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Epoxyeicosatrienoic acids (EETs) and their synthetic analogs have cardiovascular protective effects. Here, we investigated the action of a novel EET analog EET-B on the progression of post-myocardial infarction (MI) heart failure in spontaneously hypertensive rats (SHR). Adult male SHR were divided into vehicle- and EET-B (10 mg/kg/day; p.o., 9 weeks)-treated groups. After 2 weeks of treatment, rats were subjected to 30-min left coronary artery occlusion or sham operation. Systolic blood pressure (SBP) and echocardiography (ECHO) measurements were performed at the beginning of study, 4 days before, and 7 weeks after MI. At the end of the study, tissue samples were collected for histological and biochemical analyses. We demonstrated that EET-B treatment did not affect blood pressure and cardiac parameters in SHR prior to MI. Fractional shortening (FS) was decreased to 18.4 ± 1.0% in vehicle-treated MI rats compared with corresponding sham (30.6 ± 1.0%) 7 weeks following MI induction. In infarcted SHR hearts, EET-B treatment improved FS (23.7 ± 0.7%), markedly increased heme oxygenase-1 (HO-1) immunopositivity in cardiomyocytes and reduced cardiac inflammation and fibrosis (by 13 and 19%, respectively). In conclusion, these findings suggest that EET analog EET-B has beneficial therapeutic actions to reduce cardiac remodeling in SHR subjected to MI.

Epoxyeicosatrienoic acids (EETs) decrease cardiac ischemia-reperfusion injury; however, the mechanism of their protective effect remains elusive. Here, we investigated the cardioprotective action of a novel EET analog, EET-B, in reperfusion and the role of hypoxia-inducible factor (HIF)-1α in such action of EET-B. Adult male rats were subjected to 30 min of left coronary artery occlusion followed by 2 h of reperfusion. Administration of 14,15-EET (2.5 mg/kg) or EET-B (2.5 mg/kg) 5 min before reperfusion reduced infarct size expressed as a percentage of the area at risk from 64.3 ± 1.3% in control to 42.6 ± 1.9% and 46.0 ± 1.6%, respectively, and their coadministration did not provide any stronger effect. The 14,15-EET antagonist 14,15-epoxyeicosa-5( Z)-enoic acid (2.5 mg/kg) inhibited the infarct size-limiting effect of EET-B (62.5 ± 1.1%). Similarly, the HIF-1α inhibitors 2-methoxyestradiol (2.5 mg/kg) and acriflavine (2 mg/kg) completely abolished the cardioprotective effect of EET-B. In a separate set of experiments, the immunoreactivity of HIF-1α and its degrading enzyme prolyl hydroxylase domain protein 3 (PHD3) were analyzed in the ischemic areas and nonischemic septa. At the end of ischemia, the HIF-1α immunogenic signal markedly increased in the ischemic area compared with the septum (10.31 ± 0.78% vs. 0.34 ± 0.08%). After 20 min and 2 h of reperfusion, HIF-1α immunoreactivity decreased to 2.40 ± 0.48% and 1.85 ± 0.43%, respectively, in the controls. EET-B blunted the decrease of HIF-1α immunoreactivity (7.80 ± 0.69% and 6.44 ± 1.37%, respectively) and significantly reduced PHD3 immunogenic signal in ischemic tissue after reperfusion. In conclusion, EET-B provides an infarct size-limiting effect at reperfusion that is mediated by HIF-1α and downregulation of its degrading enzyme PHD3. NEW & NOTEWORTHY The present study shows that EET-B is an effective agonistic 14,15-epoxyeicosatrienoic acid analog, and its administration before reperfusion markedly reduced myocardial infarction in rats. Most importantly, we demonstrate that increased hypoxia-inducible factor-1α levels play a role in cardioprotection mediated by EET-B in reperfusion likely by mechanisms including downregulation of the hypoxia-inducible factor -1α-degrading enzyme prolyl hydroxylase domain protein 3.

Background and Purpose In light of the opioid epidemic, physicians are increasingly prescribing non-opioid analgesics to surgical patients. Transient receptor potential vanilloid 1 (TRPV1) inhibitors are potentially alternative pain therapeutics for surgery. Here we examined in rodents whether cardioprotection conferred by two occurrences during surgery, a laparotomy or morphine delivery, is mediated by TRPV1. We further tested whether an experimental analgesic peptide (known as P5) targeted against the TRPV1 C-terminus region interferes with laparotomy- or morphine-induced cardioprotection. Experimental Approach Male Sprague-Dawley rats were subjected to 30 minutes coronary occlusion followed by 120 minutes reperfusion. Before ischaemia, a laparotomy with or without capsaicin application (0.1% cream, a TRPV1 activator) was performed. Additional rats were given morphine (0.3mg·kg-1) with or without capsaicin. Further, capsazepine (3mg·kg-1, a classical TRPV1 inhibitor), or P5 (3mg·kg-1, a peptide analgesic and TRPV1 inhibitor), were given either alone or prior to a laparotomy or morphine administration. Myocardial infarct size was determined. Key Results A laparotomy, in addition to combining a laparotomy with capsaicin cream, reduced infarct size versus control (44±2*%, 40±2*%, vs. 66±1%, n=6, *P < 0.01). Morphine, in addition to combining morphine administration with capsaicin cream, also reduced infarct size versus control (37±3*%, 43±3*%, vs. 61±2%, n=6, *P < 0.01). When TRPV1 inhibitors capsazepine or P5 were given, either TRPV1 inhibitor abolished the infarct size reduction afforded by a laparotomy or morphine. Conclusion and Implications Inhibiting TRPV1 blocks laparotomy- or morphine-induced cardioprotection. Impaired organ protection may be a potential pitfall of using TRPV1 inhibitors for pain control.

Aims Fibroblast growth factor 1 (FGF1), a heparin/heparan sulfate-binding growth factor, is a potent cardioprotective agent against myocardial infarction (MI). The impact of heparin, the standard of care for MI patients entering the emergency room, on cardioprotective effects of FGF1 is unknown, however. Methods and results To address this, a rat model of MI was employed to compare cardioprotective potentials (lower infarct size and improve post-ischemic function) of native FGF1 and an engineered FGF1 (FGF1ΔHBS) with reduced heparin-binding affinity when given at the onset of reperfusion in the absence or presence of heparin. FGF1 and FGF1ΔHBS did not alter heparin's anticoagulant properties. Treatment with heparin alone or native FGF1 significantly reduced infarct size compared to saline (P

Opioids reduce injury from myocardial ischemia-reperfusion in humans. In experimental models, this mechanism involves GSK3βinhibition. HSP90 regulates mitochondrial protein import, with GSK3βinhibition increasing HSP90 mitochondrial content. Therefore, we determined whether morphine-induced cardioprotection is mediated by HSP90 and if the protective effect is downstream of GSK3βinhibition. Male Sprague-Dawley rats, aged 8–10 weeks, were subjected to anin vivomyocardial 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 GSK3βinhibitor, 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%). GSK3βinhibition 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 GSK3β, potentially via the HSP-TOM mitochondrial import pathway.

Background The transient receptor potential vanilloid 1 ( TRPV 1) mediates cellular responses to pain, heat, or noxious stimuli by calcium influx; however, the cellular localization and function of TRPV 1 in the cardiomyocyte is largely unknown. We studied whether myocardial injury is regulated by TRPV 1 and whether we could mitigate reperfusion injury by limiting the calcineurin interaction with TRPV 1. Methods and Results In primary cardiomyocytes, confocal and electron microscopy demonstrates that TRPV 1 is localized to the mitochondria. Capsaicin, the specific TRPV 1 agonist, dose‐dependently reduced mitochondrial membrane potential and was blocked by the TRPV 1 antagonist capsazepine or the calcineurin inhibitor cyclosporine. Using in silico analysis, we discovered an interaction site for TRPV 1 with calcineurin. We synthesized a peptide, V1‐cal, to inhibit the interaction between TRPV 1 and calcineurin. In an in vivo rat myocardial infarction model, V1‐cal given just prior to reperfusion substantially mitigated myocardial infarct size compared with vehicle, capsaicin, or cyclosporine (24±3% versus 61±2%, 45±1%, and 49±2%, respectively; n=6 per group; P TRPV 1 knockout rats. Conclusions TRPV 1 is localized at the mitochondria in cardiomyocytes and regulates mitochondrial membrane potential through an interaction with calcineurin. We developed a novel therapeutic, V1‐cal, that substantially reduces reperfusion injury by inhibiting the interaction of calcineurin with TRPV 1. These data suggest that TRPV 1 is an end‐effector of cardioprotection and that modulating the TRPV 1 protein interaction with calcineurin limits reperfusion injury.

