Classic Ischemic Preconditioning Research Articles

Inhibition of NOX2 or NLRP3 inflammasome prevents cardiac remote ischemic preconditioning.

Introduction: Short episodes of ischemia-reperfusion (IR) in the heart (classical ischemic preconditioning, IPC) or in a limb (remote ischemic preconditioning, RIPC) before a prolonged ischemic episode, reduce the size of the infarct. It is unknown whether IPC and RIPC share common mechanisms of protection. Animals KO for NOX2, a superoxide-producing enzyme, or KO for NLRP3, a protein component of inflammasome, are not protected by IPC. The aim of this study was to investigate if NOX2 or NLRP3 inflammasome are involved in the protection induced by RIPC. Methods: We preconditioned rats using 4 × 5min periods of IR in the limb with or without a NOX2 inhibitor (apocynin) or an NLRP3 inhibitor (Bay117082). In isolated hearts, we measured the infarct size after 30min of ischemia and 60min of reperfusion. In hearts from preconditioned rats we measured the activity of NOX2; the mRNA of Nrf2, gamma-glutamylcysteine ligase, glutathione dehydrogenase, thioredoxin reductase and sulfiredoxin by RT-qPCR; the content of glutathione; the activation of the NLRP3 inflammasome and the content of IL-1β and IL-10 in cardiac tissue. In exosomes isolated from plasma, we quantified NOX2 activity. Results: The infarct size after IR decreased from 40% in controls to 9% of the heart volume after RIPC. This protective effect was lost in the presence of both inhibitors. RIPC increased NOX2 activity in the heart and exosomes, as indicated by the increased association of p47phox to the membrane and by the increased oxidation rate of NADPH. RIPC also increased the mRNA of Nrf2 and antioxidant enzymes. Also, RIPC increased the content of glutathione and the GSH/GSSG ratio. The inflammasome proteins NLRP3, procaspase-1, and caspase-1 were all increased in the hearts of RIPC rats. At the end of RIPC protocol, IL-1β increased in plasma but decreased in cardiac tissue. At the same time, IL-10 did not change in cardiac tissue but increased by 70% during the next 50min of perfusion. Conclusion: RIPC activates NOX2 which upregulates the heart's antioxidant defenses and activates the NLRP3 inflammasome which stimulates a cardiac anti-inflammatory response. These changes may underlie the decrease in the infarct size induced by RIPC.

Remote ischemic preconditioning preserves Connexin 43 phosphorylation in the rat heart in vivo.

BackgroundRemote ischemic preconditioning (RIPC) protects the heart from ischemia and reperfusion (I/R) injury. The underlying molecular mechanisms are unclear. It has been demonstrated that Connexin 43 (Cx43) is critically involved in cardioprotective interventions including classical ischemic preconditioning. In the present study we investigated the influence of RIPC on the expression patterns of Cx43 after I/R in the rat heart in vivo.MethodsMale Wistar rats were subjected to 35 min regional myocardial ischemia followed by 2 h reperfusion with or without 4 cycles of 5 minutes bilateral hind limb ischemia and reperfusion (RIPC), to RIPC without ischemia or underwent no intervention (Sham). Infarct size was measured by TTC staining. The myocardium was divided into area at risk (AAR) and area not at risk (non AAR). Expression of Cx43-mRNA and protein was analyzed by qPCR and Western Blot analysis, respectively. Localization of Cx43 was visualized by confocal immunofluorescence staining.ResultsRIPC reduced the infarct size (I/R: 73 ± 5% vs. RIPC I/R: 34 ± 14%, p < 0.05). Expression of Cx43 mRNA did not differ between groups. I/R caused a strong decrease of relative Cx43 protein expression in the AAR that was partly abolished by RIPC. Furthermore, RIPC decreased the level of ischemia-induced dephosphorylation of Cx43. Confocal immunofluorescence staining showed that I/R caused a loss of the Cx43 signal at the intercalated discs, while the Cx43 signal at the intercalated discs was partly sustained after RIPC.ConclusionPreservation of Cx43 protein expression and phosphorylation after RIPC might protect the rat heart in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-014-0228-8) contains supplementary material, which is available to authorized users.

