Nociceptive-induced myocardial remote conditioning is mediated by neuronal gamma protein kinase C.

Deciphering the remote conditioning molecular mechanism may provide targets to develop therapeutics that can broaden the clinical application. To further investigate this, we tested whether two protein kinase C (PKC) isozymes, the ubiquitously expressed epsilon PKC (εPKC) and the neuronal-specific gamma PKC (γPKC), mediate nociceptive-induced remote myocardial conditioning. Male Sprague-Dawley rats were used for both in vivo and ex vivo myocardial ischemia-reperfusion protocols. For the in vivo studies, using a surgical abdominal incision for comparison, applying only to the abdomen either bradykinin or the εPKC activator (ψεRACK) reduced myocardial infarct size (45 ± 1, 44 ± 2 %, respectively, vs. incision: 43 ± 2 %, and control: 63 ± 2 %, P < 0.001). Western blot showed only εPKC, and not γPKC, is highly expressed in the myocardium. However, applying a selective γPKC inhibitor (γV5-3) to the abdominal skin blocked remote protection by any of these strategies. Using an ex vivo isolated heart model without an intact nervous system, only selective εPKC activation, unlike a selective classical PKC isozyme activator (activating α, ß, ßII, and γ), reduced myocardial injury. Importantly, the classical PKC isozyme activator given to the abdomen in vivo (with an intact nervous system including γPKC) during myocardial ischemia reduced infarct size as effectively as an abdominal incision or ψεRACK (45 ± 1 vs. 45 ± 2 and 47 ± 1 %, respectively). The classical PKC activator-induced protection was also blocked by spinal cord surgical transection. These findings identified potential remote conditioning mimetics, with these strategies effective even during myocardial ischemia. A novel mechanism of nociceptive-induced remote conditioning, involving γPKC, was also identified.

Acute methadone treatment reduces myocardial infarct size via the delta-opioid receptor in rats during reperfusion.

BACKGROUND: Methadone is an opioid agonist often given to manage acute and chronic pain. We sought to determine whether methadone compared with morphine dose dependently reduces myocardial infarct size (IS) and whether the mechanism is delta-opioid receptor mediated. Furthermore, we examined whether myocardial IS reduction varies with the timing of methadone administration or duration of induced ischemia. METHODS: After surgical instrumentation, we divided male Sprague-Dawley rats into 3 sets. The first set was divided into groups, which received methadone (0.03-3 mg/kg), morphine (0.03-3 mg/kg), or water (placebo) 30 min before ischemia. Some animals of the first set also received the delta-opioid antagonist naltrindole (5 mg/kg) before methadone (0.3 mg/kg), morphine (0.3 mg/kg), or placebo administration. The second set of animals was divided into groups that received methadone (0.3 mg/kg) 5 min before reperfusion or 10 s after reperfusion. These 2 sets of animals were subjected to 30 min of myocardial ischemia by left anterior descending coronary artery occlusion and then 2 h of reperfusion. The third set of animals received placebo, methadone (0.3 mg/kg), or morphine (0.3 mg/kg) 5 min before reperfusion and were subjected to 45 min of ischemia by left anterior descending coronary artery occlusion with 2 h of reperfusion. Myocardial IS was assessed by staining myocardial tissue with triphenyltetrazolium chloride and expressed as a percentage of the area at risk (mean +/- sem). RESULTS: Methadone or morphine administered before ischemia reduced myocardial IS. The greatest effect was achieved at a dose of 0.3 mg/kg (methadone, 46% +/- 1%, P < 0.001 and morphine, 47% +/- 1%, P < 0.001 versus placebo, 61% +/- 1%, respectively). Naltrindole (5 mg/kg) blocked methadone-induced (0.3 mg/kg) and morphine-induced (0.3 mg/kg) cardioprotection (naltrindole + methadone, 58% +/- 1%, P < 0.001 versus methadone; and naltrindole + morphine, 58 +/- 1%, P < 0.001 versus morphine). Methadone (0.3 mg/kg) reduced myocardial IS when given 5 min before reperfusion (46% +/- 1%, P < 0.001 versus placebo) but not 10 s after reperfusion (60% +/- 1%, P = 0.675 versus placebo). No significant myocardial IS differences were seen for placebo when comparing the 45-min ischemia group (64% +/- 1%) with the 30-min ischemia group (60% +/- 1%, P = 0.069). The longer ischemia time of 45 min abrogated methadone-induced IS reduction (64% +/- 2%, P = 0.867 versus 45-min ischemia placebo group) and morphine-induced IS reduction (65% +/- 1%, P = 0.836 versus 45-min ischemia placebo group). CONCLUSIONS: These findings demonstrate that methadone and morphine produce similar myocardial IS-sparing effects that are delta-opioid receptor mediated and that are dependent on the duration of myocardial ischemia.