Intestinal microbiota determine severity of myocardial infarction in rats. We determined whether low molecular weight metabolites derived from intestinal microbiota and transported to the systemic circulation are linked to severity of myocardial infarction. Plasma from rats treated for seven days with the non-absorbed antibiotic vancomycin or a mixture of streptomycin, neomycin, polymyxin B and bacitracin was analyzed using mass spectrometry-based metabolite profiling platforms. Antibiotic-induced changes in the abundance of individual groups of intestinal microbiota dramatically altered the host’s metabolism. Hierarchical clustering of dissimilarities separated the levels of 284 identified metabolites from treated vs. untreated rats; 193 were altered by the antibiotic treatments with a tendency towards decreased metabolite levels. Catabolism of the aromatic amino acids phenylalanine, tryptophan and tyrosine was the most affected pathway comprising 33 affected metabolites. Both antibiotic treatments decreased the severity of an induced myocardial infarction in vivo by 27% and 29%, respectively. We then determined whether microbial metabolites of the amino acids phenylalanine, tryptophan and tyrosine were linked to decreased severity of myocardial infarction. Vancomycin-treated rats were administered amino acid metabolites prior to ischemia/reperfusion studies. Oral or intravenous pretreatment of rats with these amino acid metabolites abolished the decrease in infarct size conferred by vancomycin. Inhibition of JAK-2 (AG-490, 10 μM), Src kinase (PP1, 20 μM), Akt/PI3 kinase (Wortmannin, 100 nM), p44/42 MAPK (PD98059, 10 μM), p38 MAPK (SB203580, 10 μM), or KATP channels (glibenclamide, 3 μM) abolished cardioprotection by vancomycin, indicating microbial metabolites are interacting with cell surface receptors to transduce their signals through Src kinase, cell survival pathways and KATP channels. These inhibitors have no effect on myocardial infarct size in untreated rats. This study links gut microbiota metabolites to severity of myocardial infarction and may provide future opportunities for novel diagnostic tests and interventions for the prevention of cardiovascular disease.

Opioids confer cardioprotection after myocardial ischaemia and reperfusion. The primary aim of the present study was to evaluate the cardioprotective effect of different doses of enkephalin analogue Eribis peptide 94 (EP 94) in a porcine model of ischaemia and reperfusion. A secondary aim was to analyse the impact of ischaemia and reperfusion on the expression of opioid receptor subtypes in the porcine heart. Thirty-four anesthetised pigs underwent 40 min of balloon occlusion of the left anterior descending coronary artery followed by four hours of reperfusion. Pigs were given either vehicle (0.9% NaCl) or one of four doses of EP 94 (0.2, 1, 5 or 25 ug/kg at each administration, respectively), intravenously after 26, 33 and 40 min of ischaemia. Hearts were stained to quantify area at risk and infarct size. mRNA and protein expressions of the opioid receptor subtypes were detected with RT-PCR, immunoblotting and immunohistochemistry in the control and ischaemic/reperfused areas. There was a significant dose-response relationship between higher doses of EP 94 and reduced infarct size. Expression of κ- and δ-opioid receptors was detected at both mRNA and protein levels. In ischaemic/reperfused areas, an increased expression of mRNA for both receptors was observed, whereas only protein expression for the δ subtype was up-regulated. The μ-opioid receptor was not detected.

To further characterize the functional role of cystic fibrosis transmembrane conductance regulator (CFTR) in early and late (second window) ischemic preconditioning (IPC)- and postconditioning (POC)-mediated cardioprotection against ischemia/reperfusion (I/R) injury.CFTR knockout (CFTR(-/-)) mice and age- and gender-matched wild-type (CFTR(+/+)) and heterozygous (CFTR(+/-)) mice were used. In in vivo studies, the animals were subjected to a 30-min coronary occlusion followed by a 40-min reperfusion. In ex vivo (isolate heart) studies, a 45-min global ischemia was applied. To evaluate apoptosis, the level of activated caspase 3 and TdT-mediated dUTP-X nick end labeling (TUNEL) were examined.In the in vivo I/R models, early IPC significantly reduced the myocardial infarct size in wild-type (CFTR(+/+)) (from 40.4% ± 5.3% to 10.4% ± 2.0%, n=8, P0.001) and heterozygous (CFTR(+/-)) littermates (from 39.4% ± 2.4% to 15.4% ± 5.1%, n=6, P0.001) but failed to protect CFTR knockout (CFTR(-/-)) mice from I/R induced myocardial infarction (46.9% ± 6.2% vs 55.5% ± 7.8%, n=6, P0.5). Similar results were observed in the in vivo late IPC experiments. Furthermore, in both in vivo and ex vivo I/R models, POC significantly reduced myocardial infarction in wild-type mice, but not in CFTR knockout mice. In ex vivo I/R models, targeted inactivation of CFTR gene abolished the protective effects of IPC against I/R-induced apoptosis.These results provide compelling evidence for a critical role for CFTR Cl(-) channels in IPC- and POC-mediated cardioprotection against I/R-induced myocardial injury.

Ischemic preconditioning (IPC) is a protective phenomenon in which brief ischemia renders the myocardium resistant to subsequent ischemic insults. Here, we used A(2B)AR gene knock-out (A(2B)KO)/β-galactosidase reporter gene knock-in mice and the A(2B)AR antagonist ATL-801 to investigate the potential involvement of the A(2B)AR in IPC, focusing on the acute phase of protection. Cardioprotection provided by acute IPC elicited by two 3-min occlusion/3-min reperfusion cycles was readily apparent in an isolated, Langendorff-perfused mouse heart model in studies using hearts from A(2B)KO mice. IPC equivalently improved the recovery of contractile function following 20 min of global ischemia and 45 min of reperfusion in both WT and A(2B)KO hearts by ~30-40%, and equivalently decreased the release of cardiac troponin I during the reperfusion period (from 5969 ± 925 to 1595 ± 674 ng/g and 4376 ± 739 to 2278 ± 462 ng/g using WT and A(2B)KO hearts, respectively). Similarly, the infarct size-reducing capacity of acute IPC in an in vivo model of infarction was fully manifested in experiments using A(2B)KO mice, as well as in experiments using rats pretreated with ATL-801. We did observe, however, a marked reduction in infarct size in rats following administration of the selective A(2B)AR agonist BAY 60-6583 (~25% reduction at a dose of 1.0mg/kg). While supportive of its concept as a cardioprotective receptor, these experiments indicate that the mechanism of the early phase of IPC is not dependent on signaling by the A(2B)AR. We present the idea that the A(2B)AR may contribute to the later stages of IPC dependent on the induction of stress-responsive genes.

We have previously described novel cardioprotection in response to sustained morphine exposure, efficacious in young to aged myocardium and mechanistically distinct from conventional opioid or preconditioning (PC) responses. We further investigate opioid-dependent sustained ligand-activated preconditioning (SLP), assessing duration of protection, opioid receptor involvement, additivity with conventional responses, and signaling underlying preischemic induction of the phenotype. Male C57BL/6 mice were treated with morphine (75-mg subcutaneous pellet) for 5 days followed by morphine-free periods (0, 3, 5, or 7 days) before ex vivo assessment of myocardial tolerance to 25-min ischemia/45-min reperfusion. SLP substantially reduced infarction (by ∼50%) and postischemic contractile dysfunction (eliminating contracture, doubling force development). Cardioprotection persisted for 5 to 7 days after treatment. SLP was induced specifically by δ-receptor and not κ- or μ-opioid receptor agonism, was eliminated by δ-receptor and nonselective antagonism, and was additive with adenosinergic but not acute morphine- or PC-triggered protection. Cotreatment during preischemic morphine exposure with the phosphoinositide-3 kinase (PI3K) inhibitor wortmannin, but not the protein kinase A (PKA) inhibitor myristoylated PKI-(14-22)-amide, prevented induction of SLP. This was consistent with shifts in total and phospho-Akt during the induction period. In summary, data reveal that SLP triggers sustained protection from ischemia for up to 7 days after stimulus, is δ-opioid receptor mediated, is induced in a PI3K-dependent/PKA-independent manner, and augments adenosinergic protection. Mechanisms underlying SLP may be useful targets for manipulation of ischemic tolerance in young or aged myocardium.

