B-type natriuretic peptide at early reperfusion limits infarct size in the rat isolated heart

Natriuretic peptides are regulatory autacoids in the mammalian myocardium whose functions, mediated via particulate guanylyl cyclase/cGMP, may include cytoprotection against ischaemia-reperfusion injury. Previous work has identified that B-type natriuretic peptide (BNP) limits infarct size when administered prior to and during coronary occlusion through a K_ATP channel-dependent mechanism. The present study examined the hypothesis that the protection afforded by BNP is mediated specifically at reperfusion in a postconditioning-like manner. Langendorff-perfused rat hearts were subjected to 35 min coronary artery occlusion and 120 min reperfusion, and infarct size was determined by tetrazolium staining. Postconditioning was effected by applying six 10-second periods of global ischaemia at the onset of reperfusion.Treatment with either BNP 10 nM or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) 1–10 μM was commenced 5 min prior to reperfusion and continued until 10 min after reperfusion. Control infarct size (% of ischaemic risk zone) was 40.8 ± 3.7%.BNP at reperfusion induced a significant limitation of infarct size (BNP 22.9 ± 4.1% P<0.05 vs. control). Co-treatment at reperfusion with BNP and the K_ATP channel blockers 5-hydroxydecanote (5HD, 100 μM), glibenclamide (Glib; 10 μM) or HMR1098 (10 μM) abolished the infarct-limiting effect of BNP (BNP + 5HD 41.0 ± 3.9%, BNP + Glib 39.8 ± 5.6%, BNP + HMR 1098 46.0 ± 7.1%,P < 0.05 vs. BNP). BNP given together with L-NAME (100 μM) at reperfusion resulted in a marked loss of protection (BNP + L-NAME 53.1 ± 3.8% P < 0.001 vs. BNP). In a second series of experiments, SNAP (1–10 μM) given at reperfusion was found not to be protective (SNAP 1 μM 30.2 ± 4.9%, SNAP 2 μM 27.5 ± 9.5%, SNAP 5 μM 39.2 ± 5.7%, SNAP 10 μM 33.7 ± 6.4%, not significant vs. control). In a third series of experiments, postconditioning significantly limited infarct size (14.9 ± 3.6 % vs. control 34.5 ± 4.9%, P < 0.01) and this effect of postconditioning was abolished in the presence of isatin (100 μM), a non-specific blocker of particulate guanylyl cyclases (35.1 ± 6%, P < 0.05 vs. postconditioning). In conclusion, pharmacological activation of pGC by BNP can effectively induce protection against reperfusion injury, by mechanisms involving K_ATP channel opening and endogenous NO synthase activation. Furthermore, endogenous activation of pGC could play a role in the mechanism of postconditioning.     

Long-term Follow-up of Patients Undergoing Postconditioning During ST-Elevation Myocardial Infarction

Reperfusion injury may offset the optimal salvage of myocardium achieved during primary coronary angioplasty. Thus, coronary reperfusion must be combined with cardioprotective adjunctive therapies in order to optimize myocardial salvage and minimize infarct size. Forty-three patients with their first ST-elevation myocardial infarction were randomized to myocardial postconditioning or standard of care at the time of primary coronary angioplasty. Postconditioning was performed immediately upon crossing the lesion with the guide wire and consisted of four cycles of 30 s occlusion followed by 30 s of reperfusion. End-points included infarct size, myocardial perfusion grade (MPG), left-ventricular ejection fraction (LVEF), and long-term clinical events (death and heart failure). Despite similar ischemic times (≅4.5 h) (p = 0.9) a reduction in infarct size was observed among patients treated with the postconditioning protocol. Peak creatine phosphokinase (CPK), as well as its myocardial band (MB) fraction, was significantly lower in the postconditioning group when compared with the control group (CPK—control, 2,444 ± 1,928 IU/L vs. PC, 2,182 ± 1,717 IU/L; CPK-MB—control, 242 ± 40 IU/L vs. PC, 195 ± 33 IU/L; p = 0.64 and p < 0.01, respectively). EF in the postconditioning group was improved when compared with the control group (control, 43% ± 15 vs. PC, 52% ± 9; p = 0.05). After a mean follow-up of 3.4 years, a 6-point absolute difference in LVEF was still evident in the postconditioning group (p = 0.18). MPG was better among patients treated with the postconditioning protocol compared with control (2.5 ± 0.5 vs. 2.1 ± 0.6; p = 0.02). Due to the small sample size no significant differences in clinical events were detected (p value for death = 0.9; p value for heart failure = 0.2). A simple postconditioning protocol applied at the onset of mechanical reperfusion, resulted in reduction of infarct size, better epicardial and myocardial flow, and improvement in left ventricular function. The beneficial effects of postconditioning on cardiac function persist beyond 3 years.     