Diabetes abolishes morphine-induced cardioprotection via multiple pathways upstream of glycogen synthase kinase-3beta.

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-)GSK3beta was reduced 5 min after reperfusion in DBRs compared with NDBRs (0.83 +/- 0.29 and 1.94 +/- 0.12 [P < 0.05] pg/microg tissue, respectively). The GSK3beta 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-GSK3beta, 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 GSK3beta.

Opioid-induced cardioprotection occurs via glycogen synthase kinase beta inhibition during reperfusion in intact rat hearts.

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 delta-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 microg/kg) or LY294002 (0.3 mg/kg) or the TOR inhibitor rapamycin (3 microg/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-GSKbeta 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 GSKalpha (Ser21 or Tyr279) or phosphorylation of GSKbeta (Tyr216). These data indicate that OIC occurs via the phosphorylation of GSKbeta at Ser9 during reperfusion.

Sarcolemmal K(ATP) channel triggers opioid-induced delayed cardioprotection in the rat.

Recently, the involvement of sarcolemmal K(ATP) (sarcK(ATP)) channels in ischemic and pharmacological preconditioning (IPC and PPC) has been minimized by numerous studies suggesting a primary role for mitochondrial K(ATP) (mitoK(ATP)) channels in early and delayed cardioprotection. Although the mitoK(ATP) channel has clearly been shown to be a distal effector of delayed IPC and PPC, studies implicating it as a trigger of protection in delayed IPC are lacking. Accordingly, we characterized the role of cardiac K(ATP) channels as triggers or distal effectors of delayed cardioprotection induced by opioids in rats, and the data suggest that the sarcK(ATP) channel triggers and that the mitoK(ATP) channel is a distal effector of opioid-induced delayed cardioprotection.

12-lipoxygenase in opioid-induced delayed cardioprotection: gene array, mass spectrometric, and pharmacological analyses.

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 delta-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.

Delta opioid agonists and volatile anesthetics facilitate cardioprotection via potentiation of K(ATP) channel opening.

Opioids and volatile anesthetics produce marked cardioprotective effects against myocardial infarction via the activation of ATP-sensitive potassium (K(ATP)) channels, however, the effect of combined treatment with both drugs is unknown. We examined the hypothesis that opioids and volatile anesthetics potentiate cardiac K(ATP) channel opening, thereby enhancing cardioprotection. Rats were treated with the delta opioid agonists, TAN-67 or BW373U86, or isoflurane, together or alone with and without diazoxide, a mitochondrial K(ATP) channel opener. Glibenclamide, a non-selective K(ATP) channel blocker, was used to further characterize the signaling mechanism involved. Myocardial infarct size (IS) was determined by tetrazolium staining and was expressed as a percent of the area at risk (AAR). High doses of TAN-67 (10 mg/kg), diazoxide (10 mg/kg), and isoflurane (1 MAC) produced a significant reduction in IS compared with the control group (30+/-3%, 36+/-5%, and 42+/-2 vs. 58+/-2%, respectively), whereas lower doses of the drugs had no effect except for the low dose of isoflurane (0.5 MAC). The combination of TAN-67 and diazoxide or isoflurane and diazoxide resulted in a marked reduction in IS compared with controls in the presence of high (9+/-3% and 14+/-3%) and low (17+/-7% and 31+/-7%) dose combinations, respectively. The combination of TAN-67 or BW373U86 and isoflurane also caused a striking reduction in IS/AAR (16+/-7% and 7+/-2%, respectively). To date, this is the first demonstration that opioids and volatile anesthetics work in conjunction to confer protection against myocardial infarction through potentiation of cardiac K(ATP) channel opening.

