Role of soluble epoxide hydrolase in postischemic recovery of heart contractile function

Cytochrome P450 epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which are converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (Ephx2, sEH). To examine the functional role of sEH in the heart, mice with targeted disruption of the Ephx2 gene were studied. Hearts from sEH null mice have undetectable levels of sEH mRNA and protein and cannot convert EETs to DHETs. sEH null mice have normal heart anatomy and basal contractile function, but have higher fatty acid epoxide:diol ratios in plasma and cardiomyocyte cell culture media compared with wild type (WT). sEH null hearts have improved recovery of left ventricular developed pressure (LVDP) and less infarction compared with WT hearts after 20 minutes ischemia. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 to 100 nmol/L) before ischemia abolishes this cardioprotective phenotype. Inhibitor studies demonstrate that perfusion with phosphatidylinositol-3 kinase (PI3K) inhibitors wortmannin (200 nmol/L) or LY294002 (5 micromol/L), the ATP-sensitive K+ channel (K(ATP)) inhibitor glibenclamide (1 micromol/L), the mitochondrial K(ATP) (mitoK(ATP)) inhibitor 5-hydroxydecanoate (100 to 200 micromol/L), or the Ca2+-sensitive K+ channel (K(Ca)) inhibitor paxilline (10 micromol/L) abolishes the cardioprotection in sEH null hearts. Consistent with increased activation of the PI3K cascade, sEH null mice exhibit increased cardiac expression of glycogen synthase kinase-3beta (GSK-3beta) phospho-protein after ischemia. Together, these data suggest that targeted disruption of sEH increases the availability of cardioprotective EETs that work by activating PI3K signaling pathways and K+ channels.     

Epoxyeicosatrienoic acids limit damage to mitochondrial function following stress in cardiac cells

Epoxyeicosatrienoic acids (EETs) are polyunsaturated fatty acids synthesized from arachidonic acid by CYP2J2 epoxygenase and inactivated by soluble epoxide hydrolase (sEH or Ephx2) to dihydroxyeicosatrienoic acids. Mitochondrial function following ischemic insult is a critical determinant of reperfusion-induced cell death in the myocardium. The objectives of the current study were to investigate the protective role of EETs in mitochondrial function. Mice with the targeted disruption of the Ephx2 gene, cardiomyocyte-specific overexpression of CYP2J2 or perfused with EETs all have improved postischemic LVDP recovery compared to wild-type (WT). Perfusion with the mPTP opener, atractyloside, abolished the improved postischemic functional recovery observed in CYP2J2 Tr, sEH null and EET perfused hearts. Electron micrographs demonstrated WT hearts to have increased mitochondrial fragmentation and T-tubule swelling compared to CYP2J2 Tr hearts following 20 min global ischemia and 20 min reperfusion. Direct effects of EETs on mitochondria were assessed in isolated rat cardiomyocytes and H9c2 cells. Laser-induced loss of mitochondrial membrane potential (ΔΨ_m) and mPTP opening was significantly reduced in cells treated with 14, 15-EET (1 μM). The EET protective effect was blocked by the putative EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (1 μM, 14, 15-EEZE), paxilline (10 μM, BK_Ca inhibitor) and 5HD (100 μM, K_ATP inhibitor). Our studies show that EETs can limit mitochondrial dysfunction following cellular stress via a K⁺ channel-dependent mechanism.     

Male and female mice overexpressing the beta(2)-adrenergic receptor exhibit differences in ischemia/reperfusion injury: role of nitric oxide

