Ischemia induced activation of heat shock protein 27 kinases and casein kinase 2 in the preconditioned rabbit heart.

Protein kinase C (PKC), p38 MAP kinase, and mitogen-activated protein kinase-activated kinases 2 and 3 (MAPKAPK2 and MAPKAPK3) have been implicated in ischemic preconditioning (PC) of the heart to reduce damage following a myocardial infarct. This study examined whether extracellular signal-regulated kinase (Erk) 1, p70 ribosomal S6 kinase (p70 S6K), casein kinase 2 (CK2), and other hsp27 kinases are also activated by PC, and if they are required for protection in rabbit hearts. CK2 and hsp27 kinase activities declined during global ischemia in control hearts, whereas PC with 5 min ischemia and 10 min reperfusion increased their activities during global ischemia. Resource Q chromatography resolved two distinct peaks of hsp27 phosphotransferase activities; the first peak (at 0.36 M NaCl) appeared to correspond to the 55-kDa MAPKAPK2. Erk1 activity was elevated in both control and PC hearts after post-ischemic reperfusion, but no change was observed in p70 S6K activity. Infarct size (measured by triphenyltetrazolium staining) in isolated rabbit hearts subjected to 30 min regional ischemia and 2 h reperfusion was 31.0+/-2.6% of the risk zone in controls and was 10.3+/-2.2% in PC hearts (p<0.001). Neither the CK2 inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) nor the Mek1/2 inhibitor PD98059 infused during ischemia blocked protection by PC. The activation of CK2 and Erk1 in ischemic preconditioned hearts appear to be epiphenomena and not required for the reduction of infarction from myocardial ischemia.     

Translocation of HSP27 to sarcomere induced by ischemic preconditioning in isolated rat hearts

We investigated the role of the 27-kDa heat shock protein (HSP27) in cardiac protection using Langendorff-perfused rat hearts. After preconditioning (a single episode of 5 min global ischemia followed by 5 min of reperfusion), HSP27 redistributed from the cytosol to the sarcomere and recovery of the contractile function, after 40 min of global ischemia and 50 min of reperfusion, was significantly enhanced. Both SB203580, a p38 MAP kinase inhibitor, and bisindolylmaleimide I, a protein kinase C inhibitor, prevented the effects of preconditioning. Both 2-chloro-N(6)-cyclopentyladenosine (adenosine A1 agonist) and anisomycin (activator of p38 MAP kinase and c-jun N-terminal kinase) mimicked preconditioning. These results suggest that activation of protein kinase C followed by activation of p38 MAP kinase elicits translocation of HSP27 to the sarcomere, a process which may be involved in the cardioprotective mechanism afforded by ischemic preconditioning in rat heart.     

Opening of mitochondrial K(ATP) channel occurs downstream of PKC-epsilon activation in the mechanism of preconditioning

We examined whether the mitochondrial ATP-sensitive K channel (K(ATP)) is an effector downstream of protein kinase C-epsilon (PKC-epsilon) in the mechanism of preconditioning (PC) in isolated rabbit hearts. PC with two cycles of 5-min ischemia/5-min reperfusion before 30-min global ischemia reduced infarction from 50.3 +/- 6.8% of the left ventricle to 20.3 +/- 3.7%. PC significantly increased PKC-epsilon protein in the particulate fraction from 51 +/- 4% of the total to 60 +/- 4%, whereas no translocation was observed for PKC-delta and PKC-alpha. In mitochondria separated from the other particulate fractions, PC increased the PKC-epsilon level by 50%. Infusion of 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) blocker, after PC abolished the cardioprotection of PC, whereas PKC-epsilon translocation by PC was not interfered with 5-HD. Diazoxide, a mitochondrial K(ATP) opener, infused 10 min before ischemia limited infarct size to 5.2 +/- 1.4%, but this agent neither translocated PKC-epsilon by itself nor accelerated PKC-epsilon translocation after ischemia. Together with the results of earlier studies showing mitochondrial K(ATP) opening by PKC, the present results suggest that mitochondrial K(ATP)-mediated cardioprotection occurs subsequent to PKC-epsilon activation by PC.     