Intracoronary acid infusion as an alternative to ischemic postconditioning in pigs

Previous studies suggested that prolongation of acidosis during reperfusion is protective and may be an important mechanism of postconditioning protection. The aim of this study was to analyze the therapeutic value of this intervention during in vivo coronary reperfusion, and to compare it with ischemic postconditioning. Pigs were submitted to 48 or 60 min of ischemia and 2 h of reperfusion. Animals were allocated to either intracoronary infusion of Krebs solution at dose and duration previously described as optimal in rat hears (pH 6.4 for the first 3 min of reperfusion), ischemic postconditioning (8 cycles of 30 s ischemia/reperfusion) or their respective control groups (n = 9–11 per group). Neither prolongation of acidosis nor postconditioning modified infarct size after 48 min of ischemia as compared to pooled controls. In contrast, in animals submitted to 60 min of coronary occlusion, infarct size was reduced both by infusion of acid Krebs and ischemic postconditioning (57.92 ± 18.15% and 56.91 ± 7.50 vs. 75.37 ± 9.29% in controls, P < 0.01), despite having similar areas at risk. However, an increased incidence of ventricular fibrillation was observed in pigs reperfused with acid Krebs as compared to ischemic postconditioning (11 out of 20 vs. 3 out of 19 pigs, P < 0.05). In conclusion, in pigs submitted to coronary occlusion, intracoronary acid infusion and postconditioning offered protection against cell death only after prolonged coronary occlusion. Both interventions were equally effective, but intracoronary acid infusion was associated with high risk of ventricular fibrillation. These results are strongly against translation of acidic reperfusion to patients with acute myocardial infarction.     

Postconditioning attenuates myocardial ischemia-reperfusion injury by inhibiting events in the early minutes of reperfusion

OBJECTIVE: We previously showed that brief intermittent ischemia applied during the onset of reperfusion (i.e., postconditioning) is cardioprotective in a canine model of ischemia-reperfusion. This study tested the hypothesis that the early minutes of reperfusion (R) during which postconditioning (Post-con) is applied are critical to its cardioprotection. METHODS: In anesthetized open-chest rats, the left coronary artery (LCA) was occluded for 30 min and reperfused for 3 h. All rats were randomly divided into six groups: Control (n=8): no intervention at R; Ischemic preconditioning (IPC) (n=8): the LCA was occluded for 5 min followed by 10 min of R before the index occlusion; Post-con 1 (n=8): after LCA occlusion, three cycles of 10 s R followed by 10 s LCA re-occlusion were applied during the first minute of R; Post-con 2 (n=8): Six cycles of 10 s R and 10 s re-occlusion were applied during the first 2 min of R; Delayed Post-con (n=8): the ligature was loosened for full reflow for the first minute of R, after which the three-cycle Post-con algorithm was applied; Sham (n=6): the surgical procedure was identical to other groups, but the LCA ligature was not ligated. RESULTS: Infarct size (TTC staining) was 23% smaller in Post-con 1 (40+/-2%*) than in Control (52+/-3%), confirmed by plasma creatine kinase activity (18+/-2* vs. 46+/-6 IU/g protein). There was no further reduction in infarct size with 6 cycles of Post-con (40+/-2.9%, p>0.05 vs. Post-con 1). Meanwhile, infarct size reduction was significantly greater in the IPC group (17+/-3%) than in Post-con1 (p<0.01). The plasma lipid peroxidation product malondialdehyde (MDA, microM/ml) was less after R in IPC and Post-con 1 (0.8+/-0.07* and 0.8+/-0.06*) vs. Control (1.21+/-0.08), consistent with a visual decrease in superoxide anion generation (dihydroethidium staining) in the AAR myocardium after 3 h of reperfusion. Neutrophil accumulation (myeloperoxidase activity, MPO, U/100 g tissue) in the AAR was less in IPC (1.4+/-0.3*) and Post-con 1 (2.5+/-0.3*) vs. Control (5.5+/-0.6). The reductions in infarct size, creatine kinase, MDA and DHE staining were lost with delayed Post-con, while MPO activity remained lower than in Control (3.2+/-0.4*). CONCLUSIONS: (1) Post-con at onset of R reduces myocardial injury; (2) cardioprotection may be mediated, in part, by inhibiting oxidant generation and oxidant mediated injury; (3) the first minute of R in the rat model is critical to cardioprotection by Post-con; and (4) cardioprotection by Post-con may be independent of neutrophil accumulation in AAR. *p<0.05 Post-con vs. Control.     