OBJECTIVE: Cardiac overexpression of beta(2)-adrenergic receptors (beta(2)ARs) in male mice (MTG4) results in increased contractility and increased ischemic injury. Considering recent clinical data indicating that premenopausal women are protected from cardiovascular injury, we assessed the consequences of beta(2)AR overexpression in females (FTG4). Since protection in females is mediated via estrogen, which activates endothelial and inducible nitric oxide synthases (eNOS and iNOS) we also examined the role of NOS in ischemia/reperfusion injury in male and female TG4 and wild-type (WT) mice. METHODS: Hearts from MTG4, FTG4, MWT and FWT mice were isolated and perfused in the Langendorff mode. Hearts were pretreated with either 1 micromol/l of the nonspecific NOS inhibitor, L-NAME, or 100 nmol/l of the specific iNOS inhibitor, 1400W. Control hearts received no treatment. All hearts were subjected to 20 min ischemia and 40 min reperfusion while 31P-NMR spectra were acquired. RESULTS: During ischemia, ATP and pH fell lower in MTG4 hearts than in FTG4 or WT hearts. Hearts from MTG4 mice exhibited increased ischemia/reperfusion injury as indicated by lower recoveries of postischemic contractile function, ATP and PCr than WT. Despite contractility being elevated in FTG4 hearts to the same level as MTG4 hearts, ischemia/reperfusion injury was not increased, as indicated by similar postischemic recoveries of contractile function, ATP and PCr in FTG4 hearts compared to WT. ATP and pH fell lower during ischemia in L-NAME-treated FTG4 hearts than in untreated FTG4 hearts, falling as low as untreated MTG4s. Recoveries of contractile function, ATP and PCr were as low in L-NAME-treated FTG4 hearts as in untreated MTG4 hearts and lower than untreated FTG4 hearts. In contrast, 1400W had no effect on FTG4 hearts. MTG4 hearts were unaffected by L-NAME or 1400W. CONCLUSIONS: beta(2)AR overexpression increased ischemia/reperfusion injury in males but not females, thus females were protected from the detrimental effects of beta(2)AR overexpression. Protection was abolished by treatment with L-NAME, but not 1400W, implying that protection was mediated by eNOS not iNOS.     

Difference in the cardioprotective mechanisms between ischemic preconditioning and pharmacological preconditioning by diazoxide in rat hearts.

BACKGROUND: Recent studies have implicated the opening of mitochondrial K(ATP) (mitoK(ATP)) channels and the production of reactive oxygen species (ROS) in the cardioprotective mechanism of ischemic preconditioning (IPC). METHODS AND RESULTS: The involvement of mitoK(ATP) channels and ROS in the cardioprotective effects of both IPC and the mitoK(ATP) channel opener diazoxide (DZ) was investigated in ischemic/reperfused rat hearts. The effects of IPC and DZ on myocardial high-energy phosphate concentrations and intracellular pH (pH(i)) were also examined using (31)P nuclear magnetic resonance spectroscopy. Although both the mitoK(ATP) channel inhibitor 5-hydroxydecanoate and the antioxidant N-acetylcysteine abolished the postischemic recovery of contractile function by DZ, neither of them inhibited that by IPC. IPC attenuated the decline in pHi during ischemia, but DZ did not (6.28+/-0.04 in IPC, p<0.05, and 6.02+/-0.05 in DZ vs 6.02 +/-0.06 in control hearts). DZ, but not IPC, reduced the decrease in ATP levels during ischemia (ATP levels at 20-min ischemia: 26.3+/-3.4% of initial value in DZ, p<0.05, and 8.1+/-3.0% in IPC vs 15.1+/-1.3% in control hearts). CONCLUSIONS: These results suggest that DZ-induced cardioprotection is related to ROS production and reduced ATP degradation during ischemia, whereas attenuated acidification during ischemia is involved in IPC-induced cardioprotection, which is not mediated through mitoK(ATP) channel opening or ROS production.     

Inhibition of p38 mitogen-activated protein kinase decreases cardiomyocyte apoptosis and improves cardiac function after myocardial ischemia and reperfusion

BACKGROUND: Activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in apoptotic cell death. The role of p38 MAPK in myocardial injury caused by ischemia/reperfusion, an extreme stress to the heart, is unknown. METHODS AND RESULTS: Studies were performed with isolated, Langendorff-perfused rabbit hearts. Ischemia alone caused a moderate but transient increase in p38 MAPK activity (3.5-fold increase, P<0.05 versus basal). Ischemia followed by reperfusion further activated p38 MAPK, and the maximal level of activation (6.3-fold, P<0.01) was reached 10 minutes after reperfusion. Administration of SB 203580, a p38 MAPK inhibitor, decreased myocardial apoptosis (14.7+/-3.2% versus 30.6+/-3.5% in vehicle, P<0.01) and improved postischemic cardiac function. The cardioprotective effects of SB 203580 were closely related to its inhibition of p38 MAPK. Administering SB 203580 before ischemia and during reperfusion completely inhibited p38 MAPK activation and exerted the most cardioprotective effects. In contrast, administering SB 203580 10 minutes after reperfusion (a time point when maximal MAPK activation had already been achieved) failed to convey significant cardioprotection. Moreover, inhibition of p38 MAPK attenuated myocardial necrosis after a prolonged reperfusion. CONCLUSIONS: These results demonstrate that p38 MAPK plays a pivotal role in the signal transduction pathway mediating postischemic myocardial apoptosis and that inhibiting p38 MAPK may attenuate reperfusion injury.     