The role of C-reactive protein in ischemia/reperfusion injury and preconditioning in a rat model of myocardial infarction

For the first time the involvement of C-Reactive protein (CRP) in early (acute) and delayed ischemic (IPC) and pharmacological (chemical) preconditioning (CPC) in an in vivo model of rat myocardial infarction was presented. Acute IPC was produced by three 5 minute occlusion (ischemia) periods interspersed with 5 minute reperfusion, followed by 30 minute occlusion of the left coronary artery and 2 hour reperfusion injury. Acute CPC was produced by a k-opioid receptor agonist U50488H (5 mg/kg) applied i.v. 15 minutes before 30 minute ischemia/ 2 hour reperfusion. Delayed preconditioning was produced by 30 minute ischemia/ 2 hour reperfusion, induced 24 hour after either ischemic or pharmacological preconditioning. The myocardial ischemia/reperfusion injury was evaluated on the basis of total and cardiac creatine kinase isoenzyme activity, functional recovery of the heart (ECG), infarct size (% IS/RA) and mortality at the end of the experiments. The results obtained showed that: k-opioid receptor agonist U50488H mimics both the acute and delayed IPC in the above experimental protocol; Both acute IPC and most probably CPC act by opening of K(ATP) channels (the effects were blocked by nonspecific ATP-sensitive K channel blocker glybenclamide), and via activation of protein kinase C (a selective protein kinase C inhibitor chelerythrine blocked the efects); C-reactive protein (CRP) was significantly elevated by 54% in non-preconditioned acute ischemia/reperfusion injury. The elevation was more pronounced (82% increase) 24 hour after non-preconditioned ischemia/reperfusion injury. It reflected very well the increase in cardiac isoenzymes, infarct size and mortality of the rats, and can be used as a marker of the severity of myocardial injury in this model; The increase of CRP was prevented by both IPC and CPC in early, and especially in late preconditioning. This confirms the involvement of CRP as a marker in cardiac ischemic/reperfusion injury. It was concluded that in addition to the established involvement of adenosine, bradykinin, opioid and other receptors, a suppression of myocardial CRP/complement production might be involved in the biological mechanism of preconditioning. This could be a promising perspective in clinical interventions against ischemia/reperfusion injuries of the heart.     

Inhibition of phosphatase activity enhances preconditioning and limits cell death in the ischemic/reperfused aged rat heart

Brief, nonlethal episodes of ischemia in the mammalian heart provide cardioprotection against the detrimental effects of a longer duration ischemia. The manifestation of this preconditioning (PC) phenomenon is initiated by the enhanced phosphorylation state of signal transduction proteins. We reported previously that PC is decreased in the aged rat myocardium. Although the mechanism responsible for this loss is not understood, a reduction in the phosphorylation of critical proteins associated with PC may be postulated. Experiments were conducted to investigate whether PC in the aged heart can be restored with the inhibition of endogenous protein phosphatases thereby enhancing phosphorylation of signaling proteins. Levels of phosphatase activities were also assessed with adult heart aging. Hearts from young adult (3-4 mo.) and aged (21-22 mo.) Fischer-344 rats were perfused in the presence or absence of okadaic acid (OKA; 0.1 microM). Aged adult hearts were either not preconditioned or were preconditioned with two PC cycles (5 min ischemia/5 min reperfusion). Myocardial cellular death that developed with a subsequent ischemia was determined with triphenyltetrazolium. With PC, 55% of the aged heart after ischemia was no longer viable. OKA administered before or after ischemia reduced this ischemia-induced cellular death by 29%. Without PC, OKA reduced viability 18% only when present before and after the ischemic episode. OKA in the ischemic young heart during reperfusion reduced the loss of viability 31%. The Protein Phosphatase 2A (PP2A) activity was found to be up to 82% greater in ventricular myocardium of aged rats. In conclusion, aging-induced changes in protein dephosphorylation may be one mechanism reducing the manifestation of preconditioning in the aged heart.     

吸入麻醉预处理心脑保护作用机制的研究进展

吸入麻醉预处理能减轻心脑器官的缺血/缺氧再灌注损伤,作用机制涉及多方面,现主要对麻醉预处理发挥主要作用方面的KATP通道途径,蛋白激酶的激活途径和腺苷途径进行阐述.     