We previously demonstrated that several epoxyeicosatrienoic acids (EETs) produce reductions in myocardial infarct size in rats and dogs. Since a recent study demonstrated the release of opioids in mediating the antinociceptive effect of 14,15-EET, we hypothesized that endogenous opioids may also be involved in mediating the cardioprotective effect of the EETs. To test this hypothesis, we used an in vivo rat model of infarction and a rat Langendorff model. In the infarct model, hearts were subjected to 30 min occlusion of the left coronary artery and 2 h reperfusion. Animals were treated with 11,12-EET or 14,15-EET (2.5 mg/kg) alone 15 min before occlusion or with opioid antagonists [naloxone, naltrindole, nor-binaltorphimine (nor-BNI), and d-Phe-Cys-Tyr-d-Trp-Om-Thr-Pen-Thr-NH(2) (CTOP), a nonselective, a selective delta, a selective kappa, and a selective mu receptor antagonist, respectively] 10 min before EET administration. In four separate groups, antiserum to Met- and Leu-enkephalin and dynorphin-A-(1-17) was administered 50 min before the 11,12-EET administration. Infarct size expressed as a percent of the area at risk (IS/AAR) was 63.5 + or - 1.2, 45.3 + or - 1.0, and 40.9 + or - 1.2% for control, 11,12-EET, and 14,15-EET, respectively. The protective effects of 11,12-EET were abolished by pretreatment with either naloxone (60.5 + or - 1.8%), naltrindole (60.8 + or - 1.0%), nor-BNI (62.3 + or - 2.8%), or Met-enkephalin antiserum (63.2 + or - 1.7%) but not CTOP (42.0 + or - 3.0%). In isolated heart experiments, 11,12-EET was administered to the perfusate 15 min before 20 min global ischemia followed by 45 min reperfusion in control hearts or in those pretreated with pertussis toxin (48 h). 11,12-EET increased the recovery of left ventricular developed pressure from 33 + or - 1 to 45 + or - 6% (P0.05) and reduced IS/AAR from 37 + or - 4 to 20 + or - 3% (P0.05). Both pertussis toxin and naloxone abolished these beneficial effects of 11,12-EET. Taken together, these results suggest that the major cardioprotective effects of the EETs depend on activation of a G(i/o) protein-coupled delta- and/or kappa-opioid receptor.

Background The role of endothelial nitric oxide synthase (eNOS) in isoflurane postconditioning (IsoPC)-elicited cardioprotection is poorly understood. The authors addressed this issue using eNOS mice. Methods In vivo or Langendorff-perfused mouse hearts underwent 30 min of ischemia followed by 2 h of reperfusion in the presence and absence of postconditioning produced with isoflurane 5 min before and 3 min after reperfusion. Ca+-induced mitochondrial permeability transition (MPT) pore opening was assessed in isolated mitochondria. Echocardiography was used to evaluate ventricular function. Results Postconditioning with 0.5, 1.0, and 1.5 minimum alveolar concentrations of isoflurane decreased infarct size from 56 +/- 10% (n = 10) in control to 48 +/- 10%, 41 +/- 8% (n = 8, P < 0.05), and 38 +/- 10% (n = 8, P < 0.05), respectively, and improved cardiac function in wild-type mice. Improvement in cardiac function by IsoPC was blocked by N-nitro-L-arginine methyl ester (a nonselective nitric oxide synthase inhibitor) administered either before ischemia or at the onset of reperfusion. Mitochondria isolated from postconditioned hearts required significantly higher in vitro Ca+ loading than did controls (78 +/- 29 microm vs. 40 +/- 25 microm CaCl2 per milligram of protein, n = 10, P < 0.05) to open the MPT pore. Hearts from eNOS mice displayed no marked differences in infarct size, cardiac function, and sensitivity of MPT pore to Ca+, compared with wild-type hearts. However, IsoPC failed to alter infarct size, cardiac function, or the amount of Ca+ necessary to open the MPT pore in mitochondria isolated from the eNOS hearts compared with control hearts. Conclusions IsoPC protects mouse hearts from reperfusion injury by preventing MPT pore opening in an eNOS-dependent manner. Nitric oxide functions as both a trigger and a mediator of cardioprotection produced by IsoPC.

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce marked reductions in infarct size (IS) in canine myocardium either given before an ischemic insult or at reperfusion similar to that produced in ischemic preconditioning (IPC) and postconditioning (POC) protocols. However, no studies have addressed the possibility that EETs serve a beneficial role in IPC or POC. We tested the hypothesis that EETs may play a role in these two phenomena by preconditioning dog hearts with one 5-min period of total coronary occlusion followed by 10 min of reperfusion before 60 min of occlusion and 3 h of reperfusion or by postconditioning with three 30-s periods of reperfusion interspersed with three 30-s periods of occlusion. To test for a role of EETs in IPC and POC, the selective EET antagonists 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) or its derivative, 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxy-propoxy)-ethoxy]-ethyl ester (14,15-EEZE-PEG), were administered 10 min before IPC, 5 min after IPC, or 5 min before POC. In a separate series, the selective EET synthesis inhibitor N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) was administered 10 min before IPC. Infarct size was determined by tetrazolium staining and coronary collateral blood flow at 30 min of occlusion and reperfusion flow at 3 h by radioactive microspheres. Both IPC and POC produced nearly equivalent reductions in IS expressed as a percentage of the area at risk (AAR) [Control 21.2 ± 1.2%, IPC 8.3 ± 2.2%, POC 10.1 ± 1.8% ( P < 0.001)]. 14,15-EEZE, 14,15-EEZE-PEG, and MS-PPOH markedly attenuated the cardioprotective effects of IPC and POC (14,15-EEZE and 14,15-EEZE-PEG) at doses that had no effect on IS/AAR when given alone. These results suggest a unique role for endogenous EETs in both IPC and POC.

We previously demonstrated that pharmacologic activation of AMP-activated protein kinase (AMPK) with 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR) 24 hours prior to (AICAR preconditioning; AICAR-PC) ischemia/reperfusion (I/R) prevents postischemic leukocyte-endothelial cell adhesive interactions (LEI) by a mechanism initiated by endothelial nitric oxide synthase (eNOS)-dependent NO production during the period of AICAR-PC. The major aim of this study was to examine the role of ATP-sensitive potassium (K(ATP)) channels and heme oxygenase as mediators of the antiadhesive effects of AICAR-PC during I/R 24 hours later.Intravital fluorescence microscopy was used to quantify LEI in the small intestine of AICAR-preconditioned C57BL/6J mice treated with K(ATP) channel or heme oxygenase inhibitors during I/R 24 hours after AICAR-PC in separate experiments.I/R induced marked increases in LEI relative to sham control mice, proadhesive responses that were prevented by AICAR-PC 24 hours prior to I/R. The effects of AICAR-PC to prevent postischemic LEI were abolished by K(ATP) channel or heme oxygenase inhibition during I/R.Our results indicate that the antiadhesive effects of AICAR-PC are mediated by K(ATP) channel- and heme oxygenase-dependent mechanisms during I/R.

The lanthanide cation, gadolinium (Gd) attenuates post-ischemic myocardial stunning. This study tests the hypothesis that Gd also preconditions the myocardium against infarction following ischemia-reperfusion (IR), and explores potential mechanisms underlying Gd-induced cardioprotection. Regional myocardial infarction was induced in rats by occluding the left anterior descending artery for 30 minutes and reperfusing for 120 minutes. Rats (n = 6/group) were administered intravenous Gd (1 to 100 μmol/kg) 15 minutes prior to ischemia. Hearts were excised after reperfusion to determine infarct size (IS) and area at risk (AAR). The ratio IS/AAR (%) was reduced by Gd in a “U”-shaped, dose-dependent manner. The minimum dose that reduced IS/AAR was 5 μmol/kg (52 ± 5% vs. 64 ± 4%), while the dose that reduced IS/AAR maximally was 20 μmol/kg (44 ± 4%). Gd also reduced IS/AAR when given 1 minute before reperfusion (47 ± 3%) but not when given 10 seconds after reperfusion (60 ± 3%). Cardioprotection was maintained if I-R was delayed 24–72 hours after Gd administration. Cardioprotection by Gd was abolished by inhibition of JAK-2 with AG-490, of p42/44 MAPK with PD98059 or of KATP channels with glibenclamide. None of these agents given alone altered IS/AAR compared with controls. Inhibition of JAK-2 also blocked Gd-induced delayed cardioprotection. Gd may have broad potential roles in IR, as it confered immediate cardioprotection when given prior to ischemia or prior to reperfusion, and delayed cardioprotection for up to 72 hours after administration. The mechanism underlying Gd-induced preconditioning appears to be multi-factorial, involving JAK-2, STAT-3 and p44 MAPK pathways, as well as KATP channels.

Continuing investigations of the roles of cytochrome P450 (CYP) arachidonic acid epoxygenase metabolites in the regulation of cardiovascular physiology and pathophysiology have revealed their complex and diverse biological effects. Often these metabolites demonstrate protective properties that are revealed during cardiovascular disease. In this regard, the epoxyeicosatrienoic acids (EETs) are an emerging target for pharmacological manipulation aimed at enhancing their cardiac and vascular protective mechanisms. This review will focus on the role of EETs in the regulation of vascular tone, with emphasis on the coronary circulation, their role in limiting platelet aggregation, vascular inflammation and EET contribution to preconditioning of the ischemic myocardium. Production and metabolism of EETs as well as their specific cellular signaling mechanisms are discussed.

We examined the cardioprotective profile of the new A3 adenosine receptor (AR) agonist CP-532,903 [ N 6-(2,5-dichlorobenzyl)-3′-aminoadenosine-5′- N -methylcarboxamide] in an in vivo mouse model of infarction and an isolated heart model of global ischemia/reperfusion injury. In radioligand binding and cAMP accumulation assays using human embryonic kidney 293 cells expressing recombinant mouse ARs, CP-532,903 was found to bind with high affinity to mouse A3ARs ( K i = 9.0 ± 2.5 nM) and with high selectivity versus mouse A1AR (100-fold) and A2AARs (1000-fold). In in vivo ischemia/reperfusion experiments, pretreating mice with 30 or 100 μg/kg CP-532,903 reduced infarct size from 59.2 ± 2.1% of the risk region in vehicle-treated mice to 42.5 ± 2.3 and 39.0 ± 2.9%, respectively. Likewise, treating isolated mouse hearts with CP-532,903 (10, 30, or 100 nM) concentration dependently improved recovery of contractile function after 20 min of global ischemia and 45 min of reperfusion, including developed pressure and maximal rate of contraction/relaxation. In both models of ischemia/reperfusion injury, CP-532,903 provided no benefit in studies using mice with genetic disruption of the A3AR gene, A3 knockout (KO) mice. In isolated heart studies, protection provided by CP-532,903 and ischemic preconditioning induced by three brief ischemia/reperfusion cycles were lost in Kir6.2 KO mice lacking expression of the pore-forming subunit of the sarcolemmal ATP-sensitive potassium (KATP) channel. Whole-cell patch-clamp recordings provided evidence that the A3AR is functionally coupled to the sarcolemmal KATP channel in murine cardiomyocytes. We conclude that CP-532,903 is a highly selective agonist of the mouse A3AR that protects against ischemia/reperfusion injury by activating sarcolemmal KATP channels.

Aims Thrombopoietin (Tpo) is known for its ability to stimulate platelet production. However, it is currently unknown whether Tpo plays a physiological function in the heart. Methods and results We assessed the potential protective role of Tpo in vitro and in vivo in two rat models of myocardial ischaemia/reperfusion. Tpo receptor (c-mpl) message was detected in the heart using RT-PCR, and the Tpo receptor protein was detected using western blotting and immunohistochemistry. Tpo treatment immediately before ischaemia reduced myocardial necrosis, apoptosis, and decline in ventricular function following ischaemia/reperfusion in the rat in a concentration- and dose-dependent manner with an optimal concentration of 1.0 ng/mL in vitro and an optimal dose of 0.05 µg/kg iv in vivo . Tpo also reduced infarct size when given after the onset of ischaemia or at reperfusion. Tpo activated JAK-2 (Janus kinase-2) and p44 MAPK (mitogen-activated protein kinase) during reperfusion but not prior to ischaemia. Inhibition of JAK-2 (AG-490), p42/44 MAPK (PD98059), mitochondrial KATP channels (5-HD), and sarcolemmal KATP channels (HMR 1098) abolished Tpo-induced resistance to injury from myocardial ischaemia/reperfusion. AG-490, PD98059, 5-HD, and HMR1098 alone had no effect on cardioprotection. Treatment with a single dose of Tpo (0.05 or 1.0 µg/kg iv) did not result in the elevation of platelet count or haematocrit over a 16-day period. Conclusion A single treatment of Tpo confers cardioprotection through JAK-2, p42/44 MAPK, and KATP channels, suggesting a potential therapeutic role of Tpo in the treatment of injury resulting from myocardial ischaemia and reperfusion.

Cardiovascular disease is the leading cause of death in the United States and developing world. Experimental and clinical studies have demonstrated that a number of interventions including brief periods of ischemia or hypoxia and certain endogenous factors such as opioids, bradykinin, growth factors or pharmacological agents are capable of protecting the heart against post-ischemic contractile dysfunction, arrhythmias and myocardial infarction. This conventional cardioprotection occurs via an autocrine or paracrine action in which these protective factors are released from the heart to act upon itself. Over the last ten years, a growing body of evidence indicates that a brief ischemic insult on one organ releases endogenous factors that protect other organs against a prolonged ischemic insult. This phenomenon, termed remote preconditioning or preconditioning at a distance, implicates an endocrine action, and may involve humoral or neural-endocrine signaling. This review will summarize the endocrine factors identified and implicated in this inter-organ cytoprotection.

Myocardial infarction is the leading cause of mortality in Western societies with annual expenditures of $431.8 billion spent on coronary artery disease in man. Therapeutics to combat infarction from myocardial injury, based on studies of ischemic preconditioning (IPC), are currently in progress. Hence, this review provides an update on IPC, including general and molecular mechanisms responsible for IPC and the effects of IPC in models of aging or disease. A summary of therapeutics shown to possess efficacy in preclinical and clinical trials and future directions of studies regarding cardiac IPC are also discussed.

Cardiovascular disease is the leading cause of morbidity and mortality in industrial societies, with myocardial infarction as the primary assassin. Pharmacologic agents, including the myocardial cell membrane receptor agonists adenosine, bradykinin/angiotensin-converting enzyme inhibitors, opioids and erythropoietin or the mixed cell membrane and intracellular agonists, glucose insulin potassium, and volatile anesthetics, either clinically or experimentally reduce the extent of myocardial injury when administered just prior to reperfusion. Agents that specifically target proteins, transcription factors or ion channels, including PKC agonists/antagonists, PPAR, Phosphodiesterase-5 inhibitors, 3-Hydroxy-3-methyl glutaryl coenzyme A reductase and the ATP-dependent potassium channel are also promising. However, no agent has been specifically approved to reduce reperfusion injury clinically. In this review, we will discuss the advantages and limitations of agents to combat reperfusion injury, their market development status and findings reported in both clinical and preclinical studies. The molecular pathways activated by these agents that preserve myocardium from reperfusion injury, which appear to commonly involve glycogen synthase kinase 3beta and mitochondrial permeability transition pore inhibition, are also described.