Morphine Reduces Myocardial Infarct Size via Heat Shock Protein 90 in Rodents.

Opioids reduce injury from myocardial ischemia-reperfusion in humans. In experimental models, this mechanism involves GSK3ß inhibition. HSP90 regulates mitochondrial protein import, with GSK3ß inhibition increasing HSP90 mitochondrial content. Therefore, we determined whether morphine-induced cardioprotection is mediated by HSP90 and if the protective effect is downstream of GSK3ß inhibition. Male Sprague-Dawley rats, aged 8-10 weeks, were subjected to an in vivo myocardial ischemia-reperfusion injury protocol involving 30 minutes of ischemia followed by 2 hours of reperfusion. Hemodynamics were continually monitored and myocardial infarct size determined. Rats received morphine (0.3 mg/kg), the GSK3ß inhibitor, SB216763 (0.6 mg/kg), or saline, 10 minutes prior to ischemia. Some rats received selective HSP90 inhibitors, radicicol (0.3 mg/kg), or deoxyspergualin (DSG, 0.6 mg/kg) alone or 5 minutes prior to morphine or SB216763. Morphine reduced myocardial infarct size when compared to control (42 ± 2% versus 60 ± 1%). This protection was abolished by prior treatment of radicicol or DSG (59 ± 1%, 56 ± 2%). GSK3ß inhibition also reduced myocardial infarct size (41 ± 2%) with HSP90 inhibition by radicicol or DSG partially inhibiting SB216763-induced infarct size reduction (54 ± 3%, 47 ± 1%, resp.). These data suggest that opioid-induced cardioprotection is mediated by HSP90. Part of this protection afforded by HSP90 is downstream of GSK3ß, potentially via the HSP-TOM mitochondrial import pathway.

COX-2 and iNOS in opioid-induced delayed cardioprotection in the intact rat.

Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) have been previously implicated in the late phase of cardioprotection associated with opioid-induced and ischemic preconditioning (IPC) in conscious rabbits and COX-2 in isolated rat hearts pretreated with an exogenous delta opioid agonist. However, it is not know if both iNOS and COX-2 mediate the late phase of cardioprotection induced by opioids in the intact blood-perfused rat. Therefore, we investigated the role of COX-2 and iNOS in the delayed phase of protection mediated by delta opioid receptor activation. Rats were pretreated 24 hours prior to an occlusion/reperfusion protocol with the selective non-peptide delta opioid agonists, BW373U86 (BW) and SNC-121 (SNC). NS-398, a selective COX-2 inhibitor was administered after the 24-hour recovery period just prior to index ischemia. The selective iNOS inhibitors, S-methylthiourea (SMT) and aminoguanidine (AG), were administered in conjunction with opioid pretreatment or were also given 24 hours after opioid administration just prior to index ischemia. COX-2 inhibition by NS-398 given 24 hours after opioid administration attenuated the protective effects of both BW and SNC (46 +/- 6 vs. 13 +/- 3 and 51 +/- 5 vs. 29 +/- 2, p < 0.001, respectively). Similarly, inhibition of iNOS following 24 hours of treatment with opioids also attenuated the protective effects of BW and SNC. However, the delayed protective effects of the opioids were not attenuated by pretreatment with the iNOS inhibitors 24 hours prior to the infarct protocol. These results suggest that both COX-2 and iNOS are mediators of delayed protection induced by non-peptide delta opioid agonists. It appears that the trigger effect is not dependent on the activity of iNOS or COX-2 but the late phase of cardioprotection is dependent on the upregulation of these enzymes.

Delayed cardioprotection afforded by the glycogen synthase kinase 3 inhibitor SB-216763 occurs via a KATP- and MPTP-dependent mechanism at reperfusion.