Sphingosine can pre- and post-condition heart and utilizes a different mechanism from sphingosine 1-phosphate

Consistent with previous reports, sphingosine at a high concentration (5 microM) was cardiotoxic as evidenced by increased infarct size in response to ischemia/reperfusion in an ex vivo rat heart. Sphingosine 1-phosphate (S1P) at 5 microM was cardioprotective. However, at a physiologic concentration (0.4 microM) sphingosine as well as S1P was effective in protecting the heart from ischemia/reperfusion injury both when perfused prior to 40 min of ischemia (preconditioning) or when added to reperfusion media following ischemia (postconditioning). Protection by sphingosine and S1P was evidenced with both pre- and post-conditioning by a >75% recovery of left ventricular developed pressure during reperfusion and a decrease in infarct size from 45% of the risk area to less than 8%. When VPC23019, an S1P(1and3)G-protein coupled receptor antagonist, was added to the preconditioning or postconditioning medium along with S1P, it completely blocked S1P-induced protection. However, VPC 23019 did not affect the ability of 0.4 microM sphingosine to either precondition or postcondition hearts. Studies of preconditioning revealed that inhibition of protein kinase C with GF109203X blocked preconditioning by S1P. However, GF109203X did not affect preconditioning by 0.4 microM sphingosine. Likewise, cotreatment with the PI3 kinase inhibitor wortmanin blocked preconditioning by S1P but not by sphingosine. By contrast, inhibition of protein kinase G with KT5823 had no effect on S1P preconditioning but completely eliminated preconditioning by sphingosine. Also, the protein kinase A inhibitory peptide 14-22 amide blocked preconditioning by sphingosine but not S1P. These data reveal for the first time that sphingosine is not toxic at physiologic concentrations but rather is a potent cardioprotectant that utilizes a completely different mechanism than S1P; one that is independent of G-protein coupled receptors and utilizes cyclic nucleotide-dependent pathways.     

Ceramide in the antiapoptotic effect of ischemic preconditioning

Although the mechanism by which ischemic preconditioning (PC) inhibits myocardial apoptosis during ischemia-reperfusion is unclear, evidence indicates a role for the secondary messenger ceramide. We investigated in vivo whether PC may affect ceramide and sn-1,2-diacylglycerol (DAG) production, and attenuate apoptosis during ischemia. Rabbits underwent 30 min of ischemia, followed by 4 h of reperfusion. Before this, they received either no intervention (control group) or one episode of 5 min of ischemia, followed by 5 min of reperfusion (PC group), or an intravenous administration of the sphingomyelinase inhibitor D609. Myocardial content of ceramide and DAG was measured using the DAG kinase assay at different time points of the experiment. Apoptosis was detected and quantified by a sandwich enzyme immunoassay. Both AR and infarct size were measured using blue dye injection and triphenyltetrazolium chloride staining. Control hearts exhibited a peak of ceramide production at 5 min of the prolonged ischemia, with a mean value averaging 64 +/- 5 ng/mg tissue (P < 0.05 vs. 48 +/- 4 ng/mg at baseline). In contrast, ischemic PC and D609 prevented ceramide increase during the prolonged ischemia. Myocardial DAG content was increased only in PC hearts at 30 min of ischemia. Preconditioned and D609 groups developed less apoptosis, as well as a limited infarct size, compared with the control group. These results suggest that the antiapoptotic effect of PC may be due to a reduced ceramide production during sustained ischemia in the rabbit heart.     

SAPKs regulation of ischemic preconditioning

The role of stress-activated protein kinases (SAPKs), c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase, in preconditioning (PC) was examined with the use of isolated rat hearts subjected to four cyclic episodes of 5-min ischemia and 10-min reperfusion followed by 30-min ischemia and 2-h reperfusion (I/R). A group of hearts was preperfused with 100 microM curcumin, a c-Jun and JNK1 inhibitor, or 5 microM SB 203580, a p38 MAP kinase inhibitor. Another group of hearts was preperfused with 20 microM anisomycin, a stimulator for both JNK and p38 MAP kinases. I/R increased the protein levels of JNK1, c-Jun, and p38 MAP kinase. PC also enhanced the induction of these kinases, but subsequent I/R-mediated increase was blocked by PC. Curcumin blocked I/R- and PC-mediated increase in JNK1 and c-Jun protein levels, whereas it had no effects on p38 MAP kinase. SB 203580, on the other hand, was equally effective in reducing the p38 MAP kinase activation but exerted no effects on JNK1 and c-Jun induction. I/R-mediated increased myocardial infarction was reduced by any of the following compounds: anisomycin, curcumin, and SB 203580. The cardioprotective effects of PC were abolished by either curcumin or SB 203580. The results demonstrate that PC is mediated by a signal-transduction pathway involving both JNK1 and p38 MAP kinase. Activation of SAPKs, although transient, is obligatory for PC.     