Myocardial ischemia/reperfusion (I/R) injury is partly mediated by thrombin. In support, the functional inhibition of thrombin has been shown to decrease infarct size after I/R. Several cellular responses to thrombin are mediated by a G-protein coupled protease-activated receptor 1 (PAR1).However, the role of PAR1 in myocardial I/R injury has not been well characterized. Therefore, we hypothesized that PAR1 inhibition will reduce the amount of myocardial I/R injury. After we detected the presence of PAR1 mRNA and protein in the rat heart by RT-PCR and immunoblot analysis,we assessed the potential protective role of SCH 79797, a selective PAR1 antagonist, in two rat models of myocardial I/R injury. SCH 79797 treatment immediately before or during ischemia reduced myocardial necrosis following I/R in the intact rat heart. This response was dose-dependent with the optimal dose being 25 microg/kg IV. Likewise, SCH 79797 treatment before ischemia in the isolated heart model reduced infarct size and increased ventricular recovery following I/R in the isolated heart model with an optimal concentration of 1 microM. This reduction was abolished by a PAR1 selective agonist. SCH 79797-induced resistance to myocardial ischemia was abolished by wortmannin, an inhibitor of PI3 kinase; L-NMA, a NOS inhibitor; and glibenclamide, a nonselective K(ATP) channel blocker. PAR1 activating peptide,wortmannin, L-NMA and glibenclamide alone had no effect on functional recovery or infarct size. A single treatment of SCH 79797 administered prior to or during ischemia confers immediate cardioprotection suggesting a potential therapeutic role of PAR1 antagonist in the treatment of injury resulting from myocardial ischemia and 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

Cardiomyocyte injury following ischemia–reperfusion can lead to cell death and result in cardiac dysfunction. A wide range of cardioprotective factors have been studied to date, but only recently has the cardioprotective role of fatty acids, specifically arachidonic acid (AA), been investigated. This fatty acid can be found in the membranes of cells in an inactive state and can be released by phospholipases in response to several stimuli, such as ischemia. The metabolism of AA involves the cycloxygenase (COX) and lipoxygenase (LOX) pathways, as well as the less well characterized cytochrome P450 (CYP) monooxygenase pathway. Current research suggests important differences with respect to the cardiovascular actions of specific CYP mediated arachidonic acid metabolites. For example, CYP mediated hydroxylation of AA produces 20-hydroxyeicosatetraenoic acid (20-HETE) which has detrimental effects in the heart during ischemia, pro-inflammatory effects during reperfusion and potent vasoconstrictor effects in the coronary circulation. Conversely, epoxidation of AA by CYP enzymes generates 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) that have been shown to reduce ischemia–reperfusion injury, have potent anti-inflammatory effects within the vasculature, and are potent vasodilators in the coronary circulation. This review aims to provide an overview of current data on the role of these CYP pathways in the heart with an emphasis on their involvement as mediators of ischemia–reperfusion injury. A better understanding of these relationships will facilitate identification of novel targets for the prevention and/or treatment of ischemic heart disease, a major worldwide public health problem.

Disodium disuccinate astaxanthin has potent cardioprotective effects in animals, with demonstrated preclinical efficacy in the rat, rabbit, and canine models of experimental infarction. It has been effective in subchronic and acute dosing regimens after parenteral administration, and recently published data in rats demonstrate that oral cardioprotection is also readily achieved. Myocardial salvage in the canine can reach 100% with a 4-day subchronic dosing regimen; single-dose I.V. cardioprotection, when given 2 hours before experimental coronary occlusion, is on average two-thirds of that achieved with the subchronic regimen in dogs. In conscious animals, no effects on hemodynamic parameters have been observed. Recently, the beneficial properties of this prototypical astaxanthin conjugate have been extended to include second- and third-generation compounds with improved pharmacokinetic and/or potency profiles. The primary mechanism of cardioprotection appears to be antioxidant activity: potent direct scavenging of the lynchpin radical in ischemia-reperfusion injury, superoxide anion, has been documented in appropriate model systems. In addition, modulation of serum complement activity, reduction of the levels of deposition of C-reactive protein (CRP) and the membrane attack complex (MAC) in infarcted tissue, and reduction in oxidative stress markers from the arachidonic acid and linoleic acid pathways also suggest a significant anti-inflammatory component to the mechanism of cardioprotection. Favorable plasma protein binding has been demonstrated in vitro for several astaxanthin conjugates; this binding capacity overcomes the supramolecular assembly of the compounds that occurs in aqueous solution, which in itself improves the stability and shelf-life of aqueous formulations. Astaxanthin readily populates cardiac tissue after metabolic hydrolysis of both oral and parenteral administration of the astaxanthin ester derivates, providing a reservoir of cardioprotective agent with a significant half-life due to favorable ADME in mammals. Due to the well-documented safety profile of astaxanthin in humans, disodium disuccinate astaxanthin may well find clinical utility in cardiovascular applications in humans following successful completion of preclinical and clinical pharmacology and toxicology studies in animals and humans, respectively.

A 5-day exposure to morphine exerts a profound cardioprotective phenotype in murine hearts. In the present study, we examined mechanisms by which morphine generates this effect, exploring the roles of Gi and Gs proteins, PKA, PKC, and β-adrenergic receptors (β-AR) in acute and chronic opioid preconditioning. Langendorff-perfused hearts from placebo, acute morphine (AM; 10 μmol/l)-, or chronic morphine (CM)-treated mice (75-mg pellet, 5 days) underwent 25-min ischemia and 45-min reperfusion. After reperfusion, placebo-treated hearts exhibited marked contractile and diastolic dysfunction [rate-pressure product (RPP), 40 ± 4% baseline; end-diastolic pressure (EDP), 33 ± 3 mmHg], whereas AM hearts showed significant improvement in recovery of RPP and EDP (60 ± 3% and 23 ± 4 mmHg, respectively; P < 0.05 vs. placebo). Furthermore, CM hearts demonstrated a complete return of diastolic function and significantly greater recovery of contractile function (83 ± 3%, P < 0.05 vs. both placebo and AM). Pretreatment with Gi protein inhibitor pertussis toxin abolished AM protection while partially attenuating CM recovery ( P < 0.05 vs. placebo). Treatment with Gs inhibitor NF-449 did not affect AM preconditioning yet completely abrogated CM preconditioning. Similarly, PKA inhibition significantly attenuated the ischemia-tolerant state afforded by CM, whereas it was ineffective in AM hearts. PKC inhibition with chelerythrine was ineffective in CM hearts while completely abrogating AM preconditioning. Moreover, whereas β1-AR blockade with CGP-20712A failed to alter recovery in CM hearts, the β2-AR antagonist ICI-118,551 significantly attenuated postischemic recovery. These data describe novel findings whereby CM preconditioning is mediated by a PKC-independent pathway involving PKA, β2-AR, and Gs proteins, whereas AM preconditioning is mediated via Gi proteins and PKC.

We used pharmacological agents and genetic methods to determine whether the potent A(3) adenosine receptor (AR) agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) protects against myocardial ischemia/reperfusion injury in mice via the A(3)AR or via interactions with other AR subtypes. Pretreating wild-type (WT) mice with Cl-IB-MECA reduced myocardial infarct size induced by 30 min of coronary occlusion and 24 h of reperfusion at doses (30 and 100 mug/kg) that concomitantly reduced blood pressure and stimulated systemic histamine release. The A(3)AR-selective antagonist MRS 1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate], but not the A(2A)AR antagonist ZM 241385 [4-{2-7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl}phenol], blocked the reduction in infarct size provided by Cl-IB-MECA, suggesting a mechanism involving the A(3)AR. To further examine the selectivity of Cl-IB-MECA, we assessed its cardioprotective effectiveness in A(3)AR gene "knock-out" (A(3)KO) mice. Cl-IB-MECA did not reduce myocardial infarct size in A(3)KO mice in vivo and did not protect isolated perfused hearts obtained from A(3)KO mice from injury induced by global ischemia and reperfusion. Additional studies using WT mice treated with compound 48/80 [condensation product of p-methoxyphenethyl methylamine with formaldehyde] to deplete mast cell contents excluded the possibility that Cl-IB-MECA was cardioprotective by releasing mediators from mast cells. These data demonstrate that Cl-IB-MECA protects against myocardial ischemia/reperfusion injury in mice principally by activating the A(3)AR.