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-3β (GSK-3β) 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 postconditoning effect remains to be elucidated in clinical setting.     

Effects of Levosimendan on Myocardial Infarct Size and Hemodynamics in a Closed-Chest Porcine Ischemia–Reperfusion Model

Introduction Levosimendan is a positive inotropic drug with vasodilator action and proposed myocardioprotective properties. In a canine model, levosimendan increased coronary collateral flow and reduced myocardial infarct size (IS). We investigated the effect of levosimendan on IS and hemodynamics in the closed-chest porcine ischemia–reperfusion model, which is devoid of coronary collaterals. Methods Infusion with levosimendan (0.2 μg/kg/min following a bolus of 24 μg/kg) or saline was initiated 30 min prior to ischemia in anaesthetized pigs (n = 10 in both groups). Balloon occlusion of the left anterior descending coronary artery for 45 min was followed by 2 1/2 h of reperfusion. Hemodynamics were monitored with a Swan-Ganz catheter and a left ventricular pressure micromanometer. Left ventricular systolic and diastolic function was estimated by dP/dt_max and τ, respectively. Myocardial area at risk (AAR) and IS were assessed in vivo by myocardial perfusion imaging (MPI) and ex vivo by histopathology (fluorescein staining for AAR, tetrazolium staining for IS). Results Prior to ischemia, levosimendan improved left ventricular systolic and diastolic function with coincident preload and afterload reduction. Cardiac output increased by 10 ± 4% (p = 0.04), dP/dt_max by 15 ± 5% (p = 0.01). Pulmonary capillary wedge pressure decreased by 18 ± 3% (p = 0.04), τ by 11 ± 2% (p = 0.001), and mean arterial pressure by 11 ± 2% (p < 0.001). A similar trend was observed during ischemia–reperfusion. The ratio of IS/AAR was not reduced by levosimendan compared to saline as evaluated by histopathology (76 ± 4% vs. 64 ± 7%, p = 0.12) and by MPI (94 ± 2% vs. 87 ± 5%, p = 0.14). Conclusion Levosimendan improves hemodynamics but does not reduce IS in an ischemia–reperfusion model without coronary collaterals.     

No Cardioprotective Benefit of Ischemic Postconditioning in Patients With ST‐Segment Elevation Myocardial Infarction

Background

Postconditioning is a potential cardioprotective strategy that has demonstrated conflicting and variable reductions in infarct size in human trials.

Objectives

To determine whether postconditioning could increase the extent of myocardial salvage in patients with acute ST‐segment elevation myocardial infarction undergoing primary percutaneous coronary intervention (PPCI).

Methods

One hundred two patients (aged 57 ± 11 years; 88% male) were randomly assigned to a postconditioning or standard protocol. Cardiovascular magnetic resonance imaging was performed 3 days after PPCI to measure the volumetric extent of myocardial necrosis and the area at risk.

Results

With similar time‐to‐reperfusion (170 ± 84 minutes in the postconditioning group vs. 150 ± 70 minutes in the standard group, P = 0.22), the myocardial salvage index was not significantly different between the postconditioned group and the control group, averaging 42 ± 22% vs. 33 ± 21%, respectively (P = 0.08). Furthermore, postconditioning was not associated with a smaller infarct size compared to controls (13 ± 7 g/m² vs. 15 ± 8 g/m², respectively, P = 0.18).

Conclusions

Postconditioning does not significantly increase myocardial salvage or reduce infarct size in patients with STEMI undergoing PPCI. However, the possibility of a more modest impact of postconditioning cannot be excluded with our sample size. (J Interven Cardiol 2013;26:482‐490)

     

β1-Adrenergic receptor antagonism abrogates cardioprotective effects of intermittent hypoxia

