Dexmedetomidine preconditioning activates pro-survival kinases and attenuates regional ischemia/reperfusion injury in rat heart

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α(2)-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigate whether dexmedetomidine administration activates cardiac survival kinases and induces cardioprotection against regional ischemia/reperfusion injury. In in vivo and ex vivo models, rat hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with dexmedetomidine before ischemia. The α(2)-adrenergic receptor antagonist yohimbine was also given before ischemia, alone or with dexmedetomidine. Erk1/2, Akt and eNOS phosphorylations were determined before ischemia/reperfusion. Cardioprotection after regional ischemia/reperfusion was assessed from infarct size measurement and ventricular function recovery. Localization of α(2)-adrenergic receptors in cardiac tissue was also assessed. Dexmedetomidine preconditioning increased levels of phosphorylated Erk1/2, Akt and eNOS forms before ischemia/reperfusion; being significantly reversed by yohimbine in both models. Dexmedetomidine preconditioning (in vivo model) and peri-insult protection (ex vivo model) significantly reduced myocardial infarction size, improved functional recovery and yohimbine abolished dexmedetomidine-induced cardioprotection in both models. The phosphatidylinositol 3-kinase inhibitor LY-294002 reversed myocardial infarction size reduction induced by dexmedetomidine preconditioning. The three isotypes of α(2)-adrenergic receptors were detected in the whole cardiac tissue whereas only the subtypes 2A and 2C were observed in isolated rat adult cardiomyocytes. These results show that dexmedetomidine preconditioning and dexmedetomidine peri-insult administration produce cardioprotection against regional ischemia/reperfusion injury, which is mediated by the activation of pro-survival kinases after cardiac α(2)-adrenergic receptor stimulation.     

Antioxidant activity contributes to flavonol cardioprotection during reperfusion of rat hearts

The mechanism of flavonol-induced cardioprotection is unclear. We compared the protective actions of a flavonol that inhibits calcium utilization and has antioxidant activity, 3′,4′-dihydroxyflavonol (DiOHF); a flavonol that affects only calcium activity, 4′-OH-3′-OCH₃-flavonol (4′-OH-3′-OCH₃F); and a water-soluble flavonol with selective antioxidant activity, DiOHF-6-succinamic acid (DiOHF-6-SA), in isolated, perfused rat hearts. Hearts were subjected to global ischemia for 20 min followed by 30 min reperfusion and were treated with vehicle (0.05% DMSO), DiOHF, 4′-OH-3′-OCH₃F, or DiOHF-6-SA (all 10 μM, n = 5-8 per group). Flavonols were infused for 10 min before ischemia and during reperfusion. In vehicle-treated hearts, left-ventricular (LV) + dP/dt was reduced by 60% at the end of reperfusion compared to the preischemic level. Lactate dehydrogenase (LDH) release was elevated and endothelial NO synthase (eNOS) expression was lower in vehicle-treated hearts compared to shams. In comparison, DiOHF treatment improved LV function upon reperfusion, decreased LDH, and preserved eNOS expression. The antioxidant DiOHF-6-SA also preserved contractility, reduced LDH, and preserved eNOS expression. In contrast, hearts treated with 4′-OH-3′-OCH₃F showed a degree of contractile impairment similar to that of the vehicle group. DiOHF and DiOHF-6-SA also exerted cardioprotection when given only during reperfusion and not when administered only before ischemia. Flavonol-induced cardioprotection relies on antioxidant activity and is mainly exerted during reperfusion.     

Akt and Erk1/2 activate the ornithine decarboxylase/polyamine system in cardioprotective ischemic preconditioning in rats: the role of mitochondrial permeability transition pores