The role of caveolae, membrane microenvironments enriched in signaling molecules, in myocardial ischemia is poorly defined. In the current study, we used cardiac myocytes prepared from adult rats to test the hypothesis that opioid receptors (OR), which are capable of producing cardiac protection in vivo, promote cardiac protection in cardiac myocytes in a caveolae-dependent manner. We determined protein expression and localization of delta-OR (DOR) using coimmunohistochemistry, caveolar fractionation, and immunoprecipitations. DOR colocalized in fractions with caveolin-3 (Cav-3), a structural component of caveolae in muscle cells, and could be immunoprecipitated by a Cav-3 antibody. Immunohistochemistry confirmed plasma membrane colocalization of DOR with Cav-3. Cardiac myocytes were subjected to simulated ischemia (2 h) or an ischemic preconditioning (IPC) protocol (10 min ischemia, 30 min recovery, 2 h ischemia) in the presence and absence of methyl-beta-cyclodextrin (MbetaCD, 2 mM), which binds cholesterol and disrupts caveolae. We also assessed the cardiac protective effects of SNC-121 (SNC), a selective DOR agonist, on cardiac myocytes with or without MbetaCD and MbetaCD preloaded with cholesterol. Ischemia, simulated by mineral oil layering to inhibit gas exchange, promoted cardiac myocyte cell death (trypan blue staining), a response blunted by SNC (37 +/- 3 vs. 59 +/- 3% dead cells in the presence and absence of 1 muM SNC, respectively, P0.01) or by use of the IPC protocol (35 +/- 4 vs. 62 +/- 3% dead cells, P0.01). MbetaCD treatment, which disrupted caveolae (as detected by electron microscopy), fully attenuated the protective effects of IPC or SNC, resulting in cell death comparable to that of the ischemic group. By contrast, SNC-induced protection was not abrogated in cells incubated with cholesterol-saturated MbetaCD, which maintained caveolae structure and function. These findings suggest a key role for caveolae, perhaps through enrichment of signaling molecules, in contributing to protection of cardiac myocytes from ischemic damage.

Disodium disuccinate astaxanthin (Cardax), DDA) has cardioprotective effects in the rat, rabbit, and canine models of experimental infarction. It is highly effective by parenteral administration in subchronic and acute dosing regimens. Unpublished data in rats suggest that oral cardioprotection is also readily achievable. DDA-induced myocardial salvage in the canine can reach 100% with a 4-day subchronic dosing regimen. At a single i.v. dose DDA is cardioprotective, when given 2 h before experimental coronary occlusion, but the protection is on the average two-thirds of that achieved with the subchronic regimen in dogs. In conscious animals DDA has no effects on hemodynamic parameters. The primary mechanism of cardioprotection appears to be antioxidant activity involving direct scavenging of superoxide anion, the lynchpin radical in ischemia-reperfusion injury. In addition, modulation of serum complement activity, as well as the reduction in the levels of C-reactive protein (CRP) and the membrane attack complex (MAC) in infarcted tissue suggest a significant antiinflammatory component in the mechanism of cardioprotective action of DDA. Stoichiometric binding of the meso-form of the compound to human serum albumin (HSA) has been demonstrated in vitro. This binding capacity overcomes the supramolecular assembly of the compound in aqueous solution, which by itself improves the stability and shelf life of aqueous formulations. Non-esterified astaxanthin readily enters cardiac tissue after either oral or parenteral administration, providing a reservoir of a cardioprotective agent with a significant half-life due to favorable ADME in mammals. Due to the well-documented safety profile of non-esterified astaxanthin in humans, disodium disuccinate astaxanthin may well find clinical utility in cardiovascular indications in humans following successful completion of preclinical and clinical pharmacology and toxicology studies.

The cardioprotection afforded by ischemic preconditioning (IPC) and ischemic postconditioning (PC) are receptor mediated. In this review, we will focus on the major ligand classes and receptors that contribute to IPC and PC-induced cardioprotection. Ligand classes discussed include adenosine, bradykinin, opioids, erythropoietin, adrenergics and muscarinics. The cardioprotective therapeutic window of each ligand class will also be summarized, with particular focus as to whether ligands are protective when administered at or close to the time of reperfusion. Information will primarily be directed at studies in which infarct size reduction is the gold standard to assess the efficacy of IPC and PC. Myocardial stunning is a less robust endpoint for assessing cardioprotection and the use of this endpoint is only limited to studies with human tissue where infarct size assessment is not possible. Receptor cross-talk between ligands and the common signaling pathways involved for these ligands will also be briefly discussed.

Cytochrome P-450 (CYP) ω-hydroxylases and their arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), produce a detrimental effect on ischemia-reperfusion injury in canine hearts, and the inhibition of CYP ω-hydroxylases markedly reduces myocardial infarct size expressed as a percentage of the area at risk (IS/AAR, %). In this study, we demonstrated that a specific CYP ω-hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly reduced 20-HETE production during ischemia-reperfusion and reduced myocardial infarct size compared with control [19.5 ± 1.0% (control), 9.6 ± 1.5% (0.40 mg/kg DDMS), 4.0 ± 2.0% (0.81 mg/kg DDMS), P < 0.01]. In addition, 20-hydroxyeicosa-6( Z),15( Z)-dienoic acid (20-HEDE, a putative 20-HETE antagonist) significantly reduced myocardial infarct size from control [10.3 ± 1.3% (0.032 mg/kg 20-HEDE) and 5.9 ± 1.9% (0.064 mg/kg 20-HEDE), P < 0.05]. We further demonstrated that one 5-min period of ischemic preconditioning (IPC) reduced infarct size to a similar extent as that observed with the high doses of DDMS and 20-HEDE, and the higher dose of DDMS given simultaneously with IPC augmented the infarct size reduction [9.9 ± 2.8% (IPC) to 2.5 ± 1.4% (0.81 mg/kg DDMS), P < 0.05] to a greater degree than that observed with either treatment alone. These results suggest an important negative role for endogenous CYP ω-hydroxylases and their product, 20-HETE, to exacerbate myocardial injury in canine myocardium. Furthermore, for the first time, this study demonstrates that the effect of IPC and the inhibition of CYP ω-hydroxylase synthesis (DDMS) or its actions (20-HEDE) may have additive effects in protecting the canine heart from ischemia-reperfusion injury.

Ischemia-reperfusion of the heart and other organs results in the accumulation of unesterified arachidonic acid (AA) via the action of membrane-bound phospholipases, primarily phospholipase A2. AA can be metabolized by the classical cyclooxygenase (COX) and lipoxygenase (LOX) pathways to well-characterized metabolites and their respective cardioprotective end products such as prostacyclin (PGI2) and 12-hydroxyeicosatetraenoic acid (12-HETE). However, it has only been recently recognized that another less well-characterized pathway of AA metabolism, the cytochrome P450 (CYP) pathway, may have important cardiovascular effects. Several lines of data support the possibility that certain CYP metabolites resulting from the hydroxylation of AA such as 20-hydroxyeicosatetraenoic acid (20-HETE) are potent vasoconstrictors and may produce detrimental effects in the heart during ischemia and pro-inflammatory effects during reperfusion. On the other hand, a group of regioisomers resulting from the epoxidation of AA, including 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acid (EETs), have been shown to reduce ischemic and/or reperfusion injury in the heart and vasculature. This review will discuss the detrimental and beneficial actions, including the potential cellular mechanisms responsible as a result of stimulating or inhibiting the two arms of this novel CYP pathway. The therapeutic potential of increasing EET concentrations and/or reducing 20-HETE concentrations will also be addressed.