Previous studies in our laboratory suggest that an acute inhibition of glycogen synthase kinase 3 (GSK3) by SB-216763 (SB21) is cardioprotective when administered just before reperfusion. However, it is unknown whether the GSK inhibitor SB21 administered 24 h before ischemia is cardioprotective and whether the mechanism involves ATP-sensitive potassium (K(ATP)) channels and the mitochondrial permeability transition pore (MPTP). Male Sprague-Dawley rats were administered the GSK inhibitor SB21 (0.6 mg/kg) or vehicle 24 h before ischemia. Subsequently, the rats were acutely anesthetized with Inactin and underwent 30 min of ischemia and 2 h of reperfusion followed by infarct size determination. Subsets of rats received either the sarcolemmal K(ATP) channel blocker HMR-1098 (6 mg/kg), the mitochondrial K(ATP) channel blocker 5-hydroxydecanoic acid (5-HD; 10 mg/kg), or the MPTP opener atractyloside (5 mg/kg) either 5 min before SB21 administration or 5 min before reperfusion 24 h later. The infarct size was reduced in SB21 compared with vehicle (44 +/- 2% vs. 61 +/- 2%, respectively; P < 0.01). 5-HD administered either before SB21 treatment or 5 min before reperfusion the following day abrogated SB21-induced protection (54 +/- 4% and 61 +/- 2%, respectively). HMR-1098 did not affect the SB21-induced infarct size reduction when administered before the SB21 treatment (43 +/- 1%); however, HMR-1098 partially abrogated the SB21-induced infarct size reduction when administered just before reperfusion 24 h later (52 +/- 1%). The MPTP opening either before SB21 administration or 5 min before reperfusion abrogated the infarct size reduction produced by SB21 (61 +/- 2% and 62 +/- 2%, respectively). Hence, GSK inhibition reduces infarct size when given 24 h before the administration via the opening K(ATP) channels and MPTP closure.

GSK3beta inhibition and K(ATP) channel opening mediate acute opioid-induced cardioprotection at reperfusion.

Both glycogen synthase kinase 3beta (GSK3beta) and the ATP-dependant potassium channel (K(ATP)) mediate opioid-induced cardioprotection (OIC). However, whether direct K(ATP) channel openers induce cardioprotection prior to reperfusion and their signaling cascade position with respect to GSK3beta inhibition is unknown. Therefore, we investigated the role of K(ATP) channel opening at reperfusion in OIC, and the interaction between the GSK signaling axis and K(ATP) 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 K(ATP) antagonist, HMR-1098 (6 mg/kg), or the putative mitochondrial K(ATP) antagonist, 5-HD (10 mg/kg). The putative sK(ATP) channel opener, P-1075 (1microg/kg) or the putative mK(ATP) 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 sK(ATP) and mK(ATP) channel are involved in acute OIC and the GSK signaling axis regulates cardioprotection via K(ATP) channel opening.

Darbepoetin alfa protects the rat heart against infarction: dose-response, phase of action, and mechanisms.

Erythropoietin is known to stimulate red cell production and has recently been shown to protect the heart against injury from ischemia/reperfusion. However, it is unknown whether darbepoetin alfa (Dpa), a long-acting analog of erythropoietin, can play a protective role against myocardial infarction. We assessed the potential protective role of Dpa in an in vivo rat model of myocardial ischemia/reperfusion and the underlying mechanisms. We found that a single intravenous Dpa treatment immediately before 30 minutes of regional ischemia reduced myocardial necrosis following 120 minutes of reperfusion in a dose-dependent manner. Optimal protection with Dpa against myocardial infarction was manifest at a dose of 2.5 microg/kg. Dpa conferred cardioprotection when administered after the onset of ischemia and at the start of reperfusion. Dpa (2.5 microg/kg) also reduced infarct size and Troponin I leakage 24 hours after reperfusion. Inhibition of p42/44 MAPK (PD98059), p38 MAPK (SB203580), mitochondrial ATP-dependent potassium (KATP) channels (5-HD), sarcolemmal KATP channels (HMR 1098), but not phosphatidylinositol-3 (PI3) kinase/Akt (Wortmannin and LY 294002) abolished Dpa-induced cardioprotection. Dpa confers immediate and sustained cardioprotection in rats, suggesting a potential therapeutic role of this long-acting erythropoietin analog for the treatment of acute myocardial infarction.