Role of Protein Kinase C in Ischemic “Conditioning”

Since the discovery of ischemic preconditioning (IPC) 26 years ago, numerous studies attempted to determine the mechanism of this powerful form of cardioprotection. One of the first proposed pathways of IPC suggested that the preconditioning stimulus activated phospholipase C via G-protein, and diacylglycerol released from phospholipid moieties activated protein kinase C (PKC) by translocating it from the cytosol to the sarcolemmal membranes. The major protective isoform of PKC was found to be the PKC-∈. Despite some contradictions and controversies, today even the most skeptical opponents acknowledge that PKC plays a significant role in the mechanism of IPC. During recent years, both the role and the place of PKC-∈ in the mechanism of IPC have been revised. The current review presents the evolution of the "PKC theory" and summarizes the most recent data regarding the role of PKC in IPC. In addition to classical IPC, PKC appears to play a role in the mechanisms of newer conditioning protocols, that is, remote IPC and ischemic postconditioning.

Novel Rapid‐Multiple‐Short‐Cycle Preconditioning Stimuli Induces Robust Cardioprotective Signaling Mechanisms and Protects the Heart against in vivo Ischemia Reperfusion Injury: An Effective Approach with Clinical Justification

Despite the large body of basic research, the well‐proved phenomenon of ischemic preconditioning (IPC) still lacks its clinical benefits. In clinics, balloon inflation/deflation cycles during coronary interventions are restricted to &lt;1‐min in duration; and multiple balloon inflations/deflations are required for complex lesions. Recently, we have shown that rapid brief 60‐sec cycle of ischemia/reperfusion (I/R) induces small burst of reactive oxygen species (ROS) formation that could trigger IPC like protection. Therefore, to investigate whether a novel rapid‐multiple‐short‐cycle preconditioning (RPC) stimuli induces in vivo cardioprotection, rats were subjected to in vivo regional I/R (30‐min I and 24‐hr R) preceded by RPC (6 cycles of 60‐sec I and 60‐ sec R) or classical IPC (C‐IPC; 3 cycles of 5‐min I and 5‐min R). Cardiac function, infarction, ROS generation, survival kinase and nitric oxide synthase (NOS) pathways were characterized. RPC conferred robust cardioprotection compared to C‐IPC. This was manifested by reduced myocardial infarction, improved postischemic cardiac function, reduced ROS generation, effective activation of Akt and ERK1/2, as well as enhanced NOS expression and activation. Thus, this novel IPC stimuli RPC is highly effective in preserving myocardial viability and function in the setting of acute myocardial I/R. (Funding: NIH grants HL63744, HL65608, and HL38324 to JLZ)

Clinically Useful Cardioprotection: Ischemic Preconditioning Then and Now

Ischemic preconditioning (IP) is the most effective, reproducible form of protection against myocardial cell death yet described. The mechanism of classic IP has not been identified, but recent investigations have focused on the mitochondrial permeability transition pore (mPTP). Similarly, the mechanism of the "second window of protection" (SWOP) is not known but thought to involve increased expression of important gene products. Currently, IP in the clinical arena is limited to cardiac surgery, planned angioplasty, and organ preservation protocols. To move preconditioning into a broader clinical arena will require resolution of important fundamental yet stubborn problems involving both basic and clinical science. Important unresolved issues include the mechanisms involved in the transition from reversible to irreversible injury, the amount of potential salvageable myocardium present at the onset of reperfusion, the identity and signaling of the mPTP, the optimization of protocols, the identity of end effectors (SWOP), and the identification of the best experimental model systems. From a clinical standpoint, important issues include the influence of comorbidities on cardioprotection, identification of appropriate animal models, the lack of a biologic marker of the cardioprotective state, the influence of coexistent therapeutic drugs, potential toxicity of pharmacologic mimics, and the window of opportunity for significant protection. Ischemic preconditioning has yielded promising results in other organs including the brain as well as tissue preservation for certain surgical procedures that will require definition of the underlying mechanism(s) to be fully exploited clinically. Over the past 25 years, the scientific community has learned much regarding the biology and potential mechanisms of IP and the concept has been expanded to many other organ systems in many other clinically relevant scenarios. To realize the full clinical potential will require continued investigation into the mechanism.

