Remote postconditioning by humoral factors in effluent from ischemic preconditioned rat hearts is mediated via PI3K/Akt-dependent cell-survival signaling at reperfusion

Short non-lethal ischemic episodes administered to hearts prior to (ischemic preconditioning, IPC) or directly after (ischemic postconditioning, IPost) ischemic events facilitate myocardial protection. Transferring coronary effluent collected during IPC treatment to un-preconditioned recipient hearts protects from lethal ischemic insults. We propose that coronary IPC effluent contains hydrophobic cytoprotective mediators acting via PI3K/Akt-dependent pro-survival signaling at ischemic reperfusion. Ex vivo rat hearts were subjected to 30 min of regional ischemia and 120 min of reperfusion. IPC effluent administered for 10 min prior to index ischemia attenuated infarct size by ≥55% versus control hearts (P < 0.05). Effluent administration for 10 min at immediate reperfusion (reperfusion therapy) or as a mimetic of pharmacological postconditioning (remote postconditioning, RIPost) significantly reduced infarct size compared to control (P < 0.05). The IPC effluent significantly increased Akt phosphorylation in un-preconditioned hearts when administered before ischemia or at reperfusion, while pharmacological inhibition of PI3K/Akt-signaling at reperfusion completely abrogated the cardioprotection offered by effluent administration. Fractionation of coronary IPC effluent revealed that cytoprotective humoral mediator(s) released during the conditioning phase were of hydrophobic nature as all hydrophobic fractions with molecules under 30 kDa significantly reduced infarct size versus the control and hydrophilic fraction-treated hearts (P < 0.05). The total hydrophobic effluent fraction significantly reduced infarct size independently of temporal administration (before ischemia, at reperfusion or as remote postconditioning). In conclusion, the IPC effluent retains strong cardioprotective properties, containing hydrophobic mediator(s) < 30 kDa offering cytoprotection via PI3K/Akt-dependent signaling at ischemic reperfusion.     

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.     

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.     

Treatment of reperfusion injury with intracoronary calcium channel antagonists and reduced coronary free calcium concentration in regionally ischemic, reperfused porcine hearts

The effect of intracoronary diltiazem, EGTA (ethylene-bis-(beta-aminomethylether)-N,N'-tetraacetic acid), nifedipine, verapamil and isotonic saline solution as placebo on reperfusion injury was investigated in regionally ischemic, reperfused porcine hearts. The left anterior descending coronary artery was distally occluded for 45 min and was reperfused for 3 days. Intracoronary infusion was started immediately before reperfusion and continued during 45 min of reperfusion. Infarct size was determined as the ratio of infarcted (tetrazolium stain) to ischemic myocardium (dye technique). Regional systolic shortening was assessed by sonomicrometry. Apart from left ventricular end-diastolic pressure before ischemia and during 45 min of reperfusion, global hemodynamic values in the five treatment groups did not differ; in particular, calculated left ventricular oxygen consumption before and during ischemia was equally low. Intracoronary EGTA decreased coronary venous free calcium concentration to about 70% of baseline value. Infarct size was reduced from 76 +/- 10% (control group, n = 8) to 60 +/- 10% (p less than 0.01) by intracoronary diltiazem (n = 8) and to 55 +/- 15% (p less than 0.01) by intracoronary EGTA (n = 8). Insignificant reductions in infarct size were found after treatment with intracoronary verapamil (63 +/- 18%, n = 8) and intracoronary nifedipine (68 +/- 9%, n = 7). Regional systolic shortening of the risk region, which did not differ among the groups before occlusion and during ischemia, recovered to the greatest extent in the EGTA-treated pigs (p less than 0.01 compared with values in the control group). Treatment with intracoronary calcium antagonists resulted in only marginal improvement of systolic shortening.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracoronary infusion of superoxide dismutase and reperfusion injury in the pig heart