Neuroprotective effects of preconditioning ischaemia on ischaemic brain injury through inhibition of mixed-lineage kinase 3 via NMDA receptor-mediated Akt1 activation

A number of works show that the mitogen-activated protein kinase (MAPK) signalling pathway responds actively in cerebral ischaemia and reperfusion. We undertook our present studies to clarify the role of mixed-lineage kinase 3 (MLK3), a MAPK kinase kinase (MAPKKK) in MAPK cascades, in global ischaemia and ischaemic tolerance. The mechanism concerning NMDA receptor-mediated Akt1 activation underlying ischaemic tolerance, was also investigated. Sprague-Dawley rats were subjected to 6 min of ischaemia and differing times of reperfusion. Our results showed MLK3 was activated in the hippocampal CA1 region with two peaks occurring at 30 min and 6 h, respectively. This activation returned to base level 3 days later. Both preconditioning with 3 min of sublethal ischaemia and NMDA pretreatment inhibited the 6-h peak of activation. However, pretreatment of ketamine before preconditioning reversed the inhibiting effect of preconditioning on MLK3 activation at 6 h of reperfusion. In the case of Akt1, however, preconditioning and NMDA pretreatment enhanced Akt1 activation at 10 min of reperfusion. Furthermore, ketamine pretreatment reversed preconditioning-induced increase of Akt1 activation. We also noted that pretreatment of LY294002 before preconditioning reversed both the inhibition of MLK3 activation at 6 h of reperfusion and the increase in Akt1 activation at 10 min of reperfusion. The above-mentioned results lead us to conclude that, in the hippocampal CA1 region, preconditioning inhibits MLK3 activation after lethal ischaemia and reperfusion and, furthermore, this effect is mediated by Akt1 activation through NMDA receptor stimulation.     

Biphasic response of cardiac NO synthase isoforms to ischemic preconditioning in conscious rabbits

In conscious rabbits, a sequence of six 4-min coronary occlusion/4-min reperfusion cycles, which elicits late preconditioning (PC), caused rapid activation of calcium-dependent nitric oxide (NO) synthase (NOS) [cNOS; endothelial NOS (eNOS) and/or neuronal NOS (nNOS)], whereas calcium-independent NOS [inducible NOS (iNOS)] activity remained unchanged. The enhanced cNOS activity was associated with increased myocardial levels of NO(2) and/or NO(3) (NO(x)). Twenty-four hours after ischemic PC was induced, the opposite pattern was observed, i.e., there was a pronounced increase in cytosolic iNOS activity but no change in cNOS activity. The initial burst of ischemia-induced cNOS activity was not affected by pretreatment with the antioxidant N-2-mercaptopropionyl glycine (MPG), the protein kinase C (PKC) inhibitor chelerythrine, or the tyrosine kinase inhibitor lavendustin A, indicating that it is independent of the generation of oxidant species and the activation of PKC and tyrosine kinases. In contrast, the delayed upregulation of iNOS 24 h after PC was prevented by pretreatment with N(omega)-nitro-L-arginine, MPG, or chelerythrine before the PC ischemia, indicating that it is triggered by a signaling mechanism that involves the generation of NO, the formation of oxidant species, and the activation of PKC. Taken together, these results demonstrate that, in conscious animals, ischemic PC elicits a biphasic response in cardiac NOS activity, i. e., an immediate activation of cNOS (most likely eNOS) followed 24 h later by a delayed upregulation of iNOS. To our knowledge, this is the first study to directly measure NOS activity after brief myocardial ischemia in vivo. In conjunction with previous functional studies, the data support a distinctive role of NOS isoforms in late PC, with eNOS serving as the trigger on day 1 and iNOS as the mediator on day 2.     