Metabolic fingerprint of ischaemic cardioprotection: importance of the malate-aspartate shuttle

The convergence of cardioprotective intracellular signalling pathways to modulate mitochondrial function as an end-target of cytoprotective stimuli is well described. However, our understanding of whether the complementary changes in mitochondrial energy metabolism are secondary responses or inherent mechanisms of ischaemic cardioprotection remains incomplete. In the heart, the malate-aspartate shuttle (MAS) constitutes the primary metabolic pathway for transfer of reducing equivalents from the cytosol into the mitochondria for oxidation. The flux of MAS is tightly linked to the flux of the tricarboxylic acid cycle and the electron transport chain, partly by the amino acid l-glutamate. In addition, emerging evidence suggests the MAS is an important regulator of cytosolic and mitochondrial calcium homeostasis. In the isolated rat heart, inhibition of MAS during ischaemia and early reperfusion by the aminotransferase inhibitor aminooxyacetate induces infarct limitation, improves haemodynamic responses, and modulates glucose metabolism, analogous to effects observed in classical ischaemic preconditioning. On the basis of these findings, the mechanisms through which MAS preserves mitochondrial function and cell survival are reviewed. We conclude that the available evidence is supportive of a down-regulation of mitochondrial respiration during lethal ischaemia with a gradual 'wake-up' during reperfusion as a pivotal feature of ischaemic cardioprotection. Finally, comments on modulating myocardial energy metabolism by the cardioprotective amino acids glutamate and glutamine are given.

Aldehyde Dehydrogenase Activation Prevents Reperfusion Arrhythmias by Inhibiting Local Renin Release From Cardiac Mast Cells

Renin released by ischemia/reperfusion from cardiac mast cells activates a local renin-angiotensin system (RAS). This exacerbates norepinephrine release and reperfusion arrhythmias (ventricular tachycardia and fibrillation), making RAS a new therapeutic target in myocardial ischemia. We investigated whether ischemic preconditioning (IPC) prevents cardiac RAS activation in guinea pig hearts ex vivo. When ischemia/reperfusion (20 minutes of ischemia/30 minutes of reperfusion) was preceded by IPC (two 5-minute ischemia/reperfusion cycles), renin and norepinephrine release and ventricular tachycardia and fibrillation duration were markedly decreased, a cardioprotective anti-RAS effect. Activation and blockade of adenosine A(2b)/A(3) receptors and activation and inhibition of protein kinase Cepsilon (PKCepsilon) mimicked and prevented, respectively, the anti-RAS effects of IPC. Moreover, activation of A(2b)/A(3) receptors or activation of PKCepsilon prevented degranulation and renin release elicited by peroxide in cultured mast cells (HMC-1). Activation and inhibition of mitochondrial aldehyde dehydrogenase type-2 (ALDH2) also mimicked and prevented, respectively, the cardioprotective anti-RAS effects of IPC. Furthermore, ALDH2 activation inhibited degranulation and renin release by reactive aldehydes in HMC-1. Notably, PKCepsilon and ALDH2 were both activated by A(2b)/A(3) receptor stimulation in HMC-1, and PKCepsilon inhibition prevented ALDH2 activation. The results uncover a signaling cascade initiated by A(2b)/A(3) receptors, which triggers PKCepsilon-mediated ALDH2 activation in cardiac mast cells, contributing to IPC-induced cardioprotection by preventing mast cell renin release and the dysfunctional consequences of local RAS activation. Thus, unlike classic IPC in which cardiac myocytes are the main target, cardiac mast cells are the critical site at which the cardioprotective anti-RAS effects of IPC develop.

ENOS is required for acute in vivo ischemic preconditioning of the heart: effects of ischemic duration and sex