Summary The effects of an intracoronary infusion of superoxide dismutase on infarct size were studied in 16 pigs submitted to a 48-min coronary occlusion of the mid left anterior descending coronary artery followed by reperfusion for 24 h. Areas at risk marked with fluoresccine and infarct sizes calculated with triphenyl tetrazolium chloride staining 24 h after the occlusion were similar in the five control animals with coronary reperfusion alone, in the five animals with an intracoronary infusion of lactate Ringer initiated 3 min before reperfusion and maintained for 33 min and in the six animals with superoxide dismutase added to the solution of lactate Ringer and infused at a rate of 2500 units/min. The ratios infarct size/area at risk were respectively 0.50±0.10, 0.63±0.10 and 0.65±0.04 in the three study groups (NS). The extent of intramyocardial hemorrhage, evaluated by morphometric analysis was also similar 0.90±0.29×10⁶, 0.70±0.14 and 1.62±0.42 red blood cells/mm³ of tissue (NS). The superoxide dismutase infusion, however, resulted in significantly fewer early reperfusion arrhythmias which involved 23±15 s of each minute electrocardiographic recording in the superoxide dismutase group, compared to 37±13 s in the lactate Ringer group and 45±14 s in the control group (p=0.004). The lack of an effect of intracoronary infusion of superoxide dismutase on infarct size suggests that in this experimental model, extracellular superoxide radicals generated during early reperfusion have no major role on myocardial cell necrosis and microvascular damage. Reperfusion arrhythmias were, however, reduced.Partially supported by a grant PA 86/0389 from the Comisión Interministerial de Ciencia y Tecnología (CICYT), grant 1/86 from the Sección de Cardiopatía isquemica de la Sociedad Española de Cardiología and grant 17/88 of the Convenio Madrid-Quebec.     

The intracoronary infusion of superoxide dismutase during the initial liberation of oxygen free radicals induced by reperfusion. The effect on the infarct size after 60 minutes of coronary occlusion in the pig model]

The role of oxygen free radicals (OFR) generated early during myocardial reperfusion in the genesis of myocardial necrosis was studied in 26 pigs submitted to transient coronary occlusion followed by one of three different reperfusion protocols. In group A, a selective intracoronary infusion of a Ringer solution was started after 60 min of coronary occlusion, and reperfusion was performed 4 min later. The infusion was maintained during the first 6 min of reperfusion at a rate of 3 ml/min. In group B, the Ringer solution administered during reperfusion contained a high concentration (2.778 U/ml) of superoxide dismutase (SOD). In group C, reperfusion was performed after 60 min of coronary occlusion with no intracoronary infusion. Twenty-four hours later the heart was excised and the area at risk and infarct size were measured by in vivo fluorescein injection and triphenyl-tetrazolium chloride staining respectively. The area at risk was similar in the 3 groups: 15.03 +/- 2.6%, 13.26 +/- 3.3% and 16.34 +/- 6.7% of ventricular mass in groups A, B, and C, respectively (p = 0.42). No differences between groups were observed in infarct size, either when measured as a percent of ventricular mass (10.04 +/- 3.8%, 9.31 +/- 3.8% and 10.1 +/- 2.4% in groups A, B, and C, p = 0.91) or as a percent of the area at risk (64.63 +/- 18.5%, 67.81 +/- 16.1%, and 61.35 +/- 6.7%, respectively, p = 0.72). Thus, the intracoronary administration of SOD during the early reperfusion has not beneficial effect on infarct size. This results suggest that the early burst of OFR is not a major determinant of infarct size in the pig.     

Failure of iloprost to protect the regionally ischemic, reperfused porcine heart.

The effect of iloprost (Schering AG, Berlin), a stable prostacyclin analogue, was investigated in ischemic, reperfused porcine hearts. The left anterior descending coronary artery (LAD) was distally occluded in 18 pigs for 45 min followed by 3-d of reperfusion. Nine pigs were continuously treated with iloprost at a dose of 25 ng/kg per min. Treatment was started as intracoronary infusion into the proximal LAD 10 min before occlusion. The intercoronary infusion was replaced by an intravenous infusion after 45 min of reperfusion, which was continued until the end of the experiment. Infarct size was determined as the ratio of infarcted (tetrazolium stain) to ischemic myocardium (dye technique). Regional systolic shortