Cardioprotective effects of rosiglitazone are associated with selective overexpression of type 2 angiotensin receptors and inhibition of p42/44 MAPK

Current evidence points to renin-angiotensin system as a key mediator in ischemia-reperfusion injury. Rosiglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand, has recently been shown to confer cardioprotection against ischemia-reperfusion in animal models. We sought to examine the expression of ANG II receptors during PPAR-gamma-mediated cardioprotection. Male Sprague-Dawley rats (nondiabetic) were fed either regular rat chow (control diet group, n = 9) or rosiglitazone-rich diet (rosiglitazone-rich diet group, n = 9) and were subjected to 1 h of myocardial ischemia followed by 1 h of reperfusion. A third group of rats had only thoracotomy and pericardiotomy and served as a sham control group (n = 9). Hemodynamics, infarct size, and expression of ANG II type 1 and type 2 receptors (AT1 and AT2) were measured in all groups. There was a 58% reduction of infarct size in the rosiglitazone-rich diet group (P < 0.01 vs. control diet group). Increased myocardial expression of AT(1) receptors in the ischemic-reperfused myocardium was attenuated in the rosiglitazone-rich diet group (P < 0.05 vs. control diet group). Importantly, myocardial AT2 mRNA and protein expression were significantly increased (by >100-fold) in the rosiglitazone-rich diet group (P < 0.05). These changes were accompanied by inhibition of p42/44 MAPK in the rosiglitazone-rich diet group, while the Akt1 expression, believed to mediate insulin sensitization, remained similar in all three groups. The cardioprotective effects of rosiglitazone against myocardial ischemia-reperfusion injury are independent of its insulin-sensitizing properties and are associated with significant overexpression of AT2 receptors along with inhibition of p42/44 MAPK.     

Sphingolipid therapy in myocardial ischemia-reperfusion injury

Sphingolipids are known to play a significant physiological role in cell growth, cell differentiation, and critical signal transduction pathways. Recent studies have demonstrated a significant role of sphingolipids and their metabolites in the pathogenesis of myocardial ischemia-reperfusion injury. Our laboratory has investigated the cytoprotective effects of N,N,N-trimethylsphingosine chloride (TMS), a stable N-methylated synthetic sphingolipid analogue on myocardial and hepatic ischemia-reperfusion injury in clinically relevant in vivo murine models of ischemia-reperfusion injury. TMS administered intravenously at the onset of ischemia reduced myocardial infarct size in the wild-type and obese (ob/ob) mice. Following myocardial I/R, there was an improvement in cardiac function in the wild-type mice. Additionally, TMS also decreased serum liver enzymes following hepatic I/R in wild-type mice. The cytoprotective effects did not extend to the ob/ob mice following hepatic I/R or to the db/db mice following both myocardial and hepatic I/R. Our data suggest that although TMS is cytoprotective following I/R in normal animals, the cytoprotective actions of TMS are largely attenuated in obese and diabetic animals which may be due to altered signaling mechanisms in these animal models. Here we review the therapeutic role of TMS and other sphingolipids in the pathogenesis of myocardial ischemia-reperfusion injury and their possible mechanisms of cardioprotection.     

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.     

Pathophysiology of myocardial reperfusion injury: preconditioning, postconditioning, and translational aspects of protective measures

Heart diseases due to myocardial ischemia, such as myocardial infarction or ischemic heart failure, are major causes of death in developed countries, and their number is unfortunately still growing. Preliminary exploration into the pathophysiology of ischemia-reperfusion injury, together with the accumulation of clinical evidence, led to the discovery of ischemic preconditioning, which has been the main hypothesis for over three decades for how ischemia-reperfusion injury can be attenuated. The subcellular pathophysiological mechanism of ischemia-reperfusion injury and preconditioning-induced cardioprotection is not well understood, but extensive research into components, including autacoids, ion channels, receptors, subcellular signaling cascades, and mitochondrial modulators, as well as strategies for modulating these components, has made evolutional progress. Owing to the accumulation of both basic and clinical evidence, the idea of ischemic postconditioning with a cardioprotective potential has been discovered and established, making it possible to apply this knowledge in the clinical setting after ischemia-reperfusion insult. Another a great outcome has been the launch of translational studies that apply basic findings for manipulating ischemia-reperfusion injury into practical clinical treatments against ischemic heart diseases. In this review, we discuss the current findings regarding the fundamental pathophysiological mechanisms of ischemia-reperfusion injury, the associated protective mechanisms of ischemic pre- and postconditioning, and the potential seeds for molecular, pharmacological, or mechanical treatments against ischemia-reperfusion injury, as well as subsequent adverse outcomes by modulation of subcellular signaling mechanisms (especially mitochondrial function). We also review emerging translational clinical trials and the subsistent clinical comorbidities that need to be overcome to make these trials applicable in clinical medicine.     

Exercise-induced cardioprotection against myocardial ischemia-reperfusion injury

Myocardial ischemia-reperfusion (IR) injury is a major contributor to the morbidity and mortality associated with coronary artery disease. Muscular exercise is a countermeasure to protect against IR-induced cardiac injury in both young and old animals. Specifically, regular bouts of endurance exercise protect the heart against all levels of IR-induced injury. Proposed mechanisms to explain the cardioprotective effects of exercise include alterations in coronary circulation, expression of endoplasmic reticulum stress proteins, increased cyclooxygenase-2 activity, induction of myocardial heat shock proteins, improved cardiac antioxidant capacity, and/or elevation of ATP-sensitive potassium channels on both the sarcolemmal and the mitochondrial inner membranes. Moreover, it seems possible that other, yet to be defined, mechanisms of exercise-induced cardioprotection may also exist. Of the known putative cardioprotective mechanisms, current evidence suggests that elevated myocardial levels of antioxidants and increased expression of sarcolemmal ATP-sensitive potassium channels are both contributors to exercise-induced cardioprotection against IR injury. At present, it is unclear if these two protective mediators act independently or interact to contribute to exercise-induced cardioprotection. Understanding the molecular basis for exercise-induced cardioprotection will provide the required knowledge base to develop therapeutic approaches to protect the heart during an IR insult.     

Inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia-reperfusion injury

Inhalation of hydrogen (H₂) gas has been demonstrated to limit the infarct volume of brain and liver by reducing ischemia-reperfusion injury in rodents. When translated into clinical practice, this therapy must be most frequently applied in the treatment of patients with acute myocardial infarction, since angioplastic recanalization of infarct-related occluded coronary artery is routinely performed. Therefore, we investigate whether H₂ gas confers cardioprotection against ischemia-reperfusion injury in rats. In isolated perfused hearts, H₂ gas enhances the recovery of left ventricular function following anoxia-reoxygenation. Inhaled H₂ gas is rapidly transported and can reach ‘at risk’ ischemic myocardium before coronary blood flow of the occluded infarct-related artery is reestablished. Inhalation of H₂ gas at incombustible levels during ischemia and reperfusion reduces infarct size without altering hemodynamic parameters, thereby preventing deleterious left ventricular remodeling. Thus, inhalation of H₂ gas is promising strategy to alleviate ischemia-reperfusion injury coincident with recanalization of coronary artery.     

The protein kinase C pathway mediates cardioprotection induced by cardiac-specific overexpression of fibroblast growth factor-2

Elucidation of protective mechanisms against ischemia-reperfusion injury is vital to the advancement of therapeutics for ischemic heart disease. Our laboratory has previously shown that cardiac-specific overexpression of fibroblast growth factor-2 (FGF2) results in increased recovery of contractile function and decreased infarct size following ischemia-reperfusion injury and has established a role for the mitogen-activated protein kinase (MAPK) signaling cascade in the cardioprotective effect of FGF2. We now show an additional role for the protein kinase C (PKC) signaling cascade in the mediation of FGF2-induced cardioprotection. Overexpression of FGF2 (FGF2 Tg) in the heart resulted in decreased translocation of PKC-delta but had no effect on PKC-alpha, -epsilon, or -zeta. In addition, multiple alterations in PKC isoform translocation occur during ischemia-reperfusion injury in FGF2 Tg hearts as assessed by Western blot analysis and confocal immunofluorescent microscopy. Treatment of FGF2 Tg and nontransgenic (NTg) hearts with the PKC inhibitor bisindolylmaleimide (1 micromol/l) revealed the necessity of PKC signaling for FGF2-induced reduction of contractile dysfunction and myocardial infarct size following ischemia-reperfusion injury. Western blot analysis of FGF2 Tg and NTg hearts subjected to ischemia-reperfusion injury in the presence of a PKC pathway inhibitor (bisindolylmaleimide, 1 micromol/l), an mitogen/extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK) pathway inhibitor (U-0126, 2.5 micromol/l), or a p38 pathway inhibitor (SB-203580, 2 micromol/l) revealed a complicated signaling network between the PKC and MAPK signaling cascades that may participate in FGF2-induced cardioprotection. Together, these data suggest that FGF2-induced cardioprotection is mediated via a PKC-dependent pathway and that the PKC and MAPK signaling cascades are integrally connected downstream of FGF2.     

Phosphomimetic Modulation of eNOS Improves Myocardial Reperfusion and Mimics Cardiac Postconditioning in Mice

Objective

Myocardial infarction resulting from ischemia-reperfusion injury can be reduced by cardiac postconditioning, in which blood flow is restored intermittently prior to full reperfusion. Although key molecular mechanisms and prosurvival pathways involved in postconditioning have been identified, a direct role for eNOS-derived NO in improving regional myocardial perfusion has not been shown. The objective of this study is to measure, with high temporal and spatial resolution, regional myocardial perfusion during ischemia-reperfusion and postconditioning, in order to determine the contribution of regional blood flow effects of NO to infarct size and protection.

Methods and Results

We used myocardial contrast echocardiography to measure regional myocardial blood flow in mice over time. Reperfusion after myocardial ischemia-reperfusion injury is improved by postconditioning, as well as by phosphomimetic eNOS modulation. Knock-in mice expressing a phosphomimetic S1176D form of eNOS showed improved myocardial reperfusion and significantly reduced infarct size. eNOS knock-out mice failed to show cardioprotection from postconditioning. The size of the no-reflow zone following ischemia-reperfusion is substantially reduced by postconditioning and by the phosphomimetic eNOS mutation.

Conclusions and Significance

Using myocardial contrast echocardiography, we show that temporal dynamics of regional myocardial perfusion restoration contribute to reduced infarct size after postconditioning. eNOS has direct effects on myocardial blood flow following ischemia-reperfusion, with reduction in the size of the no-reflow zone. These results have important implications for ongoing clinical trials on cardioprotection, because the degree of protective benefit may be significantly influenced by the regional hemodynamic effects of eNOS-derived NO.     

Effect of estrogen replacement treatment on ischemic preconditioning in isolated rat hearts

In the present study, we tested the effects of long-term estrogen replacement treatment on myocardial ischemia-reperfusion injury and on the cardioprotection of ischemic preconditioning in isolated hearts from ovariectomized rats. Ovariectomized rats were treated with 17beta-estradiol (30 micro g/kg/d, s.c.) for 12 weeks. Isolated rat hearts were perfused in the Langendorff mode. Heart rate, coronary flow, left ventricular pressure and its first derivative (+/-LVdp/dtmax) were recorded. Fifteen-min global ischemia and 30-min reperfusion caused a significant decrease of cardiac mechanical function, which were not affected by ovariectomy or estrogen replacement treatment. The isolated hearts in all groups could be preconditioned, and the cardioprotection afforded by preconditioning in the sham-operated rats was greater compared with ovariectomized rats with or without estrogen treatment. These results suggest that long-term estrogen replacement treatment exerts no effect on the inhibition of mechanical function after ischemia-reperfusion, and this study also suggests that estrogen does not affect ischemic preconditioning in isolated hearts of ovariectomized rats.     

Exercise,antioxidants, and HSP72: protection against myocardial ischemia/reperfusion

Endurance exercise is associated with protection against myocardial ischemia/reperfusion (I/R) injury and has been shown to increase heat shock protein 72 (HSP72). Dietary antioxidants have also been reported to decrease I/R-induced injury. Because exercise and antioxidants may provide cardioprotection via different mechanisms, combining these countermeasures could provide additive protection. Alternatively, because exercise-induced oxidant production may promote expression of HSP72, antioxidants could attenuate exercise-induced HSP72 expression and decrease exercise-related cardioprotection. These experiments examined the individual and combined effects of exercise and antioxidants on myocardial I/R injury (in vivo). Rats receiving a mixed antioxidant diet or control diet were assigned to exercise or sedentary groups and randomized to receive: (i) short I/R (myocardial stunning), (ii) long I/R (myocardial infarction), or (iii) sham surgery. Antioxidants significantly increased total antioxidant capacity and attenuated exercise-related HSP72 accumulation. Nonetheless, during short I/R, exercise-trained animals demonstrated improved left ventricular developed pressure (LVDP), independent of diet. Further, antioxidants alone resulted in improved LVDP. Finally, compared to control diet/sedentary animals, both exercise groups (control and antioxidant diets) and the antioxidant diet/sedentary group sustained smaller infarctions. We conclude that exercise and antioxidants can independently provide protection against myocardial contractile dysfunction and infarction, and the combination of these two strategies does not enhance or inhibit the protection observed with each individual countermeasure.     

Partial role of nitric oxide in infarct size limiting effect of quercetin and rutin against ischemia-reperfusion injury in normal and diabetic rats

Reperfusion injury is remarkable clinical issue that needs to be resolved as ischemia-reperfusion is a common phenomenon encountered in numerous clinical situations. The present communication report the involvement of nitric oxide (NO) in cardioprotection offered by flavonoids (rutin and quercetin) against myocardial ischemia reperfusion. Rutin produced better cardioprotection than quercetin in normal and diabetic rats. The observed cardioprotection offered with quercetin and rutin was partially abolished by prior administration of nitric oxide synthase inhibitor, L-NAME (N-nitro-L-arginine methyl ester) in both normal and diabetic rats. L-NAME abolished the cardioprotective actions of rutin more strongly than the cardioprotective actions of quercetin. However, mechanistic study with NOS inhibitor implied the possible partial role of nitric oxide in infarct size limiting effect of quercetin and rutin     

Mechanisms by which opening the mitochondrial ATP- sensitive K(+) channel protects the ischemic heart

Diazoxide opening of the mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel protects the heart against ischemia-reperfusion injury by unknown mechanisms. We investigated the mechanisms by which mitoK(ATP) channel opening may act as an end effector of cardioprotection in the perfused rat heart model, in permeabilized fibers, and in rat heart mitochondria. We show that diazoxide pretreatment preserves the normal low outer membrane permeability to nucleotides and cytochrome c and that these beneficial effects are abolished by the mitoK(ATP) channel inhibitor 5-hydroxydecanoate. We hypothesize that an open mitoK(ATP) channel during ischemia maintains the tight structure of the intermembrane space that is required to preserve the normal low outer membrane permeability to ADP and ATP. This hypothesis is supported by findings in mitochondria showing that small decreases in intermembrane space volume, induced by either osmotic swelling or diazoxide, increased the half-saturation constant for ADP stimulation of respiration and sharply reduced ATP hydrolysis. These effects are proposed to lead to preservation of adenine nucleotides during ischemia and efficient energy transfer upon reperfusion.     

Cholesterol diet leads to attenuation of ischemic preconditioning-induced cardiac protection: the role of connexin 43

Cardioprotection by ischemic preconditioning (IP) was abolished in connexin 43 (Cx43)-deficient mice due to loss of Cx43 located in mitochondria rather than at the sarcolemma. IP is lost in hyperlipidemic rat hearts as well. Since changes in mitochondrial Cx43 in hyperlipidemia have not yet been analyzed, we determined total and mitochondrial Cx43 levels in male Wistar rats fed a laboratory chow enriched with 2% cholesterol or normal chow for 12 wk. Hearts were isolated and perfused according to Langendorff. After a 10-min perfusion, myocardial tissue cholesterol, superoxide, and nitrotyrosine contents were measured and Cx43 content in whole heart homogenate and a mitochondrial fraction determined. In the cholesterol-fed group, tissue cholesterol and superoxide formation was increased (P < 0.05), while total Cx43 content remained unchanged. Mitochondrial total and dephosphorylated Cx43 content decreased. Hearts were subjected to an IP protocol (3 × 5 min ischemia-reperfusion) or time-matched aerobic perfusion followed by 30-min global ischemia and 5-min reperfusion. IP reduced infarct size in normal but not in cholesterol-fed rats. At 5-min reperfusion following 30-min global ischemia, the total and dephosphorylated mitochondrial Cx43 content was increased, which was abolished by IP in both normal and high-cholesterol diet. In conclusion, loss of cardioprotection by IP in hyperlipidemia is associated with a redistribution of both sarcolemmal and mitochondrial Cx43.     

Inhibition of Mas G-protein signaling improves coronary blood flow, reduces myocardial infarct size, and provides long-term cardioprotection

The Mas receptor is a class I G-protein-coupled receptor that is expressed in brain, testis, heart, and kidney. The intracellular signaling pathways activated downstream of Mas are still largely unknown. In the present study, we examined the expression pattern and signaling of Mas in the heart and assessed the participation of Mas in cardiac ischemia-reperfusion injury. Mas mRNA and protein were present in all chambers of human hearts, with cardiomyocytes and coronary arteries being sites of enriched expression. Expression of Mas in either HEK293 cells or cardiac myocytes resulted in constitutive coupling to the G(q) protein, which in turn activated phospholipase C and caused inositol phosphate accumulation. To generate chemical tools for use in probing the function of Mas, we performed a library screen and chemistry optimization program to identify potent and selective nonpeptide agonists and inverse agonists. Mas agonists activated G(q) signaling in a dose-dependent manner and reduced coronary blood flow in isolated mouse and rat hearts. Conversely, treatment of isolated rat hearts with Mas inverse agonists improved coronary flow, reduced arrhythmias, and provided cardioprotection from ischemia-reperfusion injury, an effect that was due, at least in part, to decreased cardiomyocyte apoptosis. Participation of Mas in ischemia-reperfusion injury was confirmed in Mas knockout mice, which had reduced infarct size relative to mice with normal Mas expression. These results suggest that activation of Mas during myocardial infarction contributes to ischemia-reperfusion injury and further suggest that inhibition of Mas-G(q) signaling may provide a new therapeutic strategy directed at cardioprotection.     

Hypolipidemic effect of diet supplementation with bacterial levan in cholesterol-fed rats

Levan polysaccharide, a type of fructan, has been shown to have industrial applications as a new industrial gum in the fields of cosmetics, foods like dietary fiber and pharmaceutical goods. The objective of this current study was to investigate the possible hypolipidemic and antioxidative effects of levan in rats fed with a high-cholesterol diet. Animals were allocated into four groups of six rats each: a normal diet group (Control), normal rats received levan (L), a high-cholesterol diet group (Chol) and a high-cholesterol diet with a daily dose of levan equivalent to 5%. Treated hypercholesterolemic rats were administrated with levan in drinking water through oral gavage for 60 days. After the treatment period, the plasma antioxidant enzymes and lipid profiles were determined. Our results show that treatment with levan polysaccharide positively changed plasma antioxidant enzyme activities and lipid profiles (total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides) in cholesterol-rats, and thus may have potential hypolipidemic and antioxidant effects. Levan could protect against oxidative stress linked atherosclerosis and decrease the atherogenic index.     

Coronary and myocardial effects of acetaminophen: protection during ischemia-reperfusion

Acetaminophen is a phenol with antioxidant properties, but little is known about its actions on the mammalian myocardium and coronary circulation. We studied isolated, perfused guinea pig hearts, and tested the hypothesis that acetaminophen-treated hearts would be protected during ischemia-reperfusion. Acetaminophen concentrations in the range of 0.3-0.6 mmol/l caused modest but significant (P < 0.05) coronary vasoconstriction and positive inotropy. The effects were more brisk during constant pressure perfusion than during constant flow. During 20 min of low-flow, global myocardial ischemia and 40 min of reperfusion, hearts treated with acetaminophen retained or recovered a greater percentage of left ventricular function than hearts treated with vehicle. Myofibrillar ultrastructure appeared to be preserved in the reperfused myocardium with acetaminophen. By using chemiluminescence and spin-trap methodologies, we investigated acetaminophen-mediated antioxidant mechanisms to help explain the cardioprotection. The burst of hydroxyl radicals seen between 0 and 10 min of reperfusion was significantly attenuated (P < 0.05) by acetaminophen but not by vehicle. The 3-morpholinosydnominine (SIN-1) generation of peroxynitrite and its oxidative interaction with luminol to produce blue light during ischemia-reperfusion was also blocked by acetaminophen. Our results show that acetaminophen provides significant functional and structural protection to the ischemic-reperfused myocardium, and the mechanism of cardioprotection seems to involve attenuation of the production of both hydroxyl radicals and peroxynitrite.     

Non-invasive limb ischemic pre-conditioning reduces oxidative stress and attenuates myocardium ischemia-reperfusion injury in diabetic rats

This study was to explore whether repeated non-invasive limb ischemic pre-conditioning (NLIP) can confer an equivalent cardioprotection against myocardial ischemia-reperfusion (I/R) injury in acute diabetic rats to the extent of conventional myocardial ischemic pre-conditioning (MIP) and whether or not the delayed protection of NLIP is mediated by reducing myocardial oxidative stress after ischemia-reperfusion. Streptozotocin-induced diabetic rats were randomized to four groups: Sham group, the I/R group, the MIP group and the NLIP group. Compared with the I/R group, both the NLIP and MIP groups showed an amelioration of ventricular arrhythmia, reduced myocardial infarct size, increased activities of total superoxide dismutase (SOD), manganese-SOD and glutathione peroxidase, increased expression of manganese-SOD mRNA and decreased xanthine oxidase activity and malondialdehyde concentration (All p < 0.05 vs I/R group). It is concluded that non-invasive limb ischemic pre-conditioning reduces oxidative stress and attenuates myocardium ischemia-reperfusion injury in diabetic rats.     

Intermittent hypobaric hypoxia improves postischemic recovery of myocardial contractile function via redox signaling during early reperfusion

Intermittent hypobaric hypoxia (IHH) protects hearts against ischemia-reperfusion (I/R) injury, but the underlying mechanisms are far from clear. ROS are paradoxically regarded as a major cause of myocardial I/R injury and a trigger of cardioprotection. In the present study, we investigated whether the ROS generated during early reperfusion contribute to IHH-induced cardioprotection. Using isolated perfused rat hearts, we found that IHH significantly improved the postischemic recovery of left ventricular (LV) contractile function with a concurrent reduction of lactate dehydrogenase release and myocardial infarct size (20.5 ± 5.3% in IHH vs. 42.1 ± 3.8% in the normoxic control, P < 0.01) after I/R. Meanwhile, IHH enhanced the production of protein carbonyls and malondialdehyde, respective products of protein oxidation and lipid peroxidation, in the reperfused myocardium and ROS generation in reperfused cardiomyocytes. Such effects were blocked by the mitochondrial ATP-sensitive K(+) channel inhibitor 5-hydroxydecanoate. Moreover, the IHH-improved postischemic LV performance, enhanced phosphorylation of PKB (Akt), PKC-ε, and glycogen synthase kinase-3β, as well as translocation of PKC-ε were not affected by applying H(2)O(2) (20 μmol/l) during early reperfusion but were abolished by the ROS scavengers N-(2-mercaptopropionyl)glycine (MPG) and manganese (III) tetrakis (1-methyl-4-pyridyl)porphyrin. Furthermore, IHH-reduced lactate dehydrogenase release and infarct size were reversed by MPG. Consistently, inhibition of Akt with wortmannin and PKC-ε with εV1-2 abrogated the IHH-improved postischemic LV performance. These findings suggest that IHH-induced cardioprotection depends on elevated ROS production during early reperfusion.