The JAK/STAT pathway is essential for opioid-induced cardioprotection: JAK2 as a mediator of STAT3, Akt, and GSK-3 beta.

We examined the role for the JAK/STAT signaling pathway in acute opioid-induced cardioprotection (OIC) and whether opioid-induced glycogen synthase kinase-3beta (GSK-3 beta) inhibition is mediated by the JAK/STAT pathway. Rats underwent 30 min of ischemia and either 5 min or 2 h of reperfusion, followed by tissue isolation for molecular analysis or infarct size assessment, respectively. Rats were treated with vehicle, morphine (300 microg/kg), the delta-opioid agonist fentanyl isothiocynate (FIT, 10 microg/kg), or the GSK inhibitor SB-216763 (SB21, 600 microg/kg) 10 min before ischemia. Five minutes before opioid or SB21 treatment, some rats received the putative JAK2 inhibitor AG-490 (3 mg/kg) or the putative JAK3 inhibitor ZM-449829 (3 mg/kg). H9C2 cardiomyoblast cells were also used to investigate FIT-induced signaling (1 microM) in vitro via molecular analysis. Morphine induced the phosphorylation of JAK2, yet not JAK1, in the area at risk. Morphine, FIT, and SB21 also reduced infarct size compared with vehicle (water) when administered before ischemia [43.0 +/- 2.8, 39.1 +/- 3.1, and 42.1 +/- 2.5 (*P < 0.001, respectively) vs. 58.1 +/- 1.3%, respectively]. AG-490 abrogated OIC, whereas ZM-449829 had no effect on OIC. Cardioprotection was afforded by SB21 even in the presence of AG-490. Morphine phosphorylated STAT3, Akt, and GSK-3beta, and phosphorylation was abrogated by AG-490. FIT stimulation of H9C2 cells also caused a time-dependent phosphorylation of STAT3, Akt, and GSK-3beta, and this effect was abrogated by AG-490. STAT3 phosphorylation was also dependent on phosphatidylinositol 3-kinase (PI3K) activation in both tissue and H9C2 cells. These data suggest that OIC occurs via the JAK2 regulation of PI3K pathway-dependent STAT3, Akt, and GSK-3 beta, with GSK-3 beta contributing a central role in acute OIC.

Extending the cardioprotective window using a novel delta-opioid agonist fentanyl isothiocyanate via the PI3-kinase pathway.

Selective delta-opioid agonists produce delayed cardioprotection that lasts for 24-48 h in rats; however, the maximum length of the cardioprotective window is unclear. In this study, we attempted to prolong the cardioprotective window using a unique delta-opioid agonist, fentanyl isothiocyanate (FIT), which binds irreversibly to the delta-receptor, and determined the role of the phosphatidylinositol 3-kinase (PI3K) pathway as a trigger or end effector of FIT-induced cardioprotection. Initially, male rats were administered FIT (10 microg/kg) 10 min before hearts were subjected to 30 min of ischemia and 2 h of reperfusion followed by infarct size (IS) assessment. Acute FIT administration reduced IS when given before ischemia, 5 min before reperfusion, or 10 s after reperfusion compared with control. IS reduction also occurred following a single dose of FIT at 48, 72, 96, and 120 h after administration vs. control, with the maximum effect observed at 96 h. FIT-induced IS reduction at 96 h was completely abolished when the irreversible PI3K inhibitor wortmannin (15 microg/kg) was given before FIT during the trigger phase; however, the effect was only partially abrogated when wortmannin was given 96 h later. These data suggest that FIT has a prolonged cardioprotective window greater than that of any previously described cardioprotective agent that requires PI3K primarily in the trigger phase but also partially, as a mediator or end effector.

Sarcolemmal KATP channel triggers delayed ischemic preconditioning in rats.

