Pharmacological Pre- and Postconditioning With Levosimendan Protect H9c2 Cardiomyoblasts From Anoxia/Reoxygenation-induced Cell Death via PI3K/Akt Signaling.

ABSTRACT: The calcium sensitizer levosimendan is indicated for the hemodynamic stabilization of patients with acutely decompensated heart failure and has been shown to be protective against reperfusion injury after myocardial infarction. However, affected forms of cell death and underlying signaling pathways remain controversial. Therefore, the aim of this study was to examine the influence of levosimendan preconditioning and postconditioning on anoxia/reoxygenation-induced apoptosis, necrosis, and autophagy in H9c2 myoblasts. To mimic conditions of myocardial ischemia/reperfusion, rat cardiac H9c2 myoblasts were exposed to anoxia/starvation, followed by reoxygenation/refeeding. Apoptosis, necrosis, autophagy, cell viability, survival signaling, and mitochondrial permeability transition pore (mPTP) opening were measured. Both, pharmacological preconditioning and postconditioning with levosimendan were capable to reduce apoptosis as well as necrosis in stressed H9c2 cells. However, preconditioning showed to have the stronger impact compared with postconditioning. Moreover, levosimendan preconditioning increased autophagy, suggesting enhanced repair processes initiated by the early presence of the drug. Underlying mechanisms differ between both interventions: Although both are associated with PI3/Akt activation and reduced mPTP opening, only postconditioning but not preconditioning depended on mKATP activation. This variation might indicate that a pharmacological treatment after the onset of reoxygenation at least in part directly addresses mitochondrial structures for protection. In conclusion, we demonstrate that both pharmacological preconditioning and postconditioning with levosimendan protect anoxia/reoxygenation-stressed cells but differ in the underlying mechanisms. These results are decisive to obtain more insights into the beneficial effects of levosimendan in the treatment of reperfusion-mediated damage.

In situ postconditioning with neuregulin-1ß is mediated by a PI3K/Akt-dependent pathway.

BACKGROUND: The myocardial infarct size can be reduced by pharmacological postconditioning using cardioprotective agents. Neuregulin-1ß is a potential candidate, but previous studies in an isolated heart model of ischemia and reperfusion displayed controversial results. An in situ model of ischemia/reperfusion was used to clarify whether the remote application of neuregulin-1ß can reduce the reperfusion injury. A second aim was to evaluate, if the effects are specific for reperfused tissue or if this is a general antiapoptotic effect. In addition, the contributing molecular mechanisms were investigated. METHODS: In an open chest model, mouse hearts were subjected to a regional ischemia (45-minute) using ligature of the left anterior descending artery. Neuregulin-1ß (80 ng/kg) was given using an intraperitoneal bolus injection 5 minutes before reopening of the ligature followed by a 30-minute reperfusion. RESULTS: Remote application of recombinant neuregulin-1ß protected the heart from reperfusion injury without influencing hemodynamics. This beneficial effect specifically targets reperfusion injury. In contrast, nonreperfused needle trauma was not reduced by neuregulin-1ß when applied remotely. Pharmacological blocking experiments and enzyme activation analysis using Western blot analysis revealed a crucial involvement of the antiapoptotic reperfusion injury salvage kinase cascade. In contrast, contribution of the survivor activating factor enhancement pathways to this early cardioprotection was not observed. CONCLUSIONS: Remote application of neuregulin-1ß protects hearts from early reperfusion injury by activation of the reperfusion injury salvage kinase pathway without relevant effects on intracardiac pressures in myocardial infarction. Besides its potential pharmacological application, neuregulin-1ß might act as an endogenously produced mediator in remote postconditioning.

Pharmacological postconditioning by bolus injection of phosphodiesterase-5 inhibitors vardenafil and sildenafil.