Ornithine decarboxylase (ODC) is the first rate-limiting enzyme in polyamine biosynthesis, which is essential for cell survival. We hypothesized that the ODC/polyamine system is involved in ischemic preconditioning (IPC)-mediated cardioprotection through the activation of Erk1/2 and Akt and through the inhibition of the mitochondrial permeability transition (mPT). Isolated rat hearts were subjected to 40 min of ischemia either with or without IPC (3 cycles of 5-min global ischemia), and ODC protein expression, polyamine content, and Akt and Erk1/2 phosphorylation were evaluated after 30 min of reperfusion. IPC significantly upregulated the ODC/polyamine pathway, promoted Erk1/2 and Akt phosphorylation, and reduced the infarct size and heart dysfunction after reperfusion. An inhibitor of ODC, α-difluoromethylornithine (DFMO), abolished the IPC-induced cardioprotection. Moreover, the inhibition of the IPC-induced activation of Erk1/2 and Akt using PD98059 or wortmannin downregulated the ODC/polyamine system. In separate studies, the Ca²⁺ load required to open the mPT pore was significantly lower in DFMO-treated cardiac mitochondria than in mitochondria from IPC hearts. Furthermore, spermine or spermidine significantly inhibited the mPT induced by CaCl₂. These results suggest that IPC upregulates the ODC/polyamine system and mediates preconditioning cardioprotection, which may depend on the phosphorylation/activation of Erk1/2 and Akt and on the inhibition of the mPT during reperfusion.     

Myocardial ischemic preconditioning and mitochondrial F1F0-ATPase activity

A short period of ischemia followed by reperfusion (ischemic preconditioning) is known to trigger mechanisms that contribute to the prevention of ATP depletion. In ischemic conditions, most of the ATP hydrolysis can be attributed to mitochondrial F₁F₀-ATPase (ATP synthase). The purpose of the present study was to examine the effect of myocardial ischemic preconditioning on the kinetics of ATP hydrolysis by F₁F₀-ATPase. Preconditioning was accomplished by three 3-min periods of global ischemia separated by 3 min of reperfusion. Steady state ATP hydrolysis rates in both control and preconditioned mitochondria were not significantly different. This suggests that a large influence of the enzyme on the preconditioning mechanism may be excluded. However, the time required by the reaction to reach the steady state rate was increased in the preconditioned group before sustained ischemia, and it was even more enhanced in the first 5 min of reperfusion (101 ± 3.0 sec in preconditioned vs. 83.4 ± 4.4 sec in controls, p 0.05). These results suggest that this transient increase in activation time may contribute to the cardioprotection by slowing the ATP depletion in the very critical early phase of post-ischemic reperfusion.     

Healing the diabetic heart: does myocardial preconditioning work?

Diabetes mellitus-associated ischemic heart disease is a major public burden in industrialized countries. Reperfusion to a previously ischemic myocardium is obligatory to reinstate its function prior to irreversible damage. However, reperfusion is considered ‘a double-edged sword’ as reperfusion per se could augment myocardial ischemic damage, known as myocardial ischemia-reperfusion (I/R) injury. The brief and repeated cycles of I/R given before a sustained ischemia and reperfusion are represented as ischemic preconditioning, which protects the heart from lethal I/R injury. Few studies have demonstrated preconditioning-mediated cardioprotection in the diabetic heart. In contrast, considerable number of studies suggests that myocardial defensive effects of preconditioning are abolished in the presence of chronic diabetes mellitus that raised questions over preconditioning effects in the diabetic heart. It is evidenced that chronic diabetes mellitus-associated deficit in survival pathways, impaired function of mito-K_ATP channels, MPTP opening and high oxidative stress play key roles in paradoxically suppressed cardioprotective effects of preconditioning in the diabetic heart. These controversial results open up a new area of research to identify potential mechanisms influencing disparities on preconditioning effects in diabetic hearts. In this review, we discussed first the discrepancies on the modulatory role of diabetes mellitus in I/R-induced myocardial injury. Following this, we addressed whether preconditioning could protect the diabetic heart against I/R-induced myocardial injury. Moreover, potential mechanisms pertaining to the attenuated cardioprotective effects of preconditioning in the diabetic heart have been delineated. These are important to be understood for better exploitation of preconditioning strategies in limiting I/R-induced myocardial injury in the diabetic heart.     

Preconditioning the hyperlipidemic myocardium: fact or fantasy?

Ischemic heart disease is a leading cause of death worldwide. Myocardial ischemia results in reduced coronary flow, followed by diminished oxygen and nutrient supply to the heart. Reperfusion to an ischemic myocardium often augments the ischemic damage, known as ischemia-reperfusion (I/R) injury. Number of studies demonstrated that the hyperlipidemic myocardium is rather sensitive and more vulnerable to I/R-induced myocardial injury. Repeated brief ischemia and reperfusion cycles, termed as ischemic preconditioning, given before a sustained ischemia is known to reduce myocardial damage occur as a result of I/R. A plethora of evidence supports the fact that preconditioning is one of the promising interventional strategies having an ability to limit I/R-induced myocardial injury. Despite this fact, the preconditioning-mediated cardioprotection is blunted in chronic hyperlipidemic condition. This suggests that preconditioning is moderately a ‘healthy heart protective phenomenon’. The mechanisms by which chronic hyperlipidemia abrogates cardioprotective effects of preconditioning are uncertain and are not completely understood. The impaired opening of mitochondrial-K_ATP channels, eNOS uncoupling and excessive generation of superoxides in the hyperlipidemic myocardium could play a role in attenuating preconditioning-mediated myocardial protection against I/R injury. Moreover, hyperlipidemia-induced loss of cardioprotective effect of preconditioning is associated with redistribution of both sarcolemmal and mitochondrial Connexin 43. We addressed, in this review, the potential mechanisms involved in hyperlipidemia-induced impairment of myocardial preconditioning. Additionally, novel pharmacologic interventions to attenuate hyperlipidemia-associated exaggerated I/R-induced myocardial injury have been discussed.     

Ischemic but not mechanical preconditioning attenuates ischemia/reperfusion induced myocardial apoptosis in anaesthetized rabbits: The role of Bcl-2 family proteins and ERK1/2

Objective: Recent studies suggest that ischemic preconditioning (IPC) inhibits myocardial apoptosis after ischemia and reperfusion. This study aimed first, to examine whether short mechanical stretch with acute pressure overload (MPC), which has been shown to reduce infarct size after ischemia/reperfusion, mimics IPC in attenuating myocardial apoptosis and second, to evaluate whether induced cardioprotection involves modulation of the expression of the Bcl-2 family proteins and phosphorylation of prosurvival kinases. Methods and Results: A model of anaesthetized rabbit was used and the preconditioning protocol included one cycle of short ischemia/reperfusion, or short mechanical stretch with acute pressure overload. Preconditioning stimuli were equally effective in reducing the infarct size, determined after 4 h reperfusion. However, IPC but not MPC attenuated myocardial apoptosis. IPC restored the decreased expression of Bcl-2 and Bcl-xL observed in hearts subjected to ischemia and reperfusion only. Bax levels were not different among the groups. ERK1/2 were activated during reperfusion in both IPC and MPC groups. Conclusions: The data provide further evidence that apoptosis and necrosis contribute independently to infarct size after ischemia and reperfusion. Inhibition of the myocardial apoptotic processes by IPC may involve modulation of the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL. ERK1/2 may be involved in the inhibition of both apoptosis and necrosis.     

Acute treatment with Danshen-Gegen decoction protects the myocardium against ischemia/reperfusion injury via the redox-sensitive PKC?/mKATP pathway in rats

Danshen-Gegen (DG) decoction, an herbal formulation comprising Radix Salvia Miltiorrhiza and Radix Puerariae Lobatae, is prescribed for the treatment of coronary heart disease in Chinese medicine. Experimental and clinical studies have demonstrated that DG decoction can reduce the extent of atherosclerosis. In the present study, using an ex vivo rat model of myocardial ischemia/reperfusion (I/R) injury, we investigated the myocardial preconditioning effect of an aqueous DG extract prepared from an optimized weight-to-weight ratio of Danshen and Gegen. Short-term treatment with DG extract at a daily dose of 1 g/kg and 2 g/kg for 3 days protected against myocardial I/R injury in rats. The cardioprotection afforded by DG pretreatment was paralleled by enhancements in mitochondrial antioxidant status and membrane structural integrity, as well as a decrease in the sensitivity of mitochondria to Ca²⁺-stimulated permeability transition in vitro, particularly under I/R conditions. Short-term treatment with the DG extract also enhanced the translocation of PKC? from the cytosol to mitochondria in rat myocardium, and this translocation was inhibited by α-tocopherol co-treatment with DG extract in rats. Short-term DG treatment may precondition the myocardium via a redox-sensitive PKC?/mK_ATP pathway, with resultant inhibition of the mitochondrial permeability transition through the opening of mitochondrial K_ATP channels. Our results suggest that clinical studies examining the effectiveness of DG extract given prophylactically in affording protection against myocardial I/R injury would be warranted.     

The timing of propofol administration affects the effectiveness of remote ischemic preconditioning induced cardioprotection in rats

The cardioprotection of remote ischemic preconditioning (RIPC) is abolished under propofol maintained anesthesia. Transient receptor potential vanilloid 1 (TRPV1) channel is present in the heart, and its activation could induce cardioprotection. Therefore, we tested whether the anesthetic propofol administration phase interfered with the RIPC-induced cardioprotection, and RIPC-induced cardioprotection via the cardiac TRPV1 channel. Male Sprague-Dawley rats were subjected to myocardial 30 minutes of ischemia followed by 2 hours of reperfusion. RIPC consisted of three cycles of 5-minute ischemia/reperfusion applied to a hindlimb. Propofol infusion at 12 mg/kg/h was commenced either at 10 minutes before the start of RIPC in the P-pre + RIPC group, or immediately after myocardial ischemia at the onset of reperfusion (P-post + RIPC) while performing RIPC. These two propofol infusion regimes were applied to another two grou bs without RIPC (P-pre and P-post groups). Infarct size (IS) was assessed by triphenyltetrazolium staining. Heart TRPV1 expression was detected by Western blot and immunofluorescence. RIPC significantly reduced myocardial IS compared with the control group (36.7 ± 3% versus 57.2 ± 4%; P < .01). When propofol was started before RIPC, the IS sparing effect of RIPC was completely abolished. However, propofol infusion starting immediately after myocardial ischemia did not affect RIPC-induced cardioprotection. TRPV1 expression significant increase after RIPC, then propofol inhibited the TRPV1 activation of RIPC if given before RIPC but not after. Our results suggest that the timing of propofol administration is critical to preserve the cardioprotection of RIPC. Propofol might cancel RIPC-induced cardioprotection via the cardiac TRPV1 receptor.     

Loss of myocardial protection from ischemic preconditioning following chronic exposure to R(-)-N6-(2-phenylisopropyl)adenosine is related to defect at the adenosine A1 receptor

Exogenously administered adenosine agonist will protect myocardium against infarction during ischemia. However, long-term exposure to adenosine agonists is associated with loss of this protection. To determine why this protection is lost, isolated, perfused rabbit hearts were studied after administration of R(-)-N⁶-(2-phenylisopropyl)adenosine (PIA), 0.25 mg/h IP, for 3-4 days to intact animals. All hearts experienced 30 min of regional ischemia and 120 min of reperfusion. Control groups 1 and 2 were untreated. In group 1 this ischemia/reperfusion was the only intervention, whereas group 2 hearts were preconditioned with a cycle of 5 min global ischemia/10 min reperfusion preceding the 30 min regional ischemia. Groups 3-5 had been chronically exposed to PIA. Group 3 hearts had 1 preconditioning ischemia/reperfusion cycle before the prolonged ischemia. Group 4 received a 5 min infusion of 0.1 mol/L phenylephrine in lieu of global ischemia, whereas group 5 was instead treated with 1 mol/L carbachol. Infarct size averaged 32% of the risk zone in group 1, whereas ischemic preconditioning limited infarction to 8.2 in group 2. Prolonged exposure of group 3 hearts to PIA resulted in the inability of preconditioning with 5 min global ischemia to protect (28.7 ± 4.4% infarction). However, protection was restored by either phenylephrine, an agonist of ₁-adrenergic receptors which couple to G_q and stimulate PKC, or carbachol, an agonist of M₂-muscarinic receptors which couple instead to G_i as do adenosine A₁ receptors (5.2 ± 1.7% and 9.2 ± 2.1% infarction, resp.). Therefore, cross tolerance to ischemic preconditioning develops after chronic PIA infusion. Since both the G_i and the PKC components of the preconditioning pathway were shown to be intact, tolerance must have been related to downregulation or desensitization of the A₁ adenosine receptor.     