Previous work from our laboratory has shown that the sarcolemmal K(ATP) channel (sK(ATP)) is required as a trigger for delayed cardioprotection upon exogenous opioid administration. We also established that the mitochondrial K(ATP) (mK(ATP)) channel is not required for triggering delayed delta-opioid-induced infarct size reduction. Because mechanistic differences have been found among delta-opioids and that due to ischemic preconditioning (IPC), we determined whether the triggering mechanism of delayed IPC-induced infarct size reduction involves either the sK(ATP) or mK(ATP). Male Sprague-Dawley rats received either sham surgery or IPC (3- to 5-min cycles of ischemia and reperfusion) 24 h before being subjected to 30 min of ischemia and 2 h of reperfusion. Infarct size was determined and expressed as a percentage of the area at risk, with significance compared with sham reported at P

Acute aspirin treatment abolishes, whereas acute ibuprofen treatment enhances morphine-induced cardioprotection: role of 12-lipoxygenase.

Patients suffering an acute myocardial infarction routinely receive morphine and nonsteroidal anti-inflammatory drugs (NSAIDs) alone or in combination. However, the importance of the dose, timing, or the combined administration of both on infarct size reduction has not been assessed. Additionally, it is not known whether morphine or NSAIDs require 12-lipoxygenase (12-LO) to mediate infarct size reduction as found previously for ischemic preconditioning. Male Sprague-Dawley rats were subjected to 30 min of ischemia and 2 h of reperfusion, followed by infarct size assessment (mean +/- S.E.M.%, **P < 0.01). Morphine (0.3 mg/kg), ibuprofen (3 mg/kg), but not aspirin (3 mg/kg) reduced infarct size when administered 5 min before reperfusion compared with vehicle (42.3 +/- 1.5**, 40.8 +/- 2.8**, 60.7 +/- 2.3 versus 59.1 +/- 1.7%, respectively); however, none of these agents reduced infarct size when administered 10 s after reperfusion. Ibuprofen (3 mg/kg) administered with morphine (0.3 mg/kg) reduced infarct size (43.7 +/- 1.3%**), whereas aspirin (1 and 3 mg/kg) abolished morphine-induced infarct size reduction. Morphine (0.2 mg/kg) and ibuprofen (0.6 mg/kg) given at doses not effective individually reduced infarct size when given together (59.0 +/- 1.4, 57.6 +/- 2.8, and 43.9 +/- 1.6%**, respectively). Morphine- and ibuprofen-induced infarct size reduction was abolished by the 12-LO inhibitor baicalein (3 mg/kg) and mimicked by the 12-LO metabolite 12-(S)-hydroxyeicosa-5Z,8Z,10Z,14Z-tetraenoic acid (45.2 +/- 2.5%**). These data suggest that morphine and ibuprofen reduce infarct size individually or at subthreshold doses in combination by 12-LO when administered 5 min before reperfusion. Furthermore, acute aspirin administration has a detrimental interaction with morphine that abrogates morphine-induced infarct size reduction.

Cardioprotection at a distance: mesenteric artery occlusion protects the myocardium via an opioid sensitive mechanism.

Preconditioning in remote organs protects the myocardium; however, mediators of the protection remain unknown. Protection of the heart is linked to opioids; therefore, we hypothesized that mesenteric preconditioning (MPC) releases endogenous opioids that protect the myocardium from ischemic injury. In an intact rat model of myocardial infarction, all rats underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion. Prior to coronary artery occlusion, control rats were subjected to sham surgery in which the mesenteric artery was isolated but not occluded both with and without naloxone (10mg/kg) pretreatment. Experimental groups underwent isolation and occlusion of the mesenteric artery for 15 min followed by a 10 min reperfusion period with and without naloxone pretreatment. At the end of 2 h of coronary reperfusion, myocardial infarct size (IS) was determined by tetrazolium staining and expressed as a percent of the area at risk (AAR). Control rats had an IS/AAR of 57.3+/-2. MPC resulted in a significant reduction in infarct size compared to controls (32.2+/-3, P<0.001). Pretreatment with naloxone significantly attenuated the protective effects of MPC (53.8+/-4, P<0.0002). Therefore, it appears that MPC releases endogenous opioids that protect the myocardium from ischemic injury.

PKC-delta inhibition does not block preconditioning-induced preservation in mitochondrial ATP synthesis and infarct size reduction in rats.

We have previously demonstrated that cardioprotection induced by the infusion of a selective delta1-opioid agonist is mediated by the specific translocation of PKC-delta to the mitochondria in in vivo rat hearts and via opening of the mitochondrial KATP channel. Ischemic preconditioning (IPC) is also thought to involve the translocation of specific isoforms of PKC and KATP channel activation. Therefore, we utilized the PKC-delta selective antagonist, rottlerin, to assess the effect of inhibition of this isozyme on cardioprotection induced by one-cycle of IPC prior to 30 minutes of ischemia and 2 hours of reperfusion. Infarct size (IS) was determined by tetrazolium chloride staining and expressed as a percent of the area at risk (AAR). Non-preconditioned control animals had an IS/AAR of 59.7 +/- 1.6. IPC significantly reduced the extent of myocardial infarction (6.3 +/- 1.4). Rottlerin, 0.3 mg/kg, did not alter IS/AAR in control animals (55.0 +/- 5.6), and had no significant effect on IS/AAR in preconditioned animals (14.4 +/- 3.8). Additionally, we demonstrated, using a luciferase-based assay to determine the rate of ATP synthesis and state of mitochondrial bioenergetics, that IPC preserves ATP synthesis in the ischemic myocardium and that this preservation is attenuated by the isoform non-selective PKC inhibitor, chelerythrine, but not by the delta-selective antagonist, rottlerin. These data suggest that PKC-delta does not play an important role in IPC and that differences in isoform importance are evident during pharmacological versus ischemia-induced preconditioning.

Cytochrome P450 omega-hydroxylase inhibition reduces infarct size during reperfusion via the sarcolemmal KATP channel.

Inhibition of 20-hydroxyeicosatrienoic acid (20-HETE), by pretreatment with pharmacological inhibitors of cytochrome P450 (CYP) omega-hydroxylase, has been shown to reduce infarct size in canines when administered prior to ischemia. However, it is unknown whether these agents reduce infarct size when administered just prior to reperfusion and if the sarcolemmal and/or mitochondrial K(ATP) channels (sK(ATP) and mK(ATP)) contribute to cardioprotection. Therefore, we determined whether specific CYP inhibitors for epoxygenases and omega-hydroxylases are cardioprotective when given either prior to ischemia or prior to reperfusion and furthermore, if selective inhibition of the sK(ATP) by HMR-1098 or mK(ATP) by 5-hydroxydecanoic acid (5-HD) could abrogate this effect. Male Sprague-Dawley rats underwent 30 minutes of ischemia followed by 2 hours of reperfusion. Groups received either miconazole (MIC, non-selective CYP inhibitor, 3 mg/kg), 17-octadecynoic acid (17-ODYA, CYP omega-hydroxylase inhibitor, 0,3 or 3 mg/kg), N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS, CYP omega-hydroxylase inhibitor, 0,4 or 4 mg/kg), N-methanesulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH, CYP epoxygenase inhibitor, 3 mg/kg), or vehicle either 10 minutes prior to ischemia or 5 minutes prior to reperfusion. Rats also received either HMR-1098 (6 mg/kg) or 5-HD (10 mg/kg) 10 minutes prior to reperfusion, with subsets of rats also receiving either MIC or 17-ODYA 5 minutes prior to reperfusion. DDMS and 17-ODYA dose dependently reduced infarct size. Rats treated with MIC, 17-ODYA and DDMS, but not MS-PPOH, produced comparable reductions in infarct size when administered prior to ischemia or reperfusion compared to vehicle. HMR-1098, but not 5-HD, also blocked the infarct size reduction afforded by MIC and 17-ODYA. These data suggest a novel cardioprotective pathway involving CYP omega-hydroxylase inhibition and subsequent activation of the sK(ATP) channel during reperfusion.