Ischemic preconditioning (IPC) is a powerful phenomenon that provides potent cardioprotection in mammalian hearts; however, the role of endothelial nitric oxide (NO) synthase (eNOS)-mediated NO in this process remains highly controversial. Questions also remain regarding this pathway as a function of sex and ischemic duration. Therefore, we performed extensive experiments in wild-type (WT) and eNOS knockout (eNOS(-/-)) mice to evaluate whether the infarct-limiting effect of IPC depends on eNOS, ischemic periods, and sex. Classical IPC was induced by three cycles of 5 min of regional coronary ischemia separated by 5 min of reperfusion and was followed by 30 or 60 min of sustained ischemia and 24 h of reperfusion. The control ischemia-reperfusion protocol had 30 or 60 min of ischemia followed by 24 h of reperfusion. Protection was evaluated by measuring the myocardial infarct size as a percentage of the area at risk. The major findings were that regardless of sex, WT mice exhibited robust IPC with significantly smaller myocardial infarction, whereas eNOS(-/-) mice did not. IPC-induced cardiac protection was absent in eNOS(-/-) mice of both Jackson and Harvard origin. In general, female WT mice had smaller infarctions compared with male WT mice. Although prolonged ischemia caused significantly larger infarctions in WT mice of both sexes, they were consistently protected by IPC. Importantly, prolonged myocardial ischemia was associated with increased mortality in eNOS(-/-) mice, and the survival rate was higher in female eNOS(-/-) mice compared with male eNOS(-/-) mice. In conclusion, IPC protects WT mice against in vivo myocardial ischemia-reperfusion injury regardless of sex and ischemic duration, but the deletion of eNOS abolishes the cardioprotective effect of classical IPC.

Amino acid transamination is crucial for ischaemic cardioprotection in normal and preconditioned isolated rat hearts – focus on l‐glutamate

We have found that cardioprotection by l-glutamate mimics protection by classical ischaemic preconditioning (IPC). We investigated whether the effect of IPC involves amino acid transamination and whether IPC modulates myocardial glutamate metabolism. In a glucose-perfused, isolated rat heart model subjected to 40 min global no-flow ischaemia and 120 min reperfusion, the effects of IPC (2 cycles of 5 min ischaemia and 5 min reperfusion) and continuous glutamate (20 mm) administration during reperfusion on infarct size and haemodynamic recovery were studied. The effect of inhibiting amino acid transamination was evaluated by adding the amino acid transaminase inhibitor amino-oxyacetate (AOA; 0.025 mm) during reperfusion. Changes in coronary effluent, interstitial (microdialysis) and intracellular glutamate ([GLUT](i)) concentrations were measured. Ischaemic preconditioning and postischaemic glutamate administration reduced infarct size to the same extent (41 and 40%, respectively; P < 0.05 for both), without showing an additive effect. Amino-oxyacetate abolished infarct reduction by IPC and glutamate, and increased infarct size in both control and IPC hearts in a dose-dependent manner. Ischaemic preconditioning increased [GLUT](i) before ischaemia (P < 0.01) and decreased the release of glutamate during the first 10 min of reperfusion (P = 0.03). A twofold reduction in [GLUT](i) from the preischaemic state to 45 min of reperfusion (P = 0.0001) suggested increased postischaemic glutamate utilization in IPC hearts. While IPC and AOA changed haemodynamics in accordance with infarct size, glutamate decreased haemodynamic recovery despite reduced infarct size. In conclusion, ischaemic cardioprotection of the normal and IPC-protected heart depends on amino acid transamination and activity of the malate-aspartate shuttle during reperfusion. Underlying mechanisms of IPC include myocardial glutamate metabolism.

Early phase of ischemic preconditioning in the mouse: effects of endothelial nitric oxide synthase (eNOS), ischemic duration, aging, and gender

Ischemic preconditioning (IPC) is a powerful phenomenon that provides effective cardioprotection in mammalian hearts, however, little is known in relation to prolonged ischemia (I), aging and gender. Importantly, the role of eNOS‐mediated nitric oxide (NO) pathway is still controversial. Therefore, we performed an extensive study in wild‐type (WT) and eNOS‐knockout (eNOS‐KO) mice to evaluate whether the infarct limiting effect of IPC depends on eNOS, ischemic periods, ages (12‐16 weeks and 12‐16 months), and gender. Classical IPC was induced by 3‐cycles of 5‐min regional coronary I separated by 5‐min reperfusion (R), and was followed by 30‐min or 60‐min of index I and 24‐hrs R. Control IR protocol had 30‐ or 60‐min I followed by 24‐hrs R. Protection was evaluated by measuring myocardial infarct size as a percentage of area at risk. The major findings are that regardless of age and gender, WT mice exhibited robust IPC effects with significantly smaller myocardial infarction, whereas, eNOS‐KO mice did not. In general, female WT mice had smaller infarction compared to male WT. Importantly, aging and prolonged I caused significantly larger infarction in both genders. Prolonged myocardial ischemia was fatal in eNOS‐KO mice. In conclusion, IPC protects WT mice against in vivo myocardial IR injury regardless of age, gender and/or ischemic duration, but, deletion of eNOS abolishes the cardioprotective effect of early IPC.

Effect of preischemic β-adrenoceptor stimulation on postischemic contractile dysfunction

Aims Short periods of preischemic β-adrenoceptor stimulation protect hearts against postischemic left ventricular dysfunction. It was the aim of this study to decide whether this procedure mimics ischemic preconditioning by the generation of preischemic hemodynamic and energetic stress or whether it represents an endogenous phenomenon and to investigate the influence of age and hypertension. Main methods Isolated rat hearts were investigated ex vivo by Langendorff perfusion and exposed to an established ischemia/reperfusion protocol (45 min no-flow ischemia and 90 min reperfusion). Left ventricular developed pressure (LVDP), rate pressure product, and ± d P/d t were analyzed. Key findings Isoprenaline concentration dependently increased LVDP up to 40 ± 15 mm Hg (approximately EC 50 of 9.9 ± 0.5 nM). Isoprenaline given prior to ischemia attenuated the subsequent postischemic ventricular dysfunction (approximately EC 50 of 1.4 ± 0.2 pM). However, concentrations high enough to improve LVDP in normoxic hearts did not improve postischemic recovery albeit a significant reduction of hypercontraction-induced cell damage. The effect on functional recovery was attenuated by atenolol, H89, and wortmannin suggesting that β-adrenoceptor stimulation, protein kinase A, and PI 3-kinase activation are involved. The effect was conserved in hearts from 13 month old rats but lost in age-matched spontaneously hypertensive rats. Significance The study identifies preischemic β-adrenoceptor stimulation as a pharmacological preconditioning protocol that does not simply mimic classical ischemic preconditioning by induction of hemodynamic or energetic stress prior to a prolonged ischemic period. The observed loss of effectiveness in hypertensives may contribute to the reduced ischemic tolerance of hypertensives.

Possible role of an ischemic preconditioning-like response mechanism in K ATP channel opener-mediated protection against streptozotocin-induced suppression of rat pancreatic islet function

Potassium channel openers (KCOs) decrease insulin secretion from β-cells. Some KCOs also protect against damage to β-cell function and type 1 diabetes in animal models. Previously we have found that the KCO NNC 55-0118 counteracted islet cell dysfunction, and this was associated with a lowering of the mitochondrial membrane potential (Δψ). Presently we aimed to explore whether inhibition of insulin secretion per se or rather inhibition of mitochondrial function correlates to counteraction of β-cell suppression. For this we used two novel KCOs (NNC 55-0321 and NNC 55-0462), which at certain concentrations have different actions regarding insulin secretion and the Δψ, with NNC 55-0321 being a potent inhibitor of Δψ and NNC 55-0462 being a potent inhibitor of insulin secretion. At 10 μM NNC 55-0321, but not with NNC 55-0462, the islet ATP content and ATP/ADP ratio was acutely decreased. This was accompanied by a complete protection against streptozotocin-induced suppression of islet insulin secretion using the former KCO. In cardiac research KCOs have been used to induce an ischemic preconditioning (IPC) response. In line with an IPC-like mechanism we found that NNC 55-0321 induced an initial free oxygen radical formation, PKC-ɛ isoform activation and a subsequent phosphorylation of the survival promoting factor Akt. Thus, KCOs may elicit mitochondrial events that resemble classical IPC seen in cardiomyocytes, and this could explain the enhanced islet cell function observed. KCOs with this property may be particularly interesting compounds to study as a rescue therapy during acute episodes of β-cell suppression/destruction.

Cardioprotection: spotlight on PKG

Classical ischaemic preconditioning, delayed or second window preconditioning and postconditioning are forms of cardioprotection that are dependent on cell surface receptors, intracellular signalling molecules and kinases that ultimately block formation of the mitochondrial permeability transition. The latter is presumed to cause myocardial necrosis as well as apoptosis, so prevention of its formation upon resumption of perfusion after a prolonged coronary occlusion should be cardioprotective. In all of these forms of cardioprotection, formation of cGMP and activation of protein kinase G (PKG) are recognized to be key steps in the signal transduction pathway. Burley et al. highlight the roles of cGMP and PKG in their comprehensive review. They describe the basic biology of PKG and emphasize its compartmentalization, which may be responsible for the frustration induced by assays for PKG in whole cell lysates and for the spurious conclusions about the role of PKG in cardioprotection. This review will be useful to both the novice and the seasoned investigator.

Exogenous nitric oxide triggers classic ischemic preconditioning by preventing intracellular Ca 2+ overload in cardiomyocytes

The involvement of nitric oxide (NO) in the late phase of ischemic preconditioning is well established. However, the role of NO as a trigger or mediator of “classic preconditioning” remains to be determined. The present study was designed to investigate the effects of NO on calcium homeostasis in cultured newborn rat cardiomyocytes in normoxia and hypoxia. We found that treatment with the NO donor, sodium nitroprusside (SNP) induced a sustained elevation of intracellular calcium level ([Ca 2+] i) followed by a decrease to control levels. Elevation of extracellular calcium, which generally occurs during ischemia, caused an immediate increase in [Ca 2+] i and arrhythmia in cultures of newborn cardiomyocytes. Treatment with SNP decreased [Ca 2+] i to control levels and re-established synchronized beating of cardiomyocytes. A decrease in extracellular [Na +], which inhibits the Na +/Ca 2+ exchanger, did not prevent [Ca 2+] i reduction by SNP. In contrast, application of thapsigargin, an inhibitor of sarcoplasmic reticulum Ca 2+-ATPase (SERCA2a), increased [Ca 2+] i, and in its presence, SNP did not reduce [Ca 2+] i, indicating that Ca 2+ reduction is achieved via activation of SERCA2a. The results obtained suggest that activation of SERCA2a by SNP increases Ca 2+ uptake into the sarcoplasmic reticulum (SR) and prevents cytosolic Ca 2+ overload, which might explain the protective effect of SNP from hypoxic damage.

TNFα is required to confer protection in an in vivo model of classical ischaemic preconditioning

Although Tumor Necrosis Factor alpha (TNFα) is used as a preconditioning mimetic in vitro, its role in ischaemic preconditioning (IPC) has not been clearly defined. Here, we propose to use an in vivo model (that takes into account the activation of leukocytes which may affect levels of TNFα) to demonstrate that i) TNFα acts as a trigger in IPC and ii) the dose-dependent nature of this cardioprotective effect of TNFα. Male Wistar rats were subjected to 30 min of left coronary artery occlusion (index ischaemia), followed by 24 h reperfusion. In the presence or absence of a soluble TNFα receptor (sTNFα-R), preconditioning was induced by 3 cycles of ischaemia (3 min)/reperfusion (5 min) (IPC) or various doses (0.05–4 μg/kg) of exogenous TNFα. Following 24 h reperfusion, infarct size (IS, expressed as % of the area at risk (AAR)) was assessed. Tissue levels of TNFα from the AAR, following IPC and TNFα stimulus were determined using Western Blot. IPC caused decrease in IS (4.5 ± 1.3% vs 30.8 ± 4.3% in ischaemic rats; P < 0.001) and increase of TNFα levels following the IPC stimulus. The protective effect of IPC was abrogated in the presence of the sTNFα-R. In addition, exogenous TNFα dose-dependently reduced IS with maximal protection at a dose of 0.1 μg/kg (IS = 12.6%, P < 0.01 vs ischaemic). In conclusion our data provide strong evidence for a role of TNFα during the trigger phase of IPC. In addition, exogenous TNFα mimics IPC by providing a dose-dependent cardioprotective effect against ischaemia–reperfusion injury in vivo.

β1-Adrenoreceptor activation contributes to ischemia-reperfusion damage as well as playing a role in ischemic preconditioning