A novel delta-receptor selective compound, ARD-353 [4-((2R,5S)-4-(R)-(4-diethylcarbamoylphenyl)(3-hydroxyphenyl)methyl)-2, 5-dimethylpiperazin-1-ylmethyl)benzoic acid], was evaluated for activity on infarct size in a rat model of acute myocardial infarction. ARD-353 was characterized as having delta receptor selectivity using radioligand binding and had no apparent selectivity between delta receptor subtypes as determined by [(3)H] cyclic [D-Pen(2),D-Pen(5)]enkephalin (delta(1)) and [(3)H]Deltorphin II (delta(2)) competition binding. ARD-353 also showed selective delta receptor agonist activity in mouse-isolated vas deferens. There was no evidence of any seizure-like convulsions when ARD-353 was administered to mice either i.v. or p.o., implying minimal penetration of the blood-brain barrier. ARD-353 decreased infarct size in a left anterior descending coronary artery (LAD) occlusion model of myocardial infarction. In animals pretreated with ARD-353 (i.v.) and then subjected to 30 min of LAD occlusion followed by 90 min of reperfusion, infarct size was reduced in a dose-dependent manner compared with vehicle-treated controls. The effects of ARD-353 on infarct size were blocked by the delta(1)-opioid selective antagonist 7-benzylidenenaltrexone, indicating a significant role for the delta(1)-opioid receptor in the cardioprotective mechanism of ARD-353. ARD-353 (0.3 mg/kg i.v.) produced significant protection when administered 5 min and 12 and 48 h before ischemic insult or when given immediately after the ischemic insult (at the start of reperfusion). Given the lack of central nervous system effects and beneficial efficacy in the rat model of myocardial ischemia, it is felt that ARD-353 is the first nonpeptide delta-receptor agonist with true potential for clinical use before surgically induced ischemia or in an emergency setting.

CGX-1051 is a synthetic version of a peptide originally isolated from the venom of cone snails. In the present studies, we tested the potential cardioprotective effect of CGX-1051 in a rat and dog model of myocardial ischemia/reperfusion. CGX-1051 was administered 5 minutes before reperfusion as intravenous bolus doses of 30, 100, and 300 microg/kg. Infarct size (IS) is reported as IS/area at risk (AAR). In the rat, the vehicle control group had an IS/AAR of 59.8+/-2.1%. Postischemic administration of CGX-1051 at doses of 30, 100, and 300 microg/kg resulted in an IS/AAR of 52.6+/-4.2%, 34.6+/-5.6% (P

Opioids, named by Acheson for compounds with morphine-like actions despite chemically distinct structures, have received much research interest, particularly for their central nervous system (CNS) actions involved in pain management, resulting in thousands of scientific papers focusing on their effects on the CNS and other organ systems. A more recent area which may have great clinical importance concerns the role of opioids, either endogenous or exogenous compounds, in limiting the pathogenesis of ischemia-reperfusion injury in heart and brain. The role of endogenous opioids in hibernation provides tantalizing evidence for the protective potential of opioids against ischemia or hypoxia. Mammalian hibernation, a distinct energy-conserving state, is associated with depletion of energy stores, intracellular acidosis and hypoxia, similar to those which occur during ischemia. However, despite the potentially detrimental cellular state induced with hibernation, the myocardium remains resilient for many months. What accounts for the hypoxia-tolerant state is of great interest. During hibernation, circulating levels of opioid peptides are increased dramatically, and indeed, are considered a "trigger" of hibernation. Furthermore, administration of opioid antagonists can effectively reverse hibernation in mammals. Therefore, it is not surprising that activation of opioid receptors has been demonstrated to preserve cellular status following a hypoxic insult, such as ischemia-reperfusion in many model systems including the intestine [Zhang, Y., Wu, Y.X., Hao, Y.B., Dun, Y. Yang, S.P., 2001. Role of endogenous opioid peptides in protection of ischemic preconditioning in rat small intestine. Life Sci. 68, 1013-1019], skeletal muscle [Addison, P.D., Neligan, P.C., Ashrafpour, H., Khan, A., Zhong, A., Moses, M., Forrest, C.R., Pang, C.Y., 2003. Noninvasive remote ischemic preconditioning for global protection of skeletal muscle against infarction. Am. J. Physiol. Heart Circ. Physiol. 285, H1435-H1443], the CNS [Borlongan, C.V., Wang, Y., Su, T.P., 2005. Delta opioid peptide (d-ala 2, d-leu 5) enkephalin: linking hiberation and neuroprotection. Front Biosci. 9, 3392-3398] and the myocardium [Romano, M.A., Seymour, E.M., Berry, J.A., McNish, R.A., Bolling, S.F., 2004. Relative contribution of endogenous opioids to myocardial ischemic tolerance. J Surg Res. 118, 32-37; Peart, J.N., Gross, G.J., 2004a. Exogenous activation of delta- and kappa-opioid receptors affords cardioprotection in isolated murine heart. Basic Res Cardiol. 99(1), 29-37]. For the purpose of this review, we will focus primarily on the protective effects of opioids against post-reperfusion myocardial stunning and infarction.

Adenosine kinase phosphorylates adenosine to AMP, the primary pathway for adenosine metabolism under basal conditions. Inhibition of adenosine kinase results in a site-specific increase in interstitial adenosine. Using a rat model of myocardial infarction, we examined the protective effects of adenosine kinase inhibition. Male Sprague-Dawley rats underwent 30 min regional occlusion followed by 90 min reperfusion. Infarct size, expressed as a percent of the area-at-risk, IS/AAR(%), was 58.0 +/- 2.1 % in untreated rats. Pretreatment with the adenosine kinase inhibitor, 5-iodotubercidin (1 mg/kg), limited infarct development to 37.5+/-3.7% (P0.001). The A(1) adenosine receptor (A(1)AR) antagonist, DPCPX (100 microg/kg), abolished the infarct-sparing effect of 5-iodotubercidin (IS, 62.8 +/- 1.3%). Similarly, the A(3) adenosine receptor (A(3)AR) antagonist, MRS-1523 (2 mg/kg), and the delta-opioid receptor (DOR) antagonist, BNTX, (1 mg/kg) abolished the reduction of IS produced by iodotubercidin. Pretreatment with the ROS scavenger, 2-MPG (20 mg/kg), or the PKC-delta antagonist, rottlerin (0.3 mg/kg) also abolished iodotubercidin-mediated cardioprotection. Furthermore, pretreatment with 5-HD, a mitochondrial K(ATP) (mitoK(ATP)) channel inhibitor, but not the sarcolemmal K(ATP) channel blocker, HMR-1098, abrogated the beneficial effects of adenosine kinase inhibition (IS, 59.5 +/- 3.8%). These data suggest that inhibition of adenosine kinase is effective in reducing infarct development via A(1)AR, A(3)AR and DOR activation. Data also suggest that this protection is mediated via ROS, PKC-delta and mitoK(ATP) channels.

Preliminary selective block of mu-, delta1-, delta2-, and kappa-opioid receptors had no effect on the incidence of ventricular arrhythmias during 10-min coronary occlusion-reperfusion in ketamine-narcotized rats. Repetitive short-term immobilization of rats for 2 weeks improved heart resistance to the arrhythmogenic action of coronary occlusion and reperfusion. Selective mu-opioid receptor antagonist CTAP completely abolished, while selective delta- and kappa-opioid receptor antagonists did not modulate the antiarrhythmic effect of adaptation. Probably, endogenous agonists of mu-opioid receptors play an important role in the adaptive improvement of heart resistance to arrhythmogenic factors, but are insignificant for the modulation of heart resistance to the arrhythmogenic action of short-term local ischemia-reperfusion in non-adapted animals.

Hypoxia from birth increases resistance to myocardial ischemia in infant rabbits. We hypothesized that increased cardioprotection in hearts chronically hypoxic from birth persists following development in a normoxic environment and involves increased activation of nitric oxide synthase (NOS) and ATP-dependent K (KATP) channels. Resistance to myocardial ischemia was determined in rabbits raised from birth to 10 days of age in a normoxic (FiO2 = 0.21) or hypoxic (FiO2 = 0.12) environment and subsequently exposed to normoxia for up to 60 days of age. Isolated hearts ( n = 8/group) were subjected to 30 min of global ischemia followed by 35 min of reperfusion. At 10 days of age, resistance to myocardial ischemia (percent recovery postischemic recovery left ventricular developed pressure) was higher in chronically hypoxic hearts (68 ± 4%) than normoxic controls (43 ± 4%). At 10 days of age, NG-nitro-l-arginine methyl ester (200 μM) and glibenclamide (3 μM) abolished the cardioprotective effects of chronic hypoxia (45 ± 4% and 46 ± 5%, respectively) but had no effect on normoxic hearts. At 30 days of age resistance to ischemia in normoxic hearts declined (36 ± 5%). However, in hearts subjected to chronic hypoxia from birth to 10 days and then exposed to normoxia until 30 days of age, resistance to ischemia persisted (63 ± 4%). l-NAME or glibenclamide abolished cardioprotection in previously hypoxic hearts (37 ± 4% and 39 ± 5%, respectively) but had no effect on normoxic hearts. Increased cardioprotection was lost by 60 days. We conclude that cardioprotection conferred by adaptation to hypoxia from birth persists on subsequent exposure to normoxia and is associated with enhanced NOS activity and activation of KATP channels.

Aging is often associated with decreased myocardial ischemic tolerance. We recently reported that chronic preconditioning produced by continuous exposure to morphine affords a profound cardioprotective phenotype in young mice. In this study, we determined if chronic exposure to morphine retained its ability to precondition the myocardium in the young or aged heart. Young (10-14 weeks) or aged (24-26 months) C57/BL6 mice were untreated, administered morphine acutely (30 microM), or implanted with a morphine pellet (75 mg) for 5 days prior to heart isolation and perfusion. Following equilibration, perfused hearts were subjected to 25 min ischemia and 45 min reperfusion. Untreated hearts from both young and aged mice displayed marked contractile dysfunction and LDH release following reperfusion. Acute infusion of morphine improved recovery of end-diastolic pressure and developed pressure in young (P < 0.05 vs. untreated) but not senescent hearts. Hearts from mice exposed to morphine for 5 days displayed a further improvement in post-ischemic contractile function (P < 0.05 vs. acute treatment), and a marked reduction in post-ischemic LDH efflux (P < 0.05 vs. untreated) in both young and senescent hearts. These data demonstrate that aged hearts maintain the ability to be preconditioned by chronic exposure to morphine in the absence of acute protection.

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, p0.001, 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.

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 P 9 in the ischemic zone compared with vehicle (181±20, 178±15 versus 75±17 DU, respectively; P 21 or Tyr 279 ) or phosphorylation of GSKβ (Tyr 216 ). These data indicate that OIC occurs via the phosphorylation of GSKβ at Ser 9 during reperfusion.

Acute cardioprotection is mediated primarily through delta opioid receptor stimulation independent of micro or kappa opioid receptor stimulation. Delayed cardioprotection is mediated by delta opioid receptor agonists but ambiguity remains about direct receptor involvement. Therefore, we investigated the potential of SNC-121, a non-peptide delta opioid agonist, to produce delayed cardioprotection and characterized the role of opioid receptors in this delayed response. All rats underwent 30 minutes of ischemia followed by 2 hours of reperfusion. SNC-121 induced a significant delayed cardioprotective effect. To determine the nature of this SNC-121-induced delayed cardioprotection, rats were treated with specific opioids receptor antagonists and underwent pertussis toxin (PT) treatment prior to opioid agonist stimulation. Control rats were injected with saline and allowed to recover for 24 hours. Pretreatment and early treatment with opioid receptor antagonists failed to inhibit the delayed protective effects of SNC-121, as did pretreatment with PT. Treatment with a free radical scavenger, 2-mercaptopropionyl glycine, at the time of opioid stimulation attenuated the delayed cardioprotective effects of SNC-121. These data suggest that delayed cardioprotection is stimulated via non-peptide delta opioid agonists by a mechanism unrelated to opioid receptor activation. The mechanism appears to be a non-opioid receptor mediated production of reactive oxygen species that triggers the signaling cascade leading to delayed cardioprotection.

Controversy exists regarding the relative roles of δ- and κ-opioid receptor activation as a potential cardioprotective mechanism. Furthermore, the function of opioid receptor activation in cardioprotection has not been examined in the murine heart. To this end, we employed various concentrations of selective δ - and κ-opioid receptor agonists in an isolated murine heart model undergoing 20 min global ischemia followed by 45 min reperfusion. Left-ventricular developed pressure (LVDP) returned to 50.7 ± 2.1% of baseline values in untreated hearts. Infusion of the non-selective opioid agonist, morphine (10 µM), lead to a marked improvement in post-ischemic contractile recovery where LVDP returned to 65.5 ± 2.4% of baseline function. The δ-opioid selective agonist BW373U86 hydrochloride elicited maximal protection at a concentration of 1 µM which afforded 63.9 ± 3.4% recovery of LVDP. This effect was blocked by the δ-opioid selective antagonist, BNTX. Furthermore, administration of the κ-opioid selective agonist U50,488 (1 µM) produced a marked improvement in contractile recovery leading to a 72.5 ± 5.3% recovery of LVDP. This degree of protection was also abolished by the κ-opioid receptor antagonist, nor-BNI. No differences were noted in LDH efflux from post-ischemic hearts. These data suggest that exogenous activation of δ- and κ-opioid receptors afford protection against myocardial stunning in the isolated murine heart, an effect attenuated by selective receptor antagonists.

Previous studies have demonstrated the importance of the sarcolemmal and/or the mitochondrial KATP channel (sKATP and mKATP, respectively) in mediating delayed cardioprotection induced by opioids, bradykinin, adenosine or the KATP-channel opener (KCO), diazoxide. However, the ability of other KCOs, such as the putative sKCO P-1075 or the putative mKCO BMS-191095, to induce delayed cardioprotection has not been tested. In this study, rats were randomly divided and treated with P-1075 (1 姯kg), BMS-191095 (BMS, 1 mg/kg), diazoxide (3.5 mg/kg) or vehicle. Selective blockers were also administered 5 min prior to KCO or vehicle. These included the sKATP antagonist (HMR-1098 (HMR, 6 mg/kg)), the mKATP antagonist (5-hydroxydecanoic acid (5-HD, 10 mg/kg)), the oxygen radical scavenger (N-2-mecaptopropionyl-glycine (2-MPG, 20 mg/kg)) or the MEK1/2 inhibitor (PD-98059 (PD, 1 mg/kg)). Twenty-four hours later, rats were subjected to 30-min ischemia and 2-h reperfusion, followed by infarct size assessment. P-1075, BMS or diazoxide treatment produced similar reductions in infarct size as compared to vehicle (37.2 ᠲ.3%*, 40.7 ᠱ.5%*, 35.6 ᠳ.6%* vs. 59.8 ᠱ.8%, P < 0.05, respectively). HMR administration before P-1075 and diazoxide abolished delayed cardioprotection, but not BMS-induced infarct size reduction (56.1 ᠱ.1%, 54.9 ᠳ.3%, 41.0 ᠱ.7%*, respectively). Administration of 5-HD, 2-MPG or PD prior to the three KATP openers abolished delayed cardioprotection. These data imply BMS-191095 has a selective mitochondrial site of action, perhaps the mKATP channel, whereas diazoxide and P-1075 affect both the sKATP and mKATP channels. All three KCO also require an initial oxygen-derived free radical (OFR) burst and MEK1/2 activation to trigger delayed cardioprotection.

The role of delta-opioid receptors in mediating ischemic preconditioning (IPC) in rats, rabbits, and pigs has been well-established; however, no studies have been performed in dogs. Therefore, the purpose of the present study was to determine if activation of delta-opioid receptors can mimic the cardioprotective effects of IPC in the canine heart and to determine if a nonselective opioid receptor antagonist could block IPC. All dogs were subjected to 60 minutes of left anterior descending (LAD) coronary artery occlusion and 3 hours of reperfusion. Ischemic preconditioning was produced by one 5-minute period of ischemia 10 minutes before LAD coronary artery occlusion. Infarct size (IS) expressed as a percent of the area at risk (AAR; IS/AAR) was determined by triphenyltetrazolium staining. Two selective delta-opioid receptor (DOR) agonists, TAN-67 and BW373U86, were administered by intracoronary infusion for 30 minutes before LAD occlusion and the opioid receptor antagonist naloxone was administered 30 minutes before IPC. Both TAN-67 and BW373U86 produced significant reductions in IS/AAR similar to that of IPC (control: 28+/-2.1; TAN: 12.3+/-2.2; IPC: 9.3+/-3.0: BW: 11.7+/-2.6). Naloxone attenuated the effect of IPC (control: 28+/-2.1; naloxone: 18.2+/-4.5). These results suggest that opioid receptors are important in IPC in dogs, and stimulation of delta-opioid receptors with selective agonists can mimic the cardioprotective effects of IPC and may have therapeutic potential.

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 δ-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 P