Melatonin does not prevent the protection of ischemic preconditioning in vivo despite its antioxidant effect against oxidative stress

Free radicals are involved in the protective mechanism of preconditioning (PC), whereas antioxidant compounds abolish this benefit. Melatonin is a hormone with antioxidant properties. The aim of our study was to evaluate the effect of melatonin on infarct size in ischemic preconditioning in vivo. We randomly divided 33 male rabbits into four groups and subjected them to 30 min of myocardial ischemia and 3 h of reperfusion with the following prior interventions: (i) no intervention, (ii) iv melatonin at a total dose of 50 mg/kg, (iii) PC with two cycles of 5 min ischemia and 10 min reperfusion, and (iv) combined melatonin and PC. In a second series of experiments, another antioxidant agent N-acetylcysteine (NAC) was used in a control and in a PC group. Myocardial infarct size was determined and blood samples were drawn at different time points for the determination of lipid peroxidation products, total superoxide dismutase (SOD) activity, and (1)H-NMR spectra to evaluate the changes in the metabolic profile. Melatonin showed no effect on myocardial infarct size in the group of sustained ischemia (42.9 +/- 3.6% vs 47.4 +/- 4.9%) and it did not attenuate the reduction of myocardial infarct size in the PC group (13.6 +/- 2.4% vs 14.0 +/- 1.7%). A similar effect was found in NAC-treated groups (44.8 +/- 3.4% vs 14.3 +/- 1.3%). Lipid peroxidation product levels were significantly elevated in the control and PC groups, whereas melatonin decreased them in both groups. The SOD activity was enhanced in the PC group compared to controls; melatonin kept SOD activity unchanged during ischemia/reperfusion and enhanced its activity when it was combined with PC. Melatonin did not change the metabolic profile of the control and PC groups. Melatonin does not prevent the beneficial effect of ischemic PC on infarct size despite its antioxidant properties.     

Delta-opioid receptor activation before ischemia reduces gap junction permeability in ischemic myocardium by PKC-epsilon-mediated phosphorylation of connexin 43

The aim of this study was to examine the hypothesis that delta-opioid receptor activation before ischemia suppresses gap junction (GJ) permeability by PKC-mediated connexin 43 (Cx43) modulation, which contributes to infarct size limitation afforded by the delta-opioid receptor activation. A delta-opioid receptor agonist, [D-Ala(2),D-Leu(5)]-enkephalin acetate (DADLE, 300 nM), was used in place of preconditioning (PC) ischemia to trigger PC mechanisms in rat hearts. GJ permeability during ischemia, which was assessed by Lucifer yellow, was reduced by DADLE to 47% of the control level, and this effect of DADLE was almost abolished by a PKC-epsilon inhibitor [PKC-epsilon translocation inhibitory peptide (PKC-epsilon-TIP)] but was not affected by a PKC-delta inhibitor (rottlerin). After DADLE infusion, PKC-epsilon, but not PKC-delta, was coimmunoprecipitated with Cx43, and the level of phosphorylation of Cx43 at a PKC-dependent site (Ser(368)) was significantly elevated during ischemia. DADLE reduced infarct size after 35 min of ischemia followed by 2 h of reperfusion by 69%, and PKC-epsilon-TIP and rottlerin eliminated 48% and 63%, respectively, of the infarct size-limiting effect of DADLE. Infusion of a GJ blocker, heptanol, before reperfusion reduced infarct size by 36%, and this protection was not enhanced by preischemic infusion of rottlerin + DADLE, which allows PKC-epsilon activation by DADLE. These results suggest that phosphorylation of Cx43 by PKC-epsilon plays a crucial role in delta-opioid-induced suppression of GJ permeability in ischemic myocardium and that this modulation of the GJ is possibly an adjunct mechanism of infarct size limitation afforded by preischemic delta-opioid receptor activation.     