Protein kinase A (PKA) activation has been implicated in early-phase ischemic preconditioning. We recently found that during ischemia PKA activation causes inactivation of cytochrome-c oxidase (CcO) and contributes to myocardial damage due to ischemia-reperfusion. It may be that beta-adrenergic stimulation during ischemia via endogenous catecholamine release activates PKA. Thus beta-adrenergic stimulation may mediate both myocardial protection and damage during ischemia. The present studies were designed to determine the role of the beta(1)-adrenergic receptor (beta(1)-AR) in myocardial ischemic damage and ischemic preconditioning. Langendorff-perfused rabbit hearts underwent 30-min ischemia by anterior coronary artery ligation followed by 2-h reperfusion. Occlusion-reperfusion damage was evaluated by delineating the nonperfused volume of myocardium at risk and volume of myocardial necrosis after 2-h reperfusion. In some hearts ischemic preconditioning was accomplished by two 5-min episodes of global low-flow ischemia separated by 10 min before coronary occlusion-reperfusion. Orthogonal electrocardiograms were recorded, and coronary flow was monitored by a drip count. Three hearts from each experimental group were used to determine mitochondrial CcO and aconitase activities. Two-hour reperfusion after occlusion caused an additional decrease in CcO activity vs. that after 30-min occlusion alone. Blocking the beta(1)-AR during occlusion-reperfusion reversed CcO activity depression and preserved myocardium at risk for necrosis. Similarly, mitochondrial aconitase activity exhibited a parallel response after occlusion-reperfusion as well as for the other interventions. Furthermore, classic ischemic preconditioning had no effect on CcO depression. However, blocking the beta(1)-AR during preconditioning eliminated the cardioprotection. If the beta(1)-AR was blocked after preconditioning, the myocardium was preserved. Interestingly, in both of the latter cases the depression in CcO activity was reversed. Thus the beta(1)-AR plays a dual role in myocardial ischemic damage. Our findings may lead to therapeutic strategies for preserving myocardium at risk for infarction, especially in coronary reperfusion intervention.

Attenuated cardioprotective response to bradykinin, but not classical ischaemic preconditioning, in DOCA-salt hypertensive left ventricular hypertrophy

Hypertensive left ventricular hypertrophy (LVH) co-exists frequently with ischaemic heart disease. While ischaemic preconditioning (IPC) is known to protect against ischaemia-reperfusion injury in LVH, it is not known if other cardioprotective manoeuvres are effective. Bradykinin, a key autacoid mediator in IPC, is protective in normal hearts but its ability to protect against ischaemia-reperfusion injury in LVH is unknown. Hypertensive LVH was induced in male rats by 4 weeks treatment with deoxycorticosterone acetate (DOCA) and salt drinking fluid. Hearts were Langendorff perfused, subjected to 35 min coronary artery occlusion and 120 min reperfusion, and infarct size (AN/RZ %) was determined by tetrazolium staining. The effects of IPC with 2 × 5 min cycles of global ischaemia or 10 min pretreatment with bradykinin were assessed. DOCA-salt rats were markedly hypertensive and left ventricle/body weight ratio was 26% greater than in normotensive controls. Baseline coronary flow and risk zone/LV ratio were similar in normotensive hearts and DOCA-salt hearts, and infarct size was similar (AN/RZ 50.6 ± 3.2% and 47.0 ± 3.1%, respectively). IPC was equally protective in normotensive and DOCA-salt hearts (AN/RZ 18.6 ± 3.3% and 18.4 ± 2.3%, respectively, P < 0.01 versus corresponding control). Bradykinin 0.1, 0.2 or 0.5 μM pretreatment produced concentration-dependent infarct limitation in normotensive hearts (bradykinin 0.5 μM AN/RZ, 9.5 ± 3.6%, P < 0.01 versus normotensive control), but the effect in DOCA-salt hearts was attenuated (bradykinin 0.5 μM AN/RZ, 23.4 ± 3.8%). Further, the pre-ischaemic coronary vasodilator response to bradykinin was abrogated in DOCA-salt hypertensive hearts. We conclude that the cardioprotective action of bradykinin is markedly attenuated in moderate LVH and coronary vasodilator effect is lost. The reasons for reduced sensitivity to bradykinin in the hypertensive heart are unknown but these findings may have implications for the application of preconditioning-mimetic interventions in LVH.

TNFα-induced cytoprotection requires the production of free radicals within mitochondria in C 2C 12 myotubes

We previously reported that tumour necrosis factor alpha (TNFα) can mimic classic ischemic preconditioning (IPC) in both cells and heart. However, the signalling pathways involved remain incompletely understood. One potential protective pathway could be TNFα-induced reactive oxygen species (ROS). We hypothesized that TNFα cytoprotection occurs through the generation of ROS which originate within the mitochondria. C 2C 12 myotubes were preconditioned with either a short period of hypoxia (IPC) or a low concentration of TNFα (0.5 ng/ml) prior to a simulated ischemic insult. ROS generation was evaluated on cells stained with dichlorofluorescin diacetate (DCFH-DA) by flow cytometry. The source of TNFα-induced ROS was examined with Mitotracker Red CM-H 2XRos. The bioenergetics of the mitochondria were evaluated by investigation of the respiratory parameters and the inner mitochondrial membrane potential. Pretreatment with TNFα improved cell viability compared with the simulated ischemic control (TNFα: 75 ± 1% versus 34 ± 1% for the control: p < 0.001). The ROS scavenger, N-2-mercaptopropionyl-glycine (MPG), reduced the viability of TNFα-stimulated cells to 15 ± 1% ( p < 0.001 versus TNFα). Similar results were obtained with IPC. TNFα stimulation increased ROS production mainly in the mitochondria, and this increase was abolished in the presence of MPG. Addition of TNFα to the cells increased State 2 respiration and modestly depolarised the membrane potential prior to the ischemic insult. In conclusion, TNFα-induced ROS generation can occur within the mitochondria, resulting in temporal mitochondrial perturbations which may initiate the cytoprotective effect of TNFα.

Preconditioning by 17β-estradiol in isolated rat heart depends on PI3-K/PKB pathway, PKC, and ROS

To study the cell signaling events leading to 17beta-estradiol (E(2))-induced acute cardioprotection, we subjected isolated rat hearts to three 5-min cycles of 10 microM E(2) before 30 min of regional ischemia, followed by 2 h of reperfusion. Protection was judged by changes in infarct size in percentage of risk zone volume. To test the importance of phosphoinositide 3-kinase (PI3-K), protein kinase C (PKC), or reactive oxygen species (ROS) in E(2)-induced protection, we combined wortmannin (1 microM), chelerythrine (2 microM), and 2-mercaptopropionylglycine (300 microM), respectively, with E(2) exposure. Changes in phosphorylation of protein kinase B (PKB) and selected PKC isoforms were tested by immunoblotting of total lysates and subcellular fractions, along with assessment of PKC translocation from soluble to membrane fraction of heart tissue homogenates. Intracellular ROS levels induced by E(2) preconditioning were investigated. E(2) preconditioning led to significant reduction in infarct size from 31.8 +/- 5.3 to 20.2 +/- 2.6% in male hearts and from 42.7 +/- 4.7 to 17.1 +/- 3.4% in female hearts (P < 0.05). Protection was abolished by wortmannin (30.0 +/- 3.2%), chelerythrine (45.1 +/- 4.4%), and 2-mercaptopropionylglycine (36.8 +/- 4.7%). E(2) preconditioning induced phosphorylation of PKB, PKCalpha, and PKCepsilon and membrane translocation of PKCepsilon and PKCdelta. Intracellular ROS levels were found elevated after transient treatment with hormone. Therefore, our data demonstrate the ability of E(2) to induce preconditioning-like cardioprotection via cell signaling events shared by classic ischemic preconditioning.

Cardiac genomic response following preconditioning stimulus

This review focuses on the genomic response following a preconditioning stimulus. Initial studies demonstrated that classical ischemic preconditioning mediated by cyclic episodes of short durations of reversible ischemia and reperfusion could result in the reprogramming of gene expression. Some of these genes are translated into proteins during the late preconditioning or so-called "second window of protection". Subsequent studies determined a unique similarity of the expressed gene profiles between diverse varieties of preconditioning including ischemic/hypoxic, heat shock, and oxidative stress. The most common genes that are expressed by virtually any kind of stress conditioning include antioxidants like superoxide dismutase, glutathione peroxidase and heme oxygenase and heat shock proteins such as HSP70. At a later date, differential display and subtractive hybridization techniques revealed the identities of many other genes including those belonging to mitochondrial respiratory chain such as ATPases. More recently, gene array profiles using gene chips determined several other genes triggered by preconditioning including the mitochondrial genes. The results of the studies present in the literature clearly indicate the existence of a strong resemblance between the patterns of gene expression profiles induced by diverse preconditioning stimuli, oxidative stress being situated at the cross-roads of all forms of the stresses. Redox signaling appears to be responsible for the conversion of the ischemia/reperfusion-induced "death signal" into preconditioning-mediated "survival signal".