Postconditioning enables cardioprotection against ischemia/reperfusion injury either by application of short, repetitive ischemic periods or by pharmacological intervention prior to reperfusion. Pharmacological postconditioning has been described for phosphodiesterase-5 inhibitors when the substances were applied as a permanent infusion. For clinical purposes, application of a bolus is more convenient. In a rat heart in situ model of ischemia reperfusion vardenafil or sildenafil were applied as a bolus prior to reperfusion. Cardioprotective effects were found over a broad dosage range. In accordance with current hypotheses on pharmacological postconditioning signaling, the protective effect was mediated by extracellular signal-regulated kinase and protein kinase C pathway. Interestingly, the extent of protection was independent of the concentration applied for both substances. Full protection comparable to ischemic postconditioning was reached with half-maximal human equivalence dose. In contrast, mean arterial pressure dropped upon bolus application in a dose-dependent manner. Taken together, the current study extends previous findings obtained in a permanent infusion model to bolus application. This is an important step toward clinical application of pharmacological postconditioning with sildenafil and vardenafil, especially because the beneficial effects were proven for concentrations with reduced hemodynamic side effects compared to the dosage applied for erectile dysfunction treatment.

Uncoupled eNOS annihilates neuregulin-1ß-induced cardioprotection: a novel mechanism in pharmacological postconditioning in myocardial infarction.

Myocardial infarct size can be limited by pharmacological postconditioning (pPC) with cardioprotective agents. Cardioprotective effects of neuregulin-1ß (NRG) via activation of protein kinase B (Akt) and downstream pathways like endothelial nitric oxide synthase (eNOS) have been postulated based on results from cell culture experiments. The purpose of this study was to investigate if eNOS may be involved in pPC with NRG. NRG application in an ex vivo mouse model (C57Bl6) of ischemia-reperfusion injury was analyzed. Unexpectedly, the infarct size increased when NRG was infused starting 5 min prior to reperfusion, even though protective Akt and GSK3ß phosphorylation were enhanced. In eNOS deficient mice, however, NRG significantly reduced the infarct size. Co-infusion of NRG and L-arginine (Arg) lead to a reduction in infarct size in wild type animals. Electron paramagnetic resonance measurements revealed that NRG treatment prior to reperfusion leads to an enhanced release of reactive oxygen species compared to controls and this effect is blunted by co-infusion of Arg. This study documents the cardioprotective mechanisms of NRG signaling to be mediated by GSK3ß inactivation. This is the first study to show that this protection fails in situations with dysfunctional eNOS. In eNOS deficient mice NRG exerts its protective effect via the GSK3ß pathway, suggesting that the eNOS can limit cardioprotection. As dysfunctional eNOS has been described in cardiovascular risk factors like diabetes, hypertension, and hypercholesterolemia these findings can help to explain lack of postconditioning performance in models of cardiovascular co-morbidities.

Cardioprotection by ischemic postconditioning is abrogated in hypertrophied myocardium of spontaneously hypertensive rats.

PURPOSE: This study examines if ischemic postconditioning is preserved in hypertrophied myocardium of spontaneously hypertensive rats (SHR). METHODS: Infarct sizes, hemodynamic, morphometric, and biochemical parameters were obtained from normotensive controls [Wistar-Kyoto (WKY) rats] and compared with SHRs. In open-chest rats, the infarct size was determined after 30 minutes of regional ischemia. Postconditioning was performed by 3 cycles of ischemia/reperfusion with 30 seconds each or 6 cycles of ischemia/reperfusion with 10 seconds each immediately after the infarction. RESULTS: Infarct size was comparable between control rats and SHRs. In WKY rats, postconditioning reduced the infarct size significantly. However, in SHRs, the postconditioning effect was completely lost for both postconditioning protocols. Even shortening of the ischemic period to 20 minutes did not restore the infarct sparing effect in SHRs. The phosphorylation of glycogen synthase kinase 3ß increased 2.1-fold by ischemic postconditioning in WKY rats; however, this increase was completely absent in SHRs with both postconditioning protocols. CONCLUSIONS: Myocardial hypertrophy inhibits the protection by postconditioning in an experimental animal model. Future studies have to clarify whether this result can be extrapolated to patients with arterial hypertension and myocardial hypertrophy.

Ischemic post-conditioning reduces infarct size of the in vivo rat heart: role of PI3-K, mTOR, GSK-3beta, and apoptosis.

Post-conditioning by repetitive cycles of reperfusion/ischemia after prolonged ischemia protects the heart from infarction. The objectives of this study were: Are kinases (PI3-kinase, mTOR, and GSK-3beta) involved in the signaling pathway of post-conditioning? Does post-conditioning result in a diminished necrosis or apoptosis? In open chest rats the infarct size was determined after 30 min of regional ischemia and 30 min of reperfusion using propidium iodide and microspheres. Post-conditioning was performed by three cycles of 30 s reperfusion and reocclusion each, immediately upon reperfusion. PI3-kinase and mTOR were blocked using wortmannin (0.6 mg/kg) or rapamycin (0.25 mg/kg), respectively. The phosphorylation of GSK-3beta and p70S6K was determined with phospho-specific antibodies. TUNEL staining and detection of apoptosis-inducing factor (AIF) were used for the determination of apoptosis. Control hearts had an infarct size of 49 +/- 3%, while post-conditioning significantly reduced it to 29 +/- 3% (P < 0.01). Wortmannin as well as rapamycin completely blocked the infarct size reduction of post-conditioning (51 +/- 2% and 54 +/- 5%, respectively). Western blot analysis revealed that post-conditioning increased the phosphorylation of GSK-3beta by 2.3 times (P < 0.01), and this increase could be blocked by wortmannin, a PI3-kinase inhibitor. Although rapamycin blocked the infarct size reduction, phosphorylation of p70S6K was not increased in post-conditioned hearts. After 2 h of reperfusion, the post-conditioned hearts had significantly fewer TUNEL-positive nuclei (35 %) compared to control hearts (53%; P < 0.001). AIF was equally reduced in post-conditioned rat hearts (P < 0.05 vs. control). Infarct size reduction by ischemic post-conditioning of the in vivo rat heart is PI3-kinase dependent and involves mTOR. Furthermore, GSK-3beta, which is thought to be a regulator of the mPTP, is part of the signaling pathway of post-conditioning. Finally, apoptosis was inhibited by post-conditioning, which was shown by two independent methods. The role of apoptosis and/or autophagy in post-conditioning has to be further elucidated to find therapeutic targets to protect the heart from the consequences of acute myocardial infarction.

Postconditioning with levosimendan reduces the infarct size involving the PI3K pathway and KATP-channel activation but is independent of PDE-III inhibition.

Reperfusion injury is strongly involved in the loss of functional heart tissue in patients after acute myocardial infarction. Various signal transduction pathways to reduce infarct size during reperfusion have been characterized. However, so far in the clinical setting no standard therapies are applied due to the lack of suitable drugs. Levosimendan, a calcium sensitizer, has been shown to improve survival in cardiogenic shock after infarction. Focus of the present study was to address the question, whether a bolus application of levosimendan prior to reperfusion is able to reduce the infarct size. A well-characterized model, the in vivo rat model, was used and levosimendan applied 5 min prior to reperfusion after 30-min occlusion of the left coronary artery followed by a 30-min reperfusion period. This pharmacological postconditioning was compared to the ischemic postconditioning with three times occlusion/reperfusion periods of 30 s each. To further address the question if in this in vivo model the phosphatidylinositol 3-kinase (PI3K) pathway may be involved, the PDE-III inhibiting property of levosimendan was compared to the PDE-III inhibitor enoximone. Ischemic postconditioning significantly reduced the infarct size from 48 +/- 2 to 32 +/- 1% of the area at risk (P < 0.05). Similarly, levosimendan decreased infarct size down to 29 +/- 3%. The combination of ischemic postconditioning and pharmacological postconditioning using levosimendan did not result in a further reduction of the infarct size. Both, the mitochondrial KATP-channel blocker 5-hydroxydecanoate (5-HD) and the PI3K inhibitor wortmannin abolished the protection afforded by levosimendan completely, while the inhibitors alone did not influence the infarct size in control hearts. Pharmacological postconditioning with enoximone did not result in any infarct size reduction. Postconditioning with levosimendan significantly increased the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3beta (GSK-3beta) at 5 min of reperfusion, an effect which could be blocked completely by the additional administration of wortmannin. In conclusion, levosimendan applied prior to reperfusion in acute myocardial infarction significantly reduces the infarct size in an in vivo rat model. This protection involves the PI3K pathway and the activation of mitochondrial KATP-channels, but is independent of PDE-III inhibition. This finding may open new possibilities for the treatment of patients with acute myocardial infarction using levosimendan, which is an already established therapy in cardiogenic shock. Whether the reduction of mortality in cardiogenic shock by levosimendan may in part be based on this postconditioning effect remains to be elucidated in clinical setting.