Protective role of gap junctions in preconditioning against myocardial infarction

The aim of the present study was to examine the hypothesis that acceleration of gap junction (GJ) closure during ischemia contributes to anti-infarct tolerance afforded by preconditioning (PC). First, the effects of PC on GJ communication during ischemia were assessed. Isolated buffer-perfused rabbit hearts were subjected to 5-min global ischemia with or without PC with two cycles of 5-min ischemia/5-min reperfusion or a GJ blocker (2 mM heptanol), and then the tissue excised from the ischemic region was incubated in anoxic buffer containing lucifer yellow (LY; 2.5 mg/ml), a tracer of GJ permeability, for 20 min at 37 degrees C. PC and heptanol significantly reduced the area to which LY was transported in the ischemic myocardium by 39% and by 54%, respectively. In the second series of experiments, three GJ blockers (heptanol, 18beta-glycyrrhetinic acid, and 2,3-butanedione monoxime) infused after the onset of ischemia reduced infarct size after 30-min ischemia/2-h reperfusion to an extent equivalent to that in the case of PC. In the third series of experiments, Western blotting for connexin43 (Cx43) showed that PC shortened the time to the onset of ischemia-induced Cx43 dephosphorylation but reduced the extent of Cx43 dephosphorylation during a 30-min period of ischemia. Calphostin C, a protein kinase C (PKC) inhibitor, abolished preservation of phosphorylated Cx43 but not the early onset of Cx43 dephosphorylation after ischemia in the preconditioned myocardium. These results suggest that PC-induced reduction of GJ permeability during ischemia, presumably by PKC-mediated Cx43 phosphorylation, contributes to infarct size limitation.     

Tyrosine kinase signaling in action potential shortening and expression of HSP72 in late preconditioning

We investigated the role of tyrosine kinase (TK) signaling in the opening of the ATP-sensitive K(+) (K(ATP)) channel and 72-kDa heat shock protein (HSP72) expression during late preconditioning. Rabbits were subjected to surgical operation (sham) or were preconditioned (PC) with four cycles of 5 min of ischemia and 10 min of reperfusion. Twenty-four hours later, animals were subjected to 30 min of ischemia and 180 min of reperfusion. Genistein (1 mg/kg ip) was used to block the receptor TK. Six groups were studied: control, sham, genistein-sham, PC, genistein-PC, and vehicle-PC group (1% dimethyl sulfoxide). Genistein or vehicle was given 30 min before the surgical procedure. Genistein pretreatment decreased the expression of HSP72 in PC hearts and suppressed action potential duration shortening during ischemia in sham and PC groups. Infarct size (%risk area) was reduced in the PC (11.6 +/- 1.0%) and vehicle-PC (19.3 +/- 2.0%) compared with the control (40.0 +/- 3.8%) or sham (46.0 +/- 2.0%) groups (P < 0.05). Genistein pretreatment increased infarct size to 46.4 +/- 4.1% in the PC hearts. We conclude that TK signaling is involved in K(ATP) channel opening and HSP72 expression during late PC.     

Myocardial stunning in exercise-induced ischemia in dogs: lack of late preconditioning

Late preconditioning (PC) against myocardial stunning develops after coronary artery occlusion (CAO) at rest and subsequent reperfusion. We investigated whether late PC occurs after exercise-induced ischemia (high-flow ischemia) in dogs. A circumflex coronary artery stenosis (by using occluders) was set up before the onset of treadmill exercise in nine chronically instrumented dogs to suppress exercise-induced increase in mean coronary blood flow velocity (CBFV, Doppler) without simultaneously affecting left ventricular (LV) wall thickening (Wth) at rest. Two similar exercises were performed 24 h apart. On day 1, LV Wth was reduced by 84 +/- 5% (P < 0.01), and exercise-induced increases in transmural myocardial blood flow (MBF, fluorescent microspheres) in the ischemic zone were blunted. LV Wth was depressed throughout the first 10 h and returned to its baseline value after 24 h. On day 2, changes in LV Wth and MBF were similar as was the time course for LV Wth recovery, indicating lack of late PC. Also, CBFV responses to acetylcholine, nitroglycerin, and reactive hyperemia (20-s CAO) were not significantly different on days 1 and 2. Similar results were obtained in a subgroup of four additional dogs with more severe stenosis during exercise. Late PC against myocardial stunning was confirmed to occur in a model of 10-min CAO followed by coronary artery reperfusion (CAR) in another four dogs. Thus in contrast with CAO at rest followed by CAR, severe myocardial ischemia in coronary flow-limited exercising dogs does not induce late PC against myocardial stunning.     