Adaptation to hypoxia lessens myocardial ischemic injury. This study tested whether hypoxia-induced β-adrenergic activity mobilizes mechanisms that protect myocardium during subsequent ischemia and reperfusion. Dogs were intermittent hypoxia conditioned (IHC) by a 20 days program of 5–8 daily, 5–10 min cycles of normobaric hypoxia (FIO₂=9.5–10%), or sham conditioned with normoxic air, and metoprolol (β₁-adrenoceptor antagonist) was administered throughout the IHC program. Twenty-four hours after the last IHC session, the left anterior descending coronary artery (LAD) was occluded for 60 min, and then reperfused for 5 h. Area at risk (AAR) and infarct size (IS) were measured. IHC lowered IS/AAR from 38±6% in sham-conditioned dogs to 1.1±0.3%, and eliminated ventricular tachycardia (VT) and fibrillation (VF) that occurred in 14 of 17 non-conditioned dogs. Metoprolol blunted IHC-evoked cardioprotection (IS/AAR=27±3%), and VT and/or VF occurred in 5 of 6 dogs. Metoprolol did not exacerbate ischemic injury in sham-conditioned dogs (IS/AAR=38±2%). Neither IHC nor metoprolol affected hematocrit or LAD collateral blood flow. A single IHC session failed to protect ischemic myocardium (IS/AAR = 36±8%), and protection was incomplete after 10 days of IHC (IS/AAR = 13±5%), suggesting that de novo protein synthesis was required for protection. Thus, episodic β₁-adrenergic activation during IHC evokes progressive development of powerful resistance to myocardial ischemia. Returned for 1st revision: 22 April 2006 1st Revision received: 24 April 2006     

Protection from postconditioning depends on the number of short ischemic insults in anesthetized pigs

Abstract Postconditioning in the early reperfusion period confers protection to the heart after a potentially lethal episode of prolonged ischemia. Protection from this novel intervention has been documented in rat, rabbit and canine hearts, but one group has reported that it is ineffective in pigs, a large-animal species that should be most relevant to humans. We hypothesized that this negative result was related to an inappropriate postconditioning protocol rather than the species. The present study, therefore, tested whether an effective postconditioning protocol could be identified that limits infarct size in anesthetized pigs. Domestic Landrace pigs weighing 25–29 kg were anesthetized, and after a mid-sternal thoracotomy and pericardiotomy the left anterior descending coronary artery was ligated for 60 min followed by 3 h of reperfusion. Three groups were studied: control group (n = 5) with no other intervention, 4–30 PostC group (n = 5) with 4 cycles of 30-s reperfusion/30-s ischemia, and 8–30 PostC group (n = 6) with 8 cycles of 30-s reperfusion/30-s ischemia. The two postconditioning protocols started immediately after termination of the 60-min coronary occlusion. Region at risk and infarct size were delineated with the aid of pre-mortem monastral blue injection and postmortem staining with triphenyltetrazolium chloride, respectively. In control hearts 33.5 ± 7.6% of the risk zone infarcted and 36.7 ± 3.7% in the 4–30 PostC group (P = NS). But there was only 10.5 ± 0.5% infarction in the 8–30 PostC group (P < 0.01 vs. the other two groups). Postconditioning confers protection in pigs but requires more than 4 ischemia/reperfusion cycles. Postconditioning may protect by inhibiting mitochondrial permeability transition pore formation by keeping the heart acidotic as it is reoxygenated. If true, then it would be difficult to employ too many occlusion cycles.     

Chronic Erythropoietin Treatment Limits Infarct-size in the Myocardium in Vitro

Summary It has been well established that erythropoietin (EPO) can limit myocardial ischemia/reperfusion injury in a variety of acute settings. However, despite EPO being used chronically to treat anemia the infarct limiting effects of long term treatment (chronic) have never been fully investigated. In this study we examined the effects of a 3 week treatment of EPO (5,000 IU/Kg) in male Sprague Dawley rats in limiting myocardial infarction after 35 min ischemia and 2 h reperfusion in an in vitro isolated heart perfusion model. Treating the animals ‘once a week’ failed to limit infarct size significantly compared to a saline control (54.1% ± 3.5 v 52.3% ± 4.4), whereas a ‘3 times a week’ regime succeeded in significantly reducing infarct size (36.2% ± 3.2 v 52.3% ± 4.4, p < 0.05). To demonstrate that the effect was not due to improved oxygen supply caused by a raised hematocrit level, we also administered EPO 24 h prior to ischemia/reperfusion. This treatment again reduced infarct size compared to a saline control (39.9% ± 4.4 v 58.4% ± 5.0, p < 0.05). To examine the mechanism of protection we used the PI3K inhibitor wortmannin and the nitric oxide synthase inhibitor L-NAME to try to abrogate EPO mediated protection. Where wortmannin failed to block the effects of EPO (31.7% ± 6.0 v 36.2% ± 3.2), L-NAME did abrogate protection (51.6% ± 5.6 v 36.2% ± 3.2, p < 0.05). We demonstrate that chronic EPO treatment limits infarct size and that it does so in a nitric oxide dependent manner.     

Cardioselective nitric oxide synthase 3 gene transfer protects against myocardial reperfusion injury

Nitric oxide modulates the severity of myocardial ischemia–reperfusion (I/R) injury. We investigated whether cardioselective nitric oxide synthase 3 (NOS3) gene transfer could confer myocardial protection against I/R injury in pigs and examined potential molecular mechanisms. I/R injury was induced by balloon occlusion of the left anterior descending artery for 45 min followed by 4 or 72 h reperfusion. Hemodynamic and pathological changes were measured in pigs in the absence (n = 11) or presence of prior intracoronary retroinfusion of human NOS3 (AdNOS3, 5 × 10¹⁰ PFU, n = 13) or control vector (AdRR5, 5 × 10¹⁰ PFU, n = 11). Retrograde NOS3 gene transfer selectively increased NOS3 expression and NO bioavailability in the area at risk (AAR) without changing endogenous NOS isoform expression. At 4 h R, LV systolic (dP/dt _max) and diastolic (dP/dt _min) function was better preserved in AdNOS3- than in AdRR5-injected pigs (2,539 ± 165 vs. 1,829 ± 156 mmHg/s, and ?2,781 ± 340 vs. ?2,062 ± 292 mmHg/s, respectively, P < 0.05 for both). Myocardial infarct size (% AAR) was significantly smaller in AdNOS3 than in control and AdRR5 and associated with a significantly greater reduction in cardiac myeloperoxidase activity, a marker of neutrophil infiltration. The latter effects were sustained at 72 h R in a subset of pigs (n = 7). In the AAR, intercellular endothelial adhesion molecule-1 expression and cardiomyocyte apoptosis were significantly lower in AdNOS3. In conclusion, single myocardial NOS3 retroinfusion attenuates I/R injury, and causes a sustained reduction in myocardial infarct size and inflammatory cell infiltration. Gene-based strategies to increase NO bioavailability may have therapeutic potential in myocardial I/R.     

The Cardioprotective Effect of Necrostatin Requires the Cyclophilin-D Component of the Mitochondrial Permeability Transition Pore

Background Necrostatin (Nec-1) protects against ischemia–reperfusion (IR) injury in both brain and heart. We have previously reported in this journal that necrostatin can delay opening of the mitochondrial permeability transition pore (MPTP) in isolated cardiomyocytes. Aim The aim of the present study was to investigate in more detail the role played by the MPTP in necrostatin-mediated cardioprotection employing mice lacking a key component of the MPTP, namely cyclophilin-D. Method Anaesthetized wild type (WT) and cyclophilin-D knockout (Cyp-D−/−) mice underwent an open-chest procedure involving 30 min of myocardial ischemia and 2 h of reperfusion, with subsequent infarct size assessed by triphenyltetrazolium staining. Nec-1, given at reperfusion, significantly limited infarct size in WT mice (17.7 ± 3% vs. 54.3 ± 3%, P < 0.05) but not in Cyp-D−/− mice (28.3 ± 7% vs. 30.8 ± 6%, P > 0.05). Conclusion The data obtained in Cyp-D−/− mice provide further evidence that Nec-1 protects against myocardial IR injury by modulating MPTP opening at reperfusion.     

Nitric oxide/cGMP signalling mediates the cardioprotective action of adrenomedullin in reperfused myocardium

We demonstrated previously that adrenomedullin (AM), when given during early reperfusion, limited infarct size in rat heart. The present study was undertaken to provide direct evidence of the NO-dependency of AM’s cardioprotective action by assessing NO biosynthesis and involvement of the soluble guanylyl cyclase (sGC) pathway. Perfused hearts from male CD-1 mice were subjected to 30-min left coronary occlusion and 60-min reperfusion. Infarct size was determined by tetrazolium staining. AM 10 nM was administered from 20 min after coronary occlusion until 10 min after reperfusion. Coronary effluent was analysed for NO₂ ⁻ and NO₃ ⁻, and myocardial samples were analysed for NO₂ ⁻, NO₃ ⁻, nitroso-adducts and cGMP concentration. To examine the role of NO/sGC signalling in the infarct-limiting action of AM, further hearts received the sGC inhibitor ODQ 2 μM. AM treatment stimulated NO synthesis, indicated by increased NO₂ ⁻ efflux in coronary effluent throughout reperfusion (summarised as area under curve, AM 29.2 ± 3.9 vs. control 14.4 ± 2.8 μmol min² mL⁻¹, P < 0.05). AM limited infarct size (35.4 ± 2.7 vs. 12.2 ± 2.3%, P < 0.01), associated with a 2.45-fold increase (P < 0.05) in myocardial cGMP concentration at 10 min after reperfusion. ODQ abolished the infarct size-limiting effect of AM (28.9 ± 4.3%). These data provide the first evidence that AM increases NO bioavailability in intact murine myocardium and confirm that the NO/sGC/cGMP pathway is central to the cytoprotective action of AM against ischaemia–reperfusion injury.     