Stimulation of adenosine A2b receptors blocks apoptosis in the non-infarcted myocardium even when administered after the onset of infarction.

OBJECTIVE: Chronic adenosine A2b receptor stimulation has been shown to prevent ventricular remodelling after myocardial infarction (MI). We hypothesized that this effect is due to the inhibition of cardiac myocyte apoptosis in the myocardium remote from the infarction. METHODS: Rats were subjected to MI by LAD ligation in situ. Some animals were pre-treated with the stable adenosine analogue 2-chloro-adenosine (CADO). After 24 h, pro- and anti-apoptotic signals (protein kinase C isoforms, p38, g proteins, Bcl-2/Bax ratio, Akt, Bad), and marker of apoptosis execution (caspase-3, TUNEL) were quantified in the remote myocardium. RESULTS: CADO prevented the occurrence of apoptosis in the remote myocardium of an infarcted heart. This effect occured not only when CADO was started before the onset of ischemia but also when it started 3 h after the infarction. The anti-apoptotic effect of CADO was blocked by simultaneous administration of the selective adenosine A2b receptor antagonist MRS1754 (1 mg/kg). The anti-apoptotic effect of CADO seems to be mediated by g(alphaq) and by the activation of survival kinases (Bad) and by inhibition of the pro-apoptotic PKC-delta/p38-MAPK-pathway. CONCLUSION: Chronic adenosine A2b receptor stimulation blocks cardiac myocyte apoptosis in the remote myocardium even when started after the onset of infarction. This may explain the anti-remodelling-effect of the A2b receptor stimulation after infarction.

The calcium channel blocker felodipine attenuates the positive hemodynamic effects of the beta-blocker metoprolol in severe dilated cardiomyopathy--a prospective, randomized, double-blind and placebo-controlled study with invasive hemodynamic assessment.

BACKGROUND: In addition to standard therapy with ACE-inhibitors, digitalis and diuretics, beta-adrenergic receptor blockers have become a widely accepted strategy in the treatment of chronic heart failure. The role of calcium antagonists in CHF however remains controversial. To evaluate if a combination therapy of metoprolol and felodipine might improve hemodynamic parameters, a randomized and placebo-controlled study was designed. METHODS AND RESULTS: Sixty-three patients with DCMP, LVEF 3 months in NYHA II-III on standard medication were prospectively treated with either a) a combination of metoprolol+felodipine (MF group, n=20), b) metoprolol+felodipine-placebo (MP group, n=23), or c) metoprolol-placebo+felodipine-placebo (PP group, n=20). Compared to baseline, LVEF and LVEDD significantly improved after 6 months in the MP group (LVEF: 36+/-2% vs 29+/-2%, p<0.01; LVEDD: 68+/-3 mm vs 64+/-3 mm, p<0.05), whereas in the other treatment groups only minor changes were observed. A significant benefit in hemodynamic parameters as determined by right heart catheterization was noted also only in the MP group with a marked reduction in PAP mean (17 vs 24 mmHg, p<0.01), PCWP (10 vs 15 mmHg, p<0.001) resulting in a significant increase in cardiac and stroke volume index at rest with no marked changes in the MF and PP group. CONCLUSION: beta-blocker treatment in CHF patients improves left ventricular function and additionally invasive hemodynamic measurements both at rest and during exercise. In contrast, the combined therapy with the long-acting calcium antagonist felodipine neutralizes these beneficial effects of metoprolol therapy to almost placebo level, providing evidence based on hemodynamic measurements that this combination should be avoided in patients with CHF.

