining, respectively. Results Isoflurane significantly (P < 0.05) decreased infarct size to 7 ± 2% of the area at risk compared with control experiments (26 ± 2%). Pertussis toxin pretreatment alone had no effects on myocardial infarct size (31 ± 4%) but blocked the beneficial effects of isoflurane (21 ± 3%). Nicorandil decreased infarct size (11 ± 2%), but, in contrast to isoflurane, this effect was independent of pertussis toxin pretreatment (11 ± 1%). Conclusion Isoflurane reduces myocardial infarct size by a Gi protein–mediated mechanism in vivo.

GSK3β inhibition and KATP channel opening mediate acute opioid-induced cardioprotection at reperfusion

Both glycogen synthase kinase 3β (GSK3β) and the ATP-dependant potassium channel (KATP) mediate opioid-induced cardioprotection (OIC). However, whether direct KATP channel openers induce cardioprotection prior to reperfusion and their signaling cascade position with respect to GSK3β inhibition is unknown. Therefore, we investigated the role of KATP channel opening at reperfusion in OIC, and the interaction between the GSK signaling axis and KATP channels in cardioprotection.Male Sprague-Dawley rats underwent 30 minutes ischemia with 2 hours of reperfusion and infarct size was determined. Rats given the nonselective opioid agonist, morphine (0.3 mg/kg), or the selective delta opioid agonist, BW373U86 (1.0 mg/kg), 5 minutes prior to reperfusion reduced infarct size (40.3±1.6*, 39.7±1.9* versus 60.0±1.1%, respectively, * P<0.001%). This protection was abrogated with prior administration of the putative sarcolemmal KATP antagonist, HMR-1098 (6 mg/kg), or the putative mitochondrial KATP antagonist, 5-HD (10 mg/kg). The putative sKATP channel opener, P-1075 (1μg/kg) or the putative mKATP channel opener, BMS-191095 (1 mg/kg) given 5 minutes prior to reperfusion also reduced infarct size (41.8±2.4*, 43.4±1.4*) and protection was abrogated by prior administration of the PI3k inhibitor wortmannin (60.0±1.7, 64.0±2.6%, respectively, * P<0.001). Cardioprotection afforded by the GSK inhibitor SB216763 (0.6 mg/kg) given 5 minutes prior to reperfusion was also partially blocked by either HMR or 5-HD and completely blocked when HMR and 5-HD were given in combination (40.8±1.6*, 50.4±1.6^; 49.4±1.7^, 61.6±1.6%, respectively, * or ^ P<0.001). These data indicate that both the sKATP and mKATP channel are involved in acute OIC and the GSK signaling axis regulates cardioprotection via KATP channel opening.

12-Lipoxygenase in Opioid-Induced Delayed Cardioprotection: Gene Array, Mass Spectrometric, and Pharmacological Analyses

Abstract— 12-Lipoxygenase (12-LO) has been shown to be a factor in acute ischemic preconditioning (IPC) in the isolated rat heart; however, no studies have been reported in delayed PC. We characterized the role of 12-LO in an intact rat model of delayed PC induced by a &dgr;-opioid agonist SNC-121 (SNC). Rats were pretreated with SNC and allowed to recover for 24 hours. They were then treated with either baicalein or phenidone, 2 selective 12-LO inhibitors. In addition, SNC-pretreated rats had plasma samples isolated at different times after ischemia-reperfusion for liquid chromatographic–mass spectrometric analysis of the major metabolic product of 12-LO, 12-HETE. Similar studies were conducted with inhibitors. Gene array data showed a significant induction of 12-LO message (P <0.05) after opioid pretreatment. This induction in 12-LO mRNA was confirmed by real-time polymerase chain reaction, and 12-LO protein expression was enhanced by SNC pretreatment at 24 hours relative to vehicle treatment. Both baicalein and phenidone attenuated the protective effects of SNC pretreatment on infarct size (50±4% and 42±3% versus 29±2%, P <0.05, respectively). No significant differences were observed in 12-HETE concentrations between baseline control and SNC-treated rats. However, 12-HETE concentrations were increased significantly at both 15 minutes during ischemia and at 1 hour of reperfusion in the SNC-treated rats compared with controls. Baicalein and phenidone attenuated the increase in 12-HETE at 1 hour of reperfusion. These data suggest that SNC-121 appears to enhance message and subsequently the activity and expression of 12-LO protein during times of stress, resulting in delayed cardioprotection.

Effect of exogenous kappa-opioid receptor activation in rat model of myocardial infarction.

The involvement of opioid receptor activation during ischemia-reperfusion is somewhat controversial. While it is generally accepted that activation of the &dgr;-opioid receptor (DOR) is cardioprotective, and may indeed be an important mediator of ischemic preconditioning, the role of the &kgr;-opioid receptor (KOR) is less well understood. To this end, we examined three different KOR agonists and their effects upon infarct size and arrhythmia development. Male Sprague-Dawley rats were subjected to 30 minutes of occlusion followed by 90 minutes of reperfusion. Opioid receptor agonists were administered 10 minutes before the onset of ischemia, while the opioid antagonists were given 20 minutes before occlusion. Untreated rats exhibited an infarct size (IS/AAR%) of 52.4 ± 2.7%. Pretreatment with the DOR agonist, BW373U86, limited infarct development to 37.2 ± 1.8%, which was reversed by the selective DOR antagonist, BNTX. All three KOR agonists studied, U50,488, ICI 204,448, and BRL 52537 significantly reduced infarct size to levels comparable to that of BW373U86. The infarct-sparing effects of U50,488 and ICI 204,448 were abolished by the selective KOR antagonist, nor-BNI. Nor-BNI failed to inhibit the cardioprotective effects of BRL 52537. Furthermore, U50,488 and BRL 52537, but not ICI 204,448, significantly reduced the incidence of arrhythmias. These effects were not blocked by nor-BNI. These data demonstrate that KOR activation provides a similar degree of infarct size reduction as DOR activation. KOR agonists also reduced arrhythmogenesis; however, these responses appear to be independent of KOR activation.