Attenuation of myocardial injury by postconditioning: role of hypoxia inducible factor-1α

Postconditioning (PostC) has regenerated interest as a mechanical intervention against myocardial ischemia/reperfusion injury, but its molecular mechanisms remain elusive. This study tested the hypothesis that hypoxia inducible factor-1α (HIF-1α) plays a role in PostC-induced cardioprotection. Male Wistar rats were subjected to 30 min ischemia followed by 3 h of reperfusion (Control). PostC with 3 cycles of 10 s reperfusion and 10 s re-occlusion was applied at the onset of reperfusion. Relative to the Sham group, HIF-1α protein level was increased by 2.9-fold in the Control group, but its level was enhanced by 5.8-fold with PostC (P < 0.01 vs. Control). However, HIF-1α protein level was further augmented by 2.0-fold and 3.3-fold, respectively, when the prolyl hydroxylase inhibitor, dimethyloxalylglycine (DMOG, 40 mg/kg, i.p.) was given at 24 h before ischemia in both Control and PostC groups. PostC reduced infarct size by 24% compared with the Control (27 ± 4.2% vs. 36 ± 5.2%, P < 0.01), consistent with significant lower levels of plasma creatine kinase activity, index of cardiomyocyte apoptosis and caspase-3 activity. Although pretreatment with DMOG significantly reduced infarct size relative to the Control, the infarct-sparing effect of PostC was remarkably enhanced when DMOG was given before PostC (18 ± 2.0% vs. 27 ± 4.2% in PostC alone, P < 0.05). There was a significant linear inverse relationship between HIF-1α protein level and infarct size (r = −0.799, P < 0.01) among all groups. Furthermore, along with up-regulated HIF-1α expression, the levels of iNOS mRNA and protein were significantly increased in the PostC alone and DMOG plus PostC groups. In conclusion, these data suggest that HIF-1α is involved in cardioprotection by PostC and pharmacological augmentation of HIF-1α expression that enhances the infarct-sparing effect of PostC; iNOS, the downstream gene of HIF-1α, may participate in signaling pathways in mediating PostC’s protection.     

Comparative study of AMP579 and adenosine in inhibition of neutrophil-mediated vascular and myocardial injury during 24 h of reperfusion

OBJECTIVE: The purpose of this study was to compare protective effects of AMP579 and adenosine (Ado) at reperfusion (R) on inhibition of polymorphonuclear neutrophil (PMN) activation, PMN-mediated injury to coronary artery endothelium, and final infarct size. METHODS: In anesthetized dogs, 1 h of left anterior descending coronary artery occlusion was followed by 24 h R and drugs were administered at R. Control (n=8, saline control), AMPI (n=7, AMP579, 50 microg/kg i.v. bolus followed by 3 microg/kg/min for 2 h), AMPII (n=7, AMP579, 50 microg/kg i.v. bolus), AMPIII (n=7, AMP579, 3 microg/kg/min i.v. for 2 h), and Ado (n=7, adenosine, 140 microg/kg/min i.v. for 2 h). RESULTS: AMP579 in vitro directly inhibited superoxide radical (O(-)(2)) generation (nM/5x10(6) PMNs) from PMNs dose-dependently (from 17+/-1* at 10 nM to 2+/-0.2* at 10 microM vs. activated 30+/-2). However, inhibition of O(-)(2) generation by Ado at each concentration was significantly less than for AMP579. The IC(50) value for AMP579 (0.09+/-0.02 microM) on O(-)(2) generation was significantly less than that of Ado (3.9+/-1. 1 microM). Adherence of unstimulated PMN to postischemic coronary artery endothelium (PMNs/mm(2)) was attenuated in AMPI and AMPIII vs. Control (60+/-3* and 58+/-3* vs. Control 110+/-4), while Ado partially attenuated PMN adherence (98+/-3*). Accordingly, endothelial-dependent vascular relaxation was significantly greater in AMPI and AMPIII vs. Ado. At 24 h R, myocardial blood flow (MBF, ml/min/g) in the area at risk (AAR), confirmed by colored microspheres, in AMPI and AMPIII was significantly improved (0.8+/-0. 1* and 0.7+/-0.1* vs. Control 0.3+/-0.04). Infarct size (IS, TTC staining) in AMPI and AMPIII was significantly reduced from 38+/-3% in Control to 21+/-4%* and 22+/-3%*, respectively, confirmed by lower plasma creatine kinase activity (I.U./g protein) in these two groups (27+/-6* and 32+/-2* vs. 49+/-3). Cardiac myeloperoxidase activity (MPO, Abs/min) in the AAR was significantly reduced in AMPI and AMPIII vs. Control (36+/-11* and 35+/-10* vs. 89+/-10). However, changes in MBF, IS and MPO were not significantly altered by Ado. CONCLUSIONS: These data suggest that continuous infusion of AMP579 at R is more potent than adenosine in attenuating R injury, and AMP579-induced cardioprotection involves inhibition of PMN-induced vascular and myocardial tissue injury. *P<0.05 vs. Control.     

Influence of the angiotensin II AT1 receptor antagonist irbesartan on ischemia/reperfusion injury in the dog heart

The aim of the present study was to investigate whether the non-peptide angiotensin II type 1 (AT₁) receptor antagonist irbesartan (SR 47436, BMS 186295, 2-n-butyl-3[2‘-(1H-tetrazol-5-yl)-biphenyl-4-yl)methyl]-1,3-diaza-spiro[4,4]non-1-en-4-one) has myocardial protective effects during regional myocardial ischemia/reperfusion in vivo. Eighteen anesthetized open-chest dogs were instrumented for measurement of left ventricular and aortic pressure (tip manometer and pressure transducer, respectively), and coronary flow (ultrasonic flowprobes). Regional myocardial function was assessed by Doppler displacement transducers as systolic wall thickening (sWT) in the antero-apical and the postero-basal wall. The animals underwent 1 h of left anterior descending coronary artery (LAD) occlusion and subsequent reperfusion for 3 hours. Irbesartan (10 mg kg⁻¹, n=9) or the vehicle (KOH, control, n=9) was injected intravenously 30 min before LAD occlusion. Regional myocardial blood flow (RMBF) was measured after irbesartan injection and at 30 min LAD occlusion using colored microspheres. Infarct size was determined by triphenyltetrazolium chloride staining after 3 h of reperfusion. There was no recovery of sWT in the LAD perfused area in both groups at the end of the experiments (systolic bulging, −15.1±6.1% of baseline (irbesartan) vs. −12.3±3.0% (control), mean±SEM). Irbesartan led to an increase in RMBF in normal myocardium (2.47±0.40 vs. 1.35±0.28 ml min⁻¹ g⁻¹, P<0.05), and also to an increase in collateral blood flow to the ischemic area (0.27±0.04 vs. 0.17±0.02 ml min⁻¹ g⁻¹, P=<0.05). Infarct size (percent of area at risk) was 24.8±3.2% in the treatment group compared with 26.9±4.8% in the control group (P=0.72). These results indicate that a blockade of angiotensin II AT₁ receptors with irbesartan before coronary artery occlusion led to an increase in RMBF, but did not result in a significant reduction of myocardial infarct size. Received: 6 October 1999 Returned for revision: 16 November 1999 Revision received: 7 February 2000 Accepted: 29 March 2000     

Nifedipine limits infarct size via NO-dependent mechanisms in dogs

Objectives Amlodipine increases NO levels in coronary vessels and aorta via bradykinin-dependent mechanisms in vitro. We have previously reported that nifedipine increases cardiac NO levels in the ischemic canine hearts, suggesting that nifedipine may also have protective effects against ischemia and reperfusion injury, because the enhancement of NO production limits infarct size. We tested whether nifedipine limits infarct size via NO-dependent mechanisms. Methods In open chest dogs, the left anterior descending coronary artery was perfused with blood through a bypass tube and occluded for 90 min followed by 6 hours of reperfusion. Infarct size was assessed at 6 hours of reperfusion. Nifedipine of 3 or 6 μg/kg/min was infused into the bypass tube between 10 min prior to the onset of ischemia and 60 min of reperfusion. Results Neither systemic blood pressure nor heart rate changed during infusion of nifedipine. Infarct size was reduced by the administration of nifedipine (3 or 6 μg/kg/min) compared with the untreated condition (25.6 plusmn; 2.6 and 19.1 ± 3.5 vs. 43.4 ± 5.6 %, respectively), which was completely blunted by L-NAME (45.0 ± 3.6 and 45.4 ± 4.2 vs. 47.9 ± 3.9 % in the nifedipine (3 or 6 μg/kg/min) with L-NAME groups vs. the L-NAME group). Myeloperoxidase activity of the myocardium increased after 6 hours of reperfusion, which was attenuated by nifedipine. The limitation of infarct size and the attenuation in myeloperoxidase activity were completely blunted by L-NAME. There were no significant differences in collateral blood flow at 45 min of ischemia between each group. Conclusions We conclude that the Ca channel blocker, nifedipine, limits infarct size via NO-dependent mechanisms. Received: 21 September 2000, Returned for 1. revision: 9 October 2000, 1. Revision received: 17 January 2001, Returned for 2. revision: 5 February 2001, 2. Revision received: 13 February 2001, Accepted: 14 February 2001     