Cardioprotection by postconditioning is lost in WOKW rats with metabolic syndrome: role of glycogen synthase kinase 3beta.

BACKGROUND AND OBJECTIVE: Postconditioning by repetitive I/R cycles immediately after onset of reperfusion protects the heart. Metabolic disorders undermine the protection of preconditioning. The present study tested whether postconditioning protects hearts from rats with established metabolic syndrome [Wistar-Ottawa-Karlsburg W rats (WOKW)]. METHODS AND RESULTS: After 28 weeks of age, WOKW rats were much heavier than DA (Dark Agouti) and Wistar control rats and showed the pattern of the metabolic syndrome. Postconditioning was performed by 3 30-second cycles of reperfusion/ischemia immediately after the regional ischemia (30 minutes). Infarct size was comparable in all control hearts from DA, Wistar, and WOKW rats (58 +/- 2%, 49 +/- 3%; 49 +/- 2%, respectively). Postconditioning significantly reduced the infarct size in DA rats (39 +/- 5%) and Wistar rats (29 +/- 3%). In WOKW rats, the infarct sparing effect of postconditioning was lost (43 +/- 4%).GSK-3beta and Erk are involved in the signaling of postconditioning. Therefore, the phosphorylation of these proteins was determined by Western blot analysis. Postconditioning significantly increased the phosphorylation of GSK-3beta in DA and Wistar rats (1.6-fold in DA rats, 2.3-fold in Wistar rats, P < 0.05) but failed to do so in WOKW rats. Similarly, a trend for an increased phosphorylation of Erk was found in DA rats but not in WOKW rats. Thus the inefficacy of postconditioning in reducing infarct size in rats with metabolic syndrome is paralleled by a lack of phosphorylation of GSK-3beta and Erk. CONCLUSION: The metabolic syndrome, as shown in this animal model, completely abrogates the postconditioning. This blockade involves the phosphorylation of GSK-3beta. Further studies have to evaluate whether this block of postconditioning makes patients with a metabolic syndrome more susceptible to myocardial damage after infarction.

Levosimendan is superior to enoximone in refractory cardiogenic shock complicating acute myocardial infarction.

OBJECTIVE: Cardiogenic shock is the leading cause of death in patients hospitalized for acute myocardial infarction. The objectives were to investigate the effects of levosimendan, a novel inodilator, compared with the phosphodiesterase-III inhibitor enoximone in refractory cardiogenic shock complicating acute myocardial infarction, on top of current therapy. DESIGN: Prospective, randomized, controlled single-center clinical trial. SETTING: Medical and coronary intensive care unit in a university hospital. PATIENTS: Thirty-two patients with refractory cardiogenic shock for at least 2 hrs requiring additional therapy. INTERVENTIONS: Infusion of either levosimendan (12 microg/kg over 10 min, followed by 0.1 microg/kg/min over 50 min, and of 0.2 microg/kg/min for the next 23 hrs) or enoximone (fractional loading dose of 0.5 mg/kg, followed by 2-10 microg/kg/min continuously) after initiation of current therapy, always including revascularization, intra-aortic balloon pump counterpulsation, and inotropes. MEASUREMENTS AND MAIN RESULTS: Survival rate at 30 days was significantly higher in the levosimendan-treated group (69%, 11 of 16) compared with the enoximone group (37%, 6 of 16, p = 0.023). Invasive hemodynamic parameters during the first 48 hrs were comparable in both groups. Levosimendan induced a trend toward higher cardiac index, cardiac power index, left ventricular stroke work index, and mixed venous oxygen saturation. In addition, lower cumulative values for catecholamines at 72 hrs and for clinical signs of inflammation were seen in the levosimendan-treated patients. Multiple organ failure leading to death occurred exclusively in the enoximone group (4 of 16 patients). CONCLUSIONS: In severe and refractory cardiogenic shock complicating acute myocardial infarction, levosimendan, added to current therapy, may contribute to improved survival compared with enoximone.

Chronic angiotensin II receptor blockade induces cardioprotection during ischemia by increased PKC-epsilon expression in the mouse heart.

INTRODUCTION: This study was performed to investigate the role of chronic pretreatment with angiotensin II type 1 receptor antagonists (ARB) and angiotensin converting enzyme inhibitors (ACE-I) in myocardial infarction (MI) and ischemic preconditioning (iPC). Little is known about molecular mechanisms of MI and iPC, especially about protein kinase C (PKC) isozyme levels induced by chronic pharmacologic pretreatment with ARB and ACE-I. To address one of the most important signal molecules in iPC, the PKC system was investigated in an ischemia/reperfusion model using isolated mouse hearts. METHODS: C57/BL6 mice were treated orally with candesartan cilexetil or ramipril for 2 weeks. Isolated perfused hearts were subjected to 60 minutes of left anterior descending occlusion and 30 minutes of reperfusion. IPC was performed by 3 cycles of 5 minutes of ischemia prior to the infarct ischemia. Infarct size was measured using the propidium iodide method, and PKC isoenzymes were detected by immunoblotting in the membrane and cytosolic fraction. RESULTS: In the control group, iPC reduced infarct size from 59.8 +/- 4.2% to 24.5 +/- 1.7%. ARB pretreatment itself reduced the infarct size significantly (38.1 +/- 3.0%) in hearts without iPC. This protection could neither be enhanced by additional iPC (40.3 +/- 3.4%) nor blocked by the AT2-receptor antagonist PD123.319 (40.7 +/- 3.7%). The ARB-induced cardio protection, however, was abolished by chelerythrine (5 micromol/L) (71.7 +/- 6.6%, n = 11, P < 0.001). Furthermore, PKC-epsilon (PKC-epsilon) was significantly increased in the particulate fraction of ARB-pretreated mice. On the contrary, chronic treatment with ACE-I completely blocked iPC (57.7 +/- 3.9%, n = 12, P < 0.001) without any effect on infarct size itself (51.5 +/- 3.0%, n = 12). PKC-epsilon expression was significantly reduced. CONCLUSION: Chronic AT1-receptor antagonism is capable of protecting the heart against myocardial infarction in a PKC-epsilon-dependent way. Furthermore, chronic treatment with ACE-I is suggested to have suppressing effects on iPC, possibly caused by reduced PKC-epsilon expression.

Transregulation of the alpha2-adrenergic signal transduction pathway by chronic beta-blockade: a novel mechanism for decreased platelet aggregation in patients.

Platelets play a pivotal role in the pathophysiology of acute coronary syndromes. Chronic beta-blockade has been shown to improve the long-term clinical outcome in coronary heart disease. Because platelets play a central role in thrombus formation, the aim of the present study was to investigate if chronic beta-blockade may transregulate the expression of alpha2-adrenergic receptors on human platelets and via this mechanism may modulate platelet activation. The densities of alpha2-adrenergic receptors of platelets were determined in healthy volunteers under chronic beta-blockade and as alpha2-adrenergic receptor-mediated function in catecholamine-induced platelet aggregation was determined. Chronic beta-blockade induced a time-dependent reduction of alpha2-adrenergic receptors. This reduction was accompanied by a decrease of the alpha-subunit of Gi proteins as demonstrated by Western blot analysis. This transregulation at both the receptor level and the G-protein level resulted in an almost complete loss of the alpha2-adrenergic receptor-mediated inhibition of adenylyl cyclase. The impairment of the alpha2-adrenergic receptor system correlated with a reduction of the catecholamine-induced activation and aggregation of human platelets. The functional transregulation of alpha2-adrenergic receptors by chronic beta-blockade in platelets and the consequent impairment of platelet activation may contribute to the therapeutic success of beta-blocker therapy.

Remote preconditioning by infrarenal aortic occlusion is operative via delta1-opioid receptors and free radicals in vivo in the rat heart.

BACKGROUND: Ischemic preconditioning (PC) is a powerful mechanism in reducing infarct size of the heart. Protection can be performed either by an ischemic stimulus of the heart itself or by ischemia of an organ distant to the heart (remote PC). We have previously shown that remote PC by infrarenal occlusion of the aorta [IOA] in the rat is as powerful as classical ischemic PC. This protection may be transmitted by humoral factors, and protein kinase C is a mediator in the signal transduction mechanism. Focus of the present study was to address the question whether remote preconditioning is dependent on the activation of the delta1-opioid receptor and/or free radicals, the infarct size was determined after either inhibition of the delta1-opioid receptor or scavenging free radicals. METHODS AND RESULTS: IOA was performed in rats by occlusion of the infrarenal aorta for 15 min followed by a 10-min reperfusion period. Infarction of the heart was induced by 30 min regional ischemia followed by 30 min of reperfusion. The area of infarct was determined by propidium iodide and the risk zone was demarcated by zinc cadmium sulfide fluorescent particles. Control hearts (30 min regional ischemia of the heart followed by 30 min of reperfusion; no IOA) had an infarct size of 54 +/- 3%, whereas classical preconditioning by three ischemia/reperfusion [I/R] cycles, 5 min each, reduced it to 12 +/- 1% of the risk zone (p<0.05). Fifteen minutes IOA with 10 min of reperfusion was highly protective and reduced the infarct size to 20 +/- 5% (p<0.05 vs. control). Inhibition of the delta1-opioid receptors by 7-benzylidenenaltrexone [BNTX] blocked the protection obtained by PC and IOA (41 +/- 4% and 44 +/- 2%, respectively; p<0.05 vs. the group without BNTX). BNTX in control hearts had no influence on infarct size (52 +/- 2%). Inhibition of endogenously released radicals by N-2-mercaptopropionyl glycine [MPG] blocked the infarct size reduction of IOA (46 +/- 3%; p<0.05 vs. IOA), but had no influence on the protection in classically preconditioned hearts protected by three cycles I/R (13 +/- 4%). Only if the number of the preconditioning stimuli was reduced to one was MPG able to overcome the protection (43 +/- 4%, p<0.05 vs. PC with one I/R cycle (21 +/- 4%)). CONCLUSION: Remote preconditioning using IOA protects the rat heart from infarction. Classical and remote PC share both the delta1-opioid-receptor and free radicals as common elements in their signal transduction pathways. MPG can block protection from IOA and from one, but not from three, classical preconditioning cycles. This indicates that the protection by remote preconditioning is comparable to classical PC with one I/R cycle.

Ischemic preconditioning promotes a transient, but not sustained translocation of protein kinase C and sensitization of adenylyl cyclase.

OBJECTIVE: Brief periods of ischemia precondition the heart and reduce the size of infarction caused by a subsequent sustained ischemia. The molecular memory of preconditioning, i.e., the molecule persistently activated by the preconditioning ischemia exhibiting protection during the infarct-inducing event, is subject to debate. Protein kinase C, adenylyl cyclase and beta-adrenergic receptors are candidates for this memory, supposedly their activation persists during several cycles of ischemia and reperfusion. The goal of this study was, therefore, to determine the activation of those signaling molecules after 1 - 3 cycles of myocardial ischemia and reperfusion. METHODS: Rat hearts were perfused according to the method of Langendorff with 1 - 3 cycles of ischemia (5 min) and reperfusion (10 min). In the particulate fraction of these hearts, densities of b-adrenergic receptors, activities of adenylyl cyclase, and the activities and isozyme distributions of PKC-alpha, PKC-beta, and PKC- epsilon were determined. RESULTS: The ischemia-induced upregulation of beta-adrenergic receptors is not influenced by preconditioning. In contrast, the sensitization of adenylyl cyclase observed after 5 min of ischemia is lost after repetitive periods of ischemia and reperfusion. Translocation of protein kinase C to the particulate fraction could be shown after 1 and 2 periods of ischemia including all major cardiac isozymes, with a rapid relocation to the cytosol after every cycle of reperfusion. A third period of ischemia was unable to promote a repeated translocation of protein kinase C. CONCLUSION: The ischemia-induced regulation process of beta-adrenergic receptors is not influenced in preconditioning. Moreover, a sustained translocation of protein kinase C and a sustained sensitization of adenylyl cyclase are obviously no prerequisite for preconditioning after various cycles of ischemia and reperfusion. Thus, those signaling molecules do not seem to be operative for the preconditioning's memory. It is suggested that the initial, synergistic burst of sensitization of the adenylyl cyclase and of protein kinase C translocation induces myocardial protection very early in ischemia and reperfusion.

Remote preconditioning by infrarenal occlusion of the aorta protects the heart from infarction: a newly identified non-neuronal but PKC-dependent pathway.

BACKGROUND: Ischemic preconditioning is a powerful mechanism in reducing infarct size of the heart. Protection can be performed either by an ischemic stimulus of the heart itself or by ischemia of an organ distant to the heart. To address the question whether this remote preconditioning is transduced by neuronal or humoral factors an in situ model of infrarenal occlusion of the aorta (IOA) in the rat was developed. Furthermore, the signal transduction pathways of classical and remote preconditioning regarding protein kinase C, which is one of the key enzymes in classical preconditioning, were compared. METHODS AND RESULTS: Controls (30 min regional ischemia followed by 2 h of reperfusion) had an infarct size of 62+/-5% whereas classical preconditioning reduced it to 10+/-3% of the risk zone (P< or =0.001). Fifteen minutes IOA without reperfusion of the aorta had no influence on infarct size (52+/-4%). When, however, IOA was performed for 15, 10, or 5 min, respectively, followed by a 10-min reperfusion period the size of myocardial infarction decreased significantly. This decrease was dependent on the duration of IOA (18+/-3%, 37+/-8%, 42+/-2%, respectively; P< or =0.001 for the time-dependent linear trend in decrease of infarct size). Fifteen minutes IOA showed the strongest protection which was comparable to classical preconditioning (18+/-3%, P< or =0.001 vs. control). Blockade of the nervous pathway by 20 mg/kg hexamethonium could not inhibit the protection afforded by IOA (14+/-4%). Using chelerythrine, a selective protein kinase C-inhibitor, at a dose of 5 mg/kg body weight, protection from remote (68+/-4%, P< or =0.001 vs. 15 min IOA followed by 10 min of reperfusion without chelerythrine) as well as from classical preconditioning (56+/-5%, P< or =0.001) was completely blocked. CONCLUSION: Protection of the heart by remote preconditioning using IOA is as powerful as classical preconditioning. Both protection methods share protein kinase C as a common element in their signal transduction pathways. Since hexamethonium could not block the protection from IOA and a reperfusion period has to be necessarily interspaced between the IOA and the infarct inducing ischemia of the heart, a neuronal signal transmission from the remote area to the heart can be excluded with certainty. A humoral factor must be responsible for the remote protection. Interestingly the production of the protecting factor is dependent on the duration of the ischemia of the lower limb. The protecting substance, which must be upstream of protein kinase C, remains to be identified.