Mitochondrial K(ATP) channel as an end effector of cardioprotection during late preconditioning: triggering role of nitric oxide.

Nitric oxide (NO) has been implicated in the "second-window" of ischemic preconditioning (PC). However, the identity of the end effector after initiation of preconditioning by NO is not known. It is likely that NO is involved in opening of mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels. We hypothesized that NO is an important trigger for the opening of mitoK(ATP) channels in the late phase of preconditioning and inducible nitric oxide synthase (iNOS) up-regulation via NF kappa B plays a critical role in diazoxide-induced cardioprotection. To examine this, diazoxide (7 mg/kg) was administered to wild-type (WT) mice and mice lacking the gene 24 hours before 40 minutes of global ischemia. Hearts were perfused in a Langendorff mode and effects of activation of mitoK(ATP) channel and other interventions on functional, biochemical and pathological changes in ischemic hearts were assessed. In hearts from WT mice treated diazoxide, left-ventricular-developed pressure, end-diastolic pressure and coronary flow were significantly improved after ischemia/reperfusion (I/R); lactate dehydrogenase (LDH) release was also significantly decreased, while ATP contents were significantly higher. Administration of 5-HD, a specific blocker of mitoK(ATP) channel or l -NAME, an inhibitor of iNOS before I/R, during diazoxide-pretreatment completely blocked the late cardioprotection against ischemia. Late cardioprotection was also blocked by inhibition of either PKC- delta by rottlerin or NF kappa B by DDTC before diazoxide pretreatment. Diazoxide pretreatment significantly increased nuclear translocation of p65 which was blocked by protein kinase C (PKC) or nitric oxide synthase (NOS) inhibition. Diazoxide was totally inefffective in iNOS knockout mice. These results suggest that diazoxide activates NF kappa B via PKC signaling pathway and that leads to iNOS up-regulation after 24 hours. NO which is generated upon ischemic stress triggers the opening of mitoK(ATP)channel as an end effector of cardioprotection during late PC.     

Epoxyeicosatrienoic acid prevents postischemic electrocardiogram abnormalities in an isolated heart model

Cytochrome P450 epoxygenases metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) which are in turn converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). The main objective of this study was to investigate the protective effects of EETs following ischemic injury using an ex vivo electrocardiogram (EKG) model. Hearts from C57Bl/6, transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 (Tr) and wildtype (WT) littermates were excised and perfused with constant pressure in a Langendorff apparatus. Electrodes were placed superficially at the right atrium and left ventricle to assess EKG waveforms. In ischemic reperfusion experiments hearts were subjected to 20 min of global no-flow ischemia followed by 20 min of reperfusion (R20). The EKG from C57Bl/6 hearts perfused with 1 μM 14,15-EET showed less QT prolongation (QTc) and ST elevation (STE) (QTc = 41 ± 3, STE = 2.3 ± 0.3; R20: QTc = 42 ± 2 ms, STE = 1.2 ± 0.2mv) than control hearts (QTc = 36 ± 2, STE = 2.3 ± 0.2; R20: QTc = 53 ± 3 ms; STE = 3.6 ± 0.4mv). Similar results of reduced QT prolongation and ST elevation were observed in EKG recording from CYP2J2 Tr mice (QTc = 35 ± 1, STE = 1.9 ± 0.1; R20: QTc = 38 ± 4 ms, STE = 1.3 ± 0.2mv) compared to WT hearts. The putative epoxygenase inhibitor MS-PPOH (50 μM) and EET antagonist 14,15-EEZE (10 μM) both abolished the cardioprotective response, implicating EETs in this process. In addition, separate exposure to the K_ATP channel blockers glibenclamide (1 μM) and HMR1098 (10 μM), or the PKA protein inhibitor H89 (50 nM) during reperfusion abolished the improved repolarization in both the models. Consistent with a role of PKA, CYP2J2 Tr mice had an enhanced activation of the PKAα regulatory II subunit in plasma membrane following IR injury. The present data demonstrate that EETs can enhance the recovery of ventricular repolarization following ischemia, potentially by facilitating activation of K⁺ channels and PKA-dependent signaling.     

Mechanisms by which epoxyeicosatrienoic acids (EETs) elicit cardioprotection in rat hearts

Cytochrome P450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce reductions in infarct size in canine myocardium following ischemia-reperfusion injury via opening of either the sarcolemmal K(ATP) (sarcK(ATP)) or mitochondrial K(ATP) (mitoK(ATP)) channel. In the present study, we subjected intact rat hearts to 30 min of left coronary artery occlusion and 2 h of reperfusion followed by tetrazolium staining to determine infarct size as a percent of the area at risk (IS/AAR, %). The results demonstrate that the two major regioisomers of the CYP epoxygenase pathway, 11,12-EET (2.5 mg/kg, iv) and 14,15-EET (2.5 mg/kg, iv) significantly reduced myocardial infarct size (IS/AAR, %) in rats as compared with control (41.9+/-2.3%, 40.9+/-1.2% versus 61.5+/-1.6%, respectively), whereas, a third regioisomer, 8,9-EET (2.5 mg/kg, iv) had no effect (55.2+/-1.4). The protective effect of pretreatment with 11,12- and 14,15-EETs was completely abolished (61.9+/-0.7%, 58.6+/-3.1%, HMR; 63.3+/-1.2%, 63.2+/-2.5%, 5-HD) in the presence of the selective sarcK(ATP) channel antagonist, HMR 1098 (6 mg/kg, iv) or the selective mitoK(ATP) channel antagonist, 5-HD (10 mg/kg, iv) given 10 min after 11,12- or 14,15-EET administration but 5 min prior to index ischemia. Furthermore, concomitant pretreatment with 11,12- or 14,15-EET in combination with the free radical scavenger, 2-mercaptopropionyl glycine (2-MPG), at a dose (20 mg/kg, iv) that had no effect on IS/AAR (57.7+/-1.3%), completely abolished the cardioprotective effect of 11,12- and 14,15-EETs (58.2+/-1.6%, 61.4+/-1.0%), respectively. These data suggest that part of the cardioprotective effects of EETs in rat hearts against infarction is the result of an initial burst of reactive oxygen species (ROS) and subsequent activation of both the sarcK(ATP) and mitoK(ATP) channel.     

Effect of a novel cardioprotective agent, JTV-519, on metabolism, contraction and relaxation in the ischemia-reperfused rabbit heart

The effect of a novel cardioprotective agent, JTV-519 on myocardial metabolism and contraction during ischemia and reperfusion was investigated by means of phosphorus 31-nuclear magnetic resonance (31P-NMR) in Langendorff rabbit hearts. Normothermic, 20-min, global ischemia was followed by 30-min of postischemic reperfusion and JTV-519 was administered from 40 min prior to the global ischemia. Adenosine triphosphate (ATP), creatine phosphate (PCr), inorganic phosphate (Pi), intracellular pH (pHi), left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) and coronary flow were measured. Fourteen hearts were divided into 2 experimental groups of 7: Group I were controls and Group II were perfused with JTV-519 (10(-6) mol/L). During ischemia, Group II showed a significant (p<0.01) inhibition of the increase in Pi and LVEDP and the decrease in ATP and pHi, compared with Group I. After postischemic reperfusion, Group II also showed a significant (p<0.01) improvement in ATP and pHi as compared with Group I. There were no differences in LVDP or coronary flow during ischemia and reperfusion between the 2 groups. In conclusion, JTV-519 had a significant beneficial effect on myocardial energy metabolism and relaxation during both myocardial ischemia and reperfusion.     

Overexpression of the cardiac beta(2)-adrenergic receptor and expression of a beta-adrenergic receptor kinase-1 (betaARK1) inhibitor both increase myocardial contractility but have differential effects on susceptibility to ischemic injury

Cardiac beta(2)-adrenergic receptor (beta(2)AR) overexpression is a potential contractile therapy for heart failure. Cardiac contractility was elevated in mice overexpressing beta(2)ARs (TG4s) with no adverse effects under normal conditions. To assess the consequences of beta(2)AR overexpression during ischemia, perfused hearts from TG4 and wild-type mice were subjected to 20-minute ischemia and 40-minute reperfusion. During ischemia, ATP and pH fell lower in TG4 hearts than wild type. Ischemic injury was greater in TG4 hearts, as indicated by lower postischemic recoveries of contractile function, ATP, and phosphocreatine. Because beta(2)ARs, unlike beta(1)ARs, couple to G(i) as well as G(s), we pretreated mice with the G(i) inhibitor pertussis toxin (PTX). PTX treatment increased basal contractility in TG4 hearts and abolished the contractile resistance to isoproterenol. During ischemia, ATP fell lower in TG4+PTX than in TG4 hearts. Recoveries of contractile function and ATP were lower in TG4+PTX than in TG4 hearts. We also studied mice that overexpressed either betaARK1 (TGbetaARK1) or a betaARK1 inhibitor (TGbetaARKct). Recoveries of function, ATP, and phosphocreatine were higher in TGbetaARK1 hearts than in wild-type hearts. Despite basal contractility being elevated in TGbetaARKct hearts to the same level as that of TG4s, ischemic injury was not increased. In summary, beta(2)AR overexpression increased ischemic injury, whereas betaARK1 overexpression was protective. Ischemic injury in the beta(2)AR overexpressors was exacerbated by PTX treatment, implying that it was G(s) not G(i) activity that enhanced injury. Unlike beta(2)AR overexpression, basal contractility was increased by betaARK1 inhibitor expression without increasing ischemic injury, thus implicating a safer potential therapy for heart failure.     

Postconditioning with glucagon like peptide-2 reduces ischemia/reperfusion injury in isolated rat hearts: role of survival kinases and mitochondrial KATP channels

We recently reported that heart expresses functional receptors for the anorexigenic glucagon-like peptide (GLP)-2. Activation of these cardiac receptors affected basal heart performance through extracellular regulated kinase (ERK1/2) activation. Since ERK1/2 is considered one of the prosurvival kinases of postconditioning cardioprotective pathways, we hypothesized that GLP-2 directly protects the heart against ischemia/reperfusion (I/R) injury via prosurvival kinases. Wistar rat hearts were retrogradely perfused on a Langendorff perfusion apparatus. After 40-min stabilization, hearts underwent 30-min global ischemia and 120-min reperfusion (I/R group). In GLP-2 group, the hearts received 20-min GLP-2 (10(-7)?M) infusion at the beginning of the 120-min reperfusion. Perfusion pressure and left ventricular pressure (LVP) were monitored. Infarct size was evaluated by nitroblue-tetrazolium staining. Compared with the I/R group, GLP-2-treated hearts showed a significant reduction of infarct size and of postischemic diastolic LVP (index of contracture), together with a sharp improvement of developed LVP recovery (index of contractility). The protective effects were abolished by co-infusion with phosphatidylinositol 3-kinase inhibitor, Wortmannin (WT), the ERK1/2 inhibitor, PD98059, or the mitochondrial K(ATP) channel blocker, 5-hydroxydecanoate. GLP-2 effects were accompanied by increased phosphorylation of protein kinase B (PKB/Akt), ERK1/2 and glycogen synthase kinase (GSK3β). After 7-min reperfusion, WT blocked Akt and GSK3β phosphorylation. After 30-min reperfusion, WT inhibited phosphorylation of all kinases. In conclusion, the data suggest that GLP-2, given in early reperfusion, as postconditioning, protects against myocardial I/R injury, limiting infarct size, and improving post-ischemic mechanical recovery. It seems that the GLP-2-protection of rat heart involves multiple prosurvival kinases and mitochondrial K(ATP) channels.     

Ischemic preconditioning activates MAPKAPK2 in the isolated rabbit heart: evidence for involvement of p38 MAPK

Recent studies suggest that p38 mitogen-activated protein kinase (MAPK) may be involved in ischemic preconditioning (PC). To further test this possibility, the regulation of MAPK-activated protein kinase 2 (MAPKAPK2), a kinase immediately downstream from p38 MAPK, and the activity of c-Jun NH(2)-terminal kinase (JNK), a second MAPK, were examined in preconditioned hearts. Isolated, perfused rabbit hearts were subjected to 20 to 30 minutes of global ischemia. Ventricular biopsies before treatment and after 20 minutes of ischemia were homogenized, and the activities of MAPKAPK2 and JNK were evaluated. For the MAPKAPK2 experiments, 7 groups were studied, as follows: control hearts; preconditioned hearts; hearts treated with 500 nmol/L R(-) N(6)-(2-phenylisopropyl) adenosine (PIA), an A(1)-adenosine receptor agonist; preconditioned hearts pretreated with 100 micromol/L 8-(p-sulfophenyl) theophylline (SPT), an adenosine receptor antagonist; preconditioned hearts also treated with SB 203580, a potent inhibitor of p38 MAPK activation; hearts treated with 50 ng/mL anisomycin (a p38 MAPK/JNK activator); and hearts treated with both anisomycin (50 ng/mL) and the tyrosine kinase inhibitor genistein (50 micromol/L). MAPKAPK2 activity was not altered in control hearts after 20 minutes of global ischemia. By contrast, there was a 3.8-fold increase in activity during ischemia in preconditioned hearts. Activation of MAPKAPK2 in preconditioned hearts was blocked by both SPT and SB 203580. MAPKAPK2 activity during ischemia increased 3.5-fold and 3.3-fold in hearts pretreated with PIA or anisomycin, respectively. MAPKAPK2 activation during ischemia in hearts pretreated with anisomycin was blocked by genistein. In separate hearts, anisomycin mimicked the anti-infarct effect of PC, and that protection was abolished by genistein. JNK activity was measured in control and preconditioned hearts. There was a comparable, modest decline in activity during 30 minutes of global ischemia in both groups. As a positive control, a third group of hearts was treated with anisomycin before global ischemia, and in these, JNK activity increased by 290% above baseline. These results confirm that the p38 MAPK/MAPKAPK2 pathway is activated during ischemia only if the heart is in a preconditioned state. These data further support p38 MAPK as an important signaling component in ischemic PC.     

Expression of activated PKC epsilon (PKC epsilon) protects the ischemic heart, without attenuating ischemic H(+) production

PKC epsilon is a PKC isoform that translocates during preconditioning and may mediate cardioprotection. To investigate whether PKC epsilon activation is cardioprotective, Langendorff-perfused hearts from wild-type (WT) mice and from mice expressing constitutively active mutant PKC epsilon were subjected to 20 min ischemia and 40 min reperfusion while(31)P NMR spectra were acquired. Pre-ischemic glycogen levels were similar in WT and PKC epsilon hearts. During ischemia, ATP fell less in PKC epsilon than in WT hearts. Ischemic intracellular pH, however, was similar in WT and PKC epsilon hearts. During reperfusion, recovery of contractile function and ATP were greater in PKC epsilon than WT hearts. In conclusion, expression of activated PKC epsilon protected hearts from post-ischemic energetic and contractile dysfunction, consistent with the proposed cardioprotective role of PKC epsilon. Protection occurred in the PKC epsilon hearts without attenuation of ischemic H(+) production, implying that, at least in this ischemic model, reduced acidification during ischemia is not necessary for cardioprotection.     

Transgenic expression of Bcl-2 modulates energy metabolism, prevents cytosolic acidification during ischemia, and reduces ischemia/reperfusion injury

The antiapoptotic protein Bcl-2 is targeted to the mitochondria, but it is uncertain whether Bcl-2 affects only myocyte survival after ischemia, or whether it also affects metabolic functions of mitochondria during ischemia. Hearts from mice overexpressing human Bcl-2 and from their wild-type littermates (WT) were subjected to 24 minutes of global ischemia followed by reperfusion. During ischemia, the decrease in pH(i) and the initial rate of decline in ATP were significantly reduced in Bcl-2 hearts compared with WT hearts (P<0.05). The reduced acidification during ischemia was dependent on the activity of mitochondrial F1F0-ATPase. In the presence of oligomycin (Oligo), an F1F0-ATPase inhibitor, the decrease in pH(i) was attenuated in WT hearts, but in Bcl-2 hearts, Oligo had no additional effect on pH(i) during ischemia. Likewise, addition of Oligo to WT hearts slowed the rate of decline in ATP during ischemia to a level similar to that observed in Bcl-2 hearts, but addition of Oligo had no significant effect on the rate of decline in ATP in Bcl-2 hearts during ischemia. These data are consistent with Bcl-2-mediated inhibition of consumption of glycolytic ATP. Furthermore, mitochondria from Bcl-2 hearts have a reduced rate of consumption of ATP on uncoupler addition. This could be accomplished by limiting ATP entry into the mitochondria through the voltage-dependent anion channel, and/or the adenine nucleotide transporter, or by direct inhibition of the F1F0-ATPase. Immunoprecipitation showed greater interaction between Bcl-2 and voltage-dependent anion channel during ischemia. These data indicate that Bcl-2 modulation of metabolism contributes to cardioprotection.     

SB 203580, an inhibitor of p38 MAPK, abolishes infarct-limiting effect of ischemic preconditioning in isolated rabbit hearts

There is debate concerning the involvement of p38 mitogen-activated protein kinase (MAPK) in ischemic preconditioning (PC). At the center of the controversy are data obtained after administration of SB 203580, a specific inhibitor of p38 MAPK. Whereas several studies have reported that SB 203580 abolishes the cardioprotective effect of PC, others claim that this compound is actually cardioprotective against ischemia. Many of these latter observations have been made in isolated myocardial cells. Accordingly the present study was designed to test the effect of SB 203580 in a model of preconditioning in intact rabbit hearts in which infarct size was the end-point. Isolated hearts experienced 30 min of regional ischemia followed by 120 min of reperfusion. Infarct size was measured with triphenyltetrazolium chloride. In control hearts infarction was 30.2 ± 3.3% of the risk zone. PC with 5 min of global ischemia and 10 min of reperfusion before the 30-min period of ischemia significantly reduced infarct size to 10.2 ± 2.4% (P < 0.05 vs. control). SB 203580 (2 μ M) added to the perfusate for 20 min starting 5 min before the index ischemia totally blocked the protection from PC (27.4 ± 3.3% infarction). SB 203580 alone had no effect on infarct size (28.6 ± 4.6% infarction). These results reveal that SB 203580 does not affect infarct size on its own, but selectively blocks preconditioning's anti-infarct effect in the intact rabbit heart. Received: 21 August 2000, Returned for revision: 30 August 2000, Revision received: 2000, Accepted: 6 September 2000     

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.     

Erythropoietin protects the infant heart against ischemia–reperfusion injury by triggering multiple signaling pathways

The immediate protective effect of erythropoietin (EPO) against ischemia in heart suggests a role beyond hematopoiesis and the treatment of anemia. We determined the role of JAK/STAT and Ras/Rac/MAPK in the protective effect of EPO against ischemia–reperfusion injury in infant rabbit heart. EPO (1.0 U/ml) administered 15 minutes prior to 30–minutes global ischemia and 35 minutes reperfusion resulted in increased recovery of postischemic ventricular developed pressure in rabbit hearts. EPO exerted its immediate cardioprotective effect via activation of multiple signaling pathways by: 1) phosphorylation and activation of JAK1/2, STAT3 and STAT5A but not of STAT1 and STAT5B, 2) phosphorylation and activation of PI₃ kinase and its downstream kinases Akt and Rac, 3) activation of PKC, Raf, MEK1/2, p42/44 MAPK and p38 MAPK. Pretreatment with Wortmannin abolished EPO–induced Akt activation and phosphorylation. Pretreatment with Chelerythrine followed by EPO treatment resulted in partial inhibition of Raf activation, and abolished PKC and p38 MAPK activation without any effect on Akt, MEK1/2 and p42/44 MAPK. PD98059 abolished MEK1/2 and p42/44 MAPK activation with no effect on Akt, Raf and p38 MAPK activation. SB203580 inhibited only p38 MAPK activation by EPO. We can conclude EPO increases immediate cardioprotection through the activation of multiple signal transduction pathways.     

Intermittent hypoxia-induced delayed cardioprotection is mediated by PKC and triggered by p38 MAP kinase and Erk1/2

We previously reported that acute intermittent hypoxia (IH) confers delayed cardioprotection against a prolonged ischemic insult in the rat, via the involvement of nitric oxide synthase and K(ATP) channels. In the present study, we investigated the role of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI3K), stress activated p38 MAP kinase (MAPK) and extracellular signal-regulated kinase (ERK1/2) using selective inhibitors of these pathways. Adult male rats were exposed to 1-min cycles of IH (10% O(2), 40 s)/normoxia (21% O(2), 20 s) during 4 h or to normoxic cycles. 24 h later, isolated hearts were perfused in Langendorff mode and subjected to a 30-min global ischemia followed by 120 min of reperfusion. Compared to normoxic conditions, IH significantly reduced infarct size (22.2+/-2.4% vs. 33.8+/-2.6%, p<0.05), improved coronary flow and decreased the contracture at reperfusion. When administered before sustained ischemia, chelerythrine (a PKC inhibitor) abolished both the IH-induced reduction in infarct size (36.1+/-4.9%) and improvement in hemodynamic parameters. In contrast, chelerythrine administration 10 min before IH, did not modify the delayed cardioprotective response. Similarly, wortmannin (a PI3K inhibitor) administration 10 min before IH was unable to block the cardioprotective effects. However, administration of SB203580 (a p38 MAPK inhibitor) and PD98059 (an Erk1/2 inhibitor), 30 min before IH abolished its delayed infarct-sparing effect (32.2+/-3.4% and 33.9+/-2.9%, respectively). In addition, 24 h after IH, a significant increase in p38 MAPK and Erk1/2 phosphorylation was observed by Western blot. These results suggest that the delayed preconditioning induced by intermittent hypoxia does not involve the PI3K signalling pathway and that is mediated by PKC and triggered by p38 MAPK and Erk1/2.     

AMP-activated protein kinase activates p38 mitogen-activated protein kinase by increasing recruitment of p38 MAPK to TAB1 in the ischemic heart

AMP-activated protein kinase (AMPK) promotes glucose transport, maintains ATP stores, and prevents injury and apoptosis during ischemia. AMPK has several direct molecular targets in the heart but also may interact with other stress-signaling pathways. This study examined the role of AMPK in the activation of the p38 mitogen-activated protein kinase (MAPK). In isolated heart muscles, the AMPK activator 5-aminoimidazole-4-carboxy-amide-1-beta-D-ribofuranoside (AICAR) increased p38 MAPK activation. In AMPK-deficient mouse hearts, expressing a kinase-dead (KD) alpha2 catalytic subunit, p38 MAPK activation was markedly reduced during low-flow ischemia (2.3- versus 7-fold in wild-type hearts, P<0.01) and was similarly reduced during severe no-flow ischemia in KD hearts (P<0.01 versus ischemic wild type). Knockout of the p38 MAPK upstream kinase, MAPK kinase 3 (MKK3), did not affect ischemic activation of either AMPK or p38 MAPK in transgenic mkk3(-/-) mouse hearts. Ischemia increased p38 MAPK recruitment to transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1), a scaffold protein that promotes p38 MAPK autophosphorylation. Moreover, TAB1 was associated with the alpha2 catalytic subunit of AMPK. p38 MAPK recruitment to TAB1/AMPK complexes required AMPK activation and was reduced in ischemic AMPK-deficient transgenic mouse hearts. The potential role of p38 MAPK in mediating the downstream action of AMPK to promote glucose transport was also assessed. The p38 MAPK inhibitor SB203580 partially inhibited both AICAR- and hypoxia-stimulated glucose uptake and GLUT4 translocation. Activation of p38 MAPK by anisomycin also increased glucose transport in heart muscles. Thus, AMPK has an important role in promoting p38 MAPK activation in the ischemic heart by inducing p38 MAPK autophosphorylation through interaction with the scaffold protein TAB1.     

Long-term thyroxine administration protects the heart in a pattern similar to ischemic preconditioning.

We have previously shown that long-term thyroxine administration can protect the heart against ischemia. In the present study, we investigated whether thyroxine-induced cardioprotection can mimic the pattern of protection that is afforded by a well-established cardioprotective means such as ischemic preconditioning. In a Langendorff-perfused rat heart preparation, after an initial stabilization, normal and thyroxine-treated hearts were subjected to 20 minutes of zero-flow global ischemia followed by 45 minutes of reperfusion. In thyroxine-treated hearts, phospho-p38 mitogen-activated protein kinase (MAPK) was found to be less at the end of the ischemic period, whereas ischemic contracture was accelerated and postischemic recovery was increased in comparison to normal hearts. In addition, normal hearts were subjected to a four-cycle preconditioning protocol before ischemia. Phospho-p38 MAPK was found to be less at the end of the ischemic period in preconditioned hearts, whereas ischemic contracture was accelerated and postischemic functional recovery was increased in those hearts in comparison to nonpreconditioned hearts. An increase in basal expression and phosphorylation of PKCdelta was also found to occur after long-term thyroxine administration. We conclude that long-term thyroxine administration can protect the heart from ischemic injury through a pattern of protection that closely resembles that of ischemic preconditioning.