Insulin like growth factor-1 selectively regulates the expression of matrix metalloproteinase-2 in malignant H-ras transformed cells

The present study is designed to investigate the effect of myocardial preconditioning with oxidative stress induced by pyrogallol or H₂O₂, on ischaemia-reperfusion induced myocardial injuiry. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Four episodes of preconditioning induced by pyrogallol or hydrogen peroxide (H₂O₂) or ischaemia markedly reduced LDH and CK release in coronary effluent and decreased myocardial infarct size. Administration of polymyxin B, a protein kinase C (PKC) inhibitor, during pyrogallol, H₂O₂ or ischaemic preconditioning markedly attenuated the cardioprotective effect of preconditioning produced with oxidative stress or ischaemia. These results suggest that preconditioning with oxidative stress may provide cardioprotection similar to ischaemic preconditioning, against ischaemia-reperfusion injury and this cardioprotective effect may be mediated through activation of PKC.     

Attenuation of Post-ischemia Reperfusion Injury by Thaliporphine and Morphine in Rat Hearts

Summary Pretreatment with thaliporphine before ischemia affords cardioprotective effects against reperfusion injury via antioxidant activity. This study evaluated whether thaliporphine administered at a certain period after myocardial ischemia conferred the same cardioprotection and assessed its possible new mechanism. The left main coronary artery of anaesthetized rats was occluded for 1 h and then reperfused for 2 h. Thaliporphine was administered at 10 min before reperfusion. Controls received saline only. Morphine, a nonselective opioid receptor agonist, was used as reference compound at 0.3 mg/kg. Thaliporphine at 0.05 and 0.5 mg/kg were found to reduce the infarct size. Recovery of cardiac function was higher in thaliporphine (0.5 mg/kg) group, as assessed by a significant improvement in the rates of pressure development (+dp/dt _max). This compound also reduced plasma creatine kinase and cardiac MPO activity. These protective effects afforded by thaliporphine were diminished by the opioid receptor antagonists (naloxone or naltrexone) and by the mitochondrial K_ATP blocker 5HD. In comparison, morphine reduced infarct size and MPO activity in the myocardium but produced slightly improvement in cardiac function after ischemia-reperfusion. These results demonstrate that reperfusion therapy with thaliporphine protect cardiac injury through further mechanism via activation of opioid receptor and opening of mitochondrial K_ATP channels as morphine but with stronger activity.     

Beetroot juice reduces infarct size and improves cardiac function following ischemia–reperfusion injury: Possible involvement of endogenous H2S

Ingestion of high dietary nitrate in the form of beetroot juice (BRJ) has been shown to exert antihypertensive effects in humans through increasing cyclic guanosine monophosphate (cGMP) levels. Since enhanced cGMP protects against myocardial ischemia–reperfusion (I/R) injury through upregulation of hydrogen sulfide (H₂S), we tested the hypothesis that BRJ protects against I/R injury via H₂S. Adult male CD-1 mice received either regular drinking water or those dissolved with BRJ powder (10 g/L, containing ∼0.7 mM nitrate). Seven days later, the hearts were explanted for molecular analyses. Subsets of mice were subjected to I/R injury by occlusion of the left coronary artery for 30 min and reperfusion for 24 h. A specific inhibitor of H₂S producing enzyme – cystathionine-γ-lyase (CSE), dl-propargylglycine (PAG, 50 mg/kg) was given i.p. 30 min before ischemia. Myocardial infarct size was significantly reduced in BRJ-fed mice (15.8 ± 3.2%) versus controls (46.5 ± 3.5%, mean ± standard error [SE], n = 6/group, P < .05). PAG completely blocked the infarct-limiting effect of BRJ. Moreover, BRJ significantly preserved ventricular function following I/R. Myocardial levels of H₂S and its putative protein target – vascular endothelial growth factor receptor 2 (VEGFR2) were significantly increased by BRJ intake, whereas CSE mRNA and protein content did not change. Interestingly, the BRJ-induced cardioprotection was not associated with elevated blood nitrate–nitrite levels following I/R nor induction of cardiac peroxiredoxin 5, a mitochondrial antioxidant enzyme previously linked to nitrate-induced cardioprotection. We conclude that BRJ ingestion protects against post-I/R myocardial infarction and ventricular dysfunction possibly through CSE-mediated endogenous H₂S generation. BRJ could be a promising natural and inexpensive nutraceutical supplement to reduce cardiac I/R injury in patients.     

AMPK-Regulated and Akt-Dependent Enhancement of Glucose Uptake Is Essential in Ischemic Preconditioning-Alleviated Reperfusion Injury

Aims

Ischemic preconditioning (IPC) is a potent form of endogenous protection. However, IPC-induced cardioprotective effect is significantly blunted in insulin resistance-related diseases and the underlying mechanism is unclear. This study aimed to determine the role of glucose metabolism in IPC-reduced reperfusion injury.

Methods

Normal or streptozotocin (STZ)-treated diabetic rats subjected to 2 cycles of 5 min ischemia/5 min reperfusion prior to myocardial ischemia (30 min)/reperfusion (3 h). Myocardial glucose uptake was determined by ¹⁸F-fluorodeoxyglucose-positron emission tomography (PET) scan and gamma-counter biodistribution assay.

Results

IPC exerted significant cardioprotection and markedly improved myocardial glucose uptake 1 h after reperfusion (P<0.01) as evidenced by PET images and gamma-counter biodistribution assay in ischemia/reperfused rats. Meanwhile, myocardial translocation of glucose transporter 4 (GLUT4) to plasma membrane together with myocardial Akt and AMPK phosphorylation were significantly enhanced in preconditioned hearts. Intramyocardial injection of GLUT4 siRNA markedly decreased GLUT4 expression and blocked the cardioprotection of IPC as evidence by increased myocardial infarct size. Moreover, the PI3K inhibitor wortmannin significantly inhibited activation of Akt and AMPK, reduced GLUT4 translocation, glucose uptake and ultimately, depressed IPC-induced cardioprotection. Furthermore, IPC-afforded antiapoptotic effect was markedly blunted in STZ-treated diabetic rats. Exogenous insulin supplementation significantly improved glucose uptake via co-activation of myocardial AMPK and Akt and alleviated ischemia/reperfusion injury as evidenced by reduced myocardial apoptosis and infarction size in STZ-treated rats (P<0.05).

Conclusions

The present study firstly examined the role of myocardial glucose metabolism during reperfusion in IPC using direct genetic modulation in vivo. Augmented glucose uptake via co-activation of myocardial AMPK and Akt in reperfused myocardium is essential to IPC-alleviated reperfusion injury. This intrinsic metabolic modulation and cardioprotective capacity are present in STZ-treated hearts and can be triggered by insulin.     

Cardioprotective effect of polydatin against ischemia/reperfusion injury: Roles of protein kinase C and mito KATP activation

Polydatin preconditioning (PPC) has been reported to be protective against brain and intestine ischemia/reperfusion injury (I/R injury), but whether polydatin exerts cardioprotective effect against myocardial ischemia/reperfusion and the underlying mechanisms remain unclear. Previous studies have demonstrated that oxidative stress plays an important role in the process of I/R. Elevation of oxidative agents and decline in anti-oxidant substance would promote I/R. Meanwhile, the activation of PKC signaling seems to mediate the cardioprotective effects of many drugs by alleviating Ca²⁺ influx. In the present study, we reported for the first time that intravenous administration of polydatin before I/R significantly limited the infarct size, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) leakage from the damaged myocardium after I/R. The activity of SOD and the content of MDA remarkably changed in the presence of polydatin as well. However, the cardiac function-preserving and myocardial enzymes leakage-limiting effects of polydatin vanished in the presence of PKC inhibitors and mito K_ATP channel blockers. But there was not a significant change in the activity of SOD and MDA content. We therefore conclude that PPC exerts cardioprotective effect by the activation of PKC-K_ATP-dependent signaling and the direct anti-oxidative stress mechanisms.     

Cardioprotection: A radical view: Free radicals in pre and postconditioning

A series of brief (a few minutes) ischemia/reperfusion cycles (ischemic preconditioning, IP) limits myocardial injury produced by a subsequent prolonged period of coronary artery occlusion and reperfusion. Postconditioning (PostC), which is a series of brief (a few seconds) reperfusion/ischemia cycles at reperfusion onset, attenuates also ischemia/reperfusion injury. In recent years the main idea has been that reactive oxygen species (ROS) play an essential, though double-edged, role in cardioprotection: they may participate in reperfusion injury or may play a role as signaling elements of protection in the pre-ischemic phase. It has been demonstrated that preconditioning triggering is redox-sensitive, using either ROS scavengers or ROS generators. We have shown that nitroxyl triggers preconditioning via pro-oxidative, and/or nitrosative stress-related mechanism(s). Several metabolites, including acetylcholine, bradykinin, opioids and phenylephrine, trigger preconditioning-like protection via a mitochondrial K_ATP-ROS-dependent mechanism. Intriguingly, and contradictory to the above mentioned theory of ROS as an obligatory part of reperfusion-induced damage, some studies suggest the possibility that some ROS at low concentrations could protect ischemic hearts against reperfusion injury. Yet, we demonstrated that ischemic PostC is also a cardioprotective phenomenon that requires the intervention of redox signaling to be protective. Emerging evidence suggests that in a preconditioning scenario a redox signal is required during the first few minutes of myocardial reperfusion following the index ischemic period. Intriguingly, the ROS signaling in the early reperfusion appear crucial to both preconditioning- and postconditioning-induced protection. Therefore, our and others' results suggest that the role of ROS in reperfusion may be reconsidered as they are not only deleterious.     

The obligatory role of COX-2 expression for induction of HO-1 in ischemic preconditioned rat brain

The molecular mechanisms of preconditioning-induced ischemic tolerance (PCIT) have yet to be elucidated. We investigated whether minimal expression levels of COX-2 induced by preconditioning trigger HO-1, thereby inducing the synthesis of cytoprotective proteins. We show that both COX-2 and HO-1 are induced in rat brains subjected to preconditioning by middle cerebral artery (MCA) occlusion for 10 min followed by different amounts of reperfusion time (1-24 h). Although preconditioning significantly reduced the brain infarct size against severe ischemia (24 h MCA occlusion), pretreatment with the COX-2-selective inhibitor rofecoxib increased infarct size and abolished PCIT-induced COX-2 and HO-1 expression in vivo. We also found that PGE₂ increased the phosphorylation of Akt, which was significantly inhibited by the PI3 kinase inhibitor LY294002. Taken together, we conclude that the kinetic changes in COX-2 induction during the reperfusion period following preconditioning may be important for ischemic tolerance.     

A humanin analog decreases oxidative stress and preserves mitochondrial integrity in cardiac myoblasts

A potent analog (HNG) of the endogenous peptide humanin protects against myocardial ischemia–reperfusion (MI–R) injury in vivo, decreasing infarct size and improving cardiac function. Since oxidative stress contributes to the damage from MI–R we tested the hypotheses that: (1) HNG offers cardioprotection through activation of antioxidant defense mechanisms leading to preservation of mitochondrial structure and that, (2) the activity of either of a pair of non-receptor tyrosine kinases, c-Abl and Arg is required for this protection. Rat cardiac myoblasts (H₉C₂ cells) were exposed to nanomolar concentrations of HNG and to hydrogen peroxide (H₂O₂). Cells treated with HNG in the presence of H₂O₂ demonstrated reduced intracellular reactive oxygen species (ROS), preserved mitochondrial membrane potential, ATP levels and mitochondrial structure. HNG induced activation of catalase and glutathione peroxidase (GPx) within 5 min and decreased the ratio of oxidized to reduced glutathione within 30 min. siRNA knockdown of both Abl and Arg, but neither alone, abolished the HNG-mediated reduction of ROS in myoblasts exposed to H₂O₂. These findings demonstrate an HNG-mediated, Abl- and Arg-dependent, rapid and sustained activation of critical cellular defense systems and attenuation of oxidative stress, providing mechanistic insights into the observed HNG-mediated cardioprotection in vivo.     

The role of mitochondrial membrane potential in ischemic heart failure

The molecular events occurring during myocardial infarction and cardioprotection are described with an emphasis on the changes of the mitochondrial membrane potential (ΔΨ_m). The low ΔΨ_m values of the normal beating heart (100–140 mV) are explained by the allosteric ATP-inhibition of cytochrome c oxidase (CcO) through feedback inhibition by ATP at high [ATP]/[ADP] ratios. During ischemia the mechanism is reversibly switched off by signaling through reactive oxygen species (ROS). At reperfusion high ΔΨ_m values cause a burst of ROS production leading to apoptosis and/or necrosis. Ischemic preconditioning is suggested to cause additional phosphorylation of CcO, protecting the enzyme from immediate dephosphorylation via ROS signaling.