Signalling mechanisms of cardioprotective effect of hypothermic preconditioning

The presented data demonstrate that hypothermic preconditioning prevents cardiomyocyte necrosis in response to ischemia-reperfusion, improves pump function of the heart during reperfusion period, and exerts an antiarrhythmic effect. The hypothermic preconditioning exerts more pronounced cardioprotective effect than ischemic preconditioning. The protective impact of hypothermic preconditioning depends upon 3-adrenergic receptor stimulation, an increase in cAMP levels, activation of protein kinase A and protein kinase C, AMP-activated protein kinase (AMPK) and mitochondrial permeability transition pore blocking. The hypothermic preconditioning had no effect on the phosphorylation of GSK-3J3 (glycogen synthase kinase-3beta) and Akt-kinase. The reactive oxygen species end endogenous catecholamines are triggers or mediators of hypothermic preconditioning of heart.     

Opioid receptor contributes to ischemic preconditioning through protein kinase C activation in rabbits

Recent studies have reported that protection from ischemic preconditioning (PC) is blocked by the opioid receptor antagonist naloxone (NAL). We tested whether an opioid agonist could mimic PC in the rabbit heart, whether that protection involved protein kinase C (PKC) activation, and whether opioid receptors act in concert with other PKC-coupled receptors. Rabbit hearts were subjected to 30min coronary occlusions and were reperfused for either 3 (in situ) or 2 (in vitro) h. Infarct size was determined by staining with triphenyltetrazolium chloride. In untreated in situ hearts 38.5 ± 1.6% of the risk zone infarcted. PC with 5 min ischemia/10 min reperfusion significantly limited infarction to 12.7 ± 2.9% (p < 0.01). NAL infusion did not modify infarction (39.6 ± 1.6%) in non-PC hearts, but blocked the effect of one cycle of PC (34.4 ± 3.6% infarction). NAL, however, could not block cardioprotection when PC was amplified with 3 cycles of ischemia/reperfusion (9.9 ± 1.4% infarction, p < 0.01 vs. control). Morphine could also mimic ischemic preconditioning, but only at a dose much higher than would be used clinically (3 mg/kg). In isolated hearts pretreatment with morphine (0.3 M) significantly limited infarction to 9.3 ± 1.2% (p < 0.01 vs. 32.0 ± 3.1% in controls). This cardioprotective effect of morphine could be blocked by either the PKC inhibitor chelerythrine (30.4 ± 2.6% infarction) or NAL (34.0 ± 2.6% infarction). Neither chelerythrine nor NAL by itself modified infarction in non-PC hearts. NAL could not block protection from one cycle of PC in isolated hearts indicating that an intact innervation may be required for endogenous opioid production. Thus, opioid receptors, like other PKC-coupled receptors, participate in the triggering PC in the rabbit heart.     

Isoflurane Preconditioning Confers Cardioprotection by Activation of ALDH2

The volatile anesthetic, isoflurane, protects the heart from ischemia/reperfusion (I/R) injury. Aldehyde dehydrogenase 2 (ALDH2) is thought to be an endogenous mechanism against ischemia-reperfusion injury possibly through detoxification of toxic aldehydes. We investigated whether cardioprotection by isoflurane depends on activation of ALDH2.Anesthetized rats underwent 40 min of coronary artery occlusion followed by 120 min of reperfusion and were randomly assigned to the following groups: untreated controls, isoflurane preconditioning with and without an ALDH2 inhibitor, the direct activator of ALDH2 or a protein kinase C (PKCε) inhibitor. Pretreatment with isoflurane prior to ischemia reduced LDH and CK-MB levels and infarct size, while it increased phosphorylation of ALDH2, which could be blocked by the ALDH2 inhibitor, cyanamide. Isolated neonatal cardiomyocytes were treated with hypoxia followed by reoxygenation. Hypoxia/reoxygenation (H/R) increased cardiomyocyte apoptosis and injury which were attenuated by isoflurane and forced the activation of ALDH2. In contrast, the effect of isoflurane-induced protection was almost abolished by knockdown of ALDH2. Activation of ALDH2 and cardioprotection by isoflurane were substantially blocked by the PKCε inhibitor. Activation of ALDH2 by mitochondrial PKCε plays an important role in the cardioprotection of isoflurane in myocardium I/R injury.