Long-term protection and mechanism of pacing-induced postconditioning in the heart

Brief periods of ventricular pacing during the early reperfusion phase (pacing-induced postconditioning, PPC) have been shown to reduce infarct size as measured after 2 h of reperfusion. In this study, we investigated (1) whether PPC leads to maintained reduction in infarct size, (2) whether abnormal mechanical load due to asynchronous activation is the trigger for PPC and (3) the signaling pathways that are involved in PPC. Rabbit hearts were subjected to 30 min of coronary occlusion in vivo, followed by 6 weeks of reperfusion. PPC consisted of ten 30-s intervals of left ventricular (LV) pacing, starting at reperfusion. PPC reduced infarct size (TTC staining) normalized to area at risk, from 49.0 ± 3.3% in control to 22.9 ± 5.7% in PPC rabbits. In isolated ejecting rabbit hearts, replacing LV pacing by biventricular pacing abolished the protective effect of PPC, whereas ten 30-s periods of high preload provided a protective effect similar to PPC. The protective effect of PPC was neither affected by the adenosine receptor blocker 8-SPT nor by the angiotensin II receptor blocker candesartan, but was abrogated by the cytoskeletal microtubule-disrupting agent colchicine. Blockers of the mitochondrial K_ATP channel (5HD), PKC (chelerythrine) and PI3-kinase (wortmannin) all abrogated the protection provided by PPC. In the in situ pig heart, PPC reduced infarct size from 35 ± 4 to 16 ± 12%, a protection which was abolished by the stretch-activated channel blocker gadolinium. No infarct size reduction was achieved if PPC application was delayed by 5 min or if only five pacing cycles were used. The present study indicates that (1) PPC permanently reduces myocardial injury, (2) abnormal mechanical loading is a more likely trigger for PPC than electrical stimulation or G-coupled receptor stimulation and (3) PPC may share downstream pathways with other modes of cardioprotection.     

Dose-Dependent Cardioprotection of Moderate (32°C) Versus Mild (35°C) Therapeutic Hypothermia in Porcine Acute Myocardial Infarction

Objectives

The study investigated whether a dose response exists between myocardial salvage and the depth of therapeutic hypothermia.

A novel role for mitochondrial sphingosine-1-phosphate produced by sphingosine kinase-2 in PTP-mediated cell survival during cardioprotection

Although mitochondria are key determinants of myocardial injury during ischemia–reperfusion (I/R), their interaction with critical cytoprotective signaling systems is not fully understood. Sphingosine-1-phosphate (S1P) produced by sphingosine kinase-1 protects the heart from I/R damage. Recently a new role for mitochondrial S1P produced by a second isoform of sphingosine kinase, SphK2, was described to regulate complex IV assembly and respiration via interaction with mitochondrial prohibitin-2. Here we investigated the role of SphK2 in cardioprotection by preconditioning. Littermate (WT) and sphk2 ^−/− mice underwent 45 min of in vivo ischemia and 24 h reperfusion. Mice received no intervention (I/R) or preconditioning (PC) via 5 min I/R before the index ischemia. Despite the activation of PC-cytoprotective signaling pathways in both groups, infarct size in sphk2 ^−/− mice was not reduced by PC (42 ± 3% PC vs. 43 ± 4% I/R, p = ns) versus WT (24 ± 3% PC vs. 43 ± 3% I/R, p < 0.05). sphk2 ^−/− mitochondria exhibited decreased oxidative phosphorylation and increased susceptibility to permeability transition (PTP). Unlike WT, PC did not prevent ischemic damage to electron transport or the increased susceptibility to PTP. To evaluate the direct contribution to the resistance of mitochondria to cytoprotection, SphK2, PHB2 or cytochrome oxidase subunit IV was depleted in cardiomyoblasts. PC protection was abolished by each knockdown concomitant with decreased PTP resistance. These results point to a new action of S1P in cardioprotection and suggest that the mitochondrial S1P produced by SphK2 is required for the downstream protective modulation of PTP as an effector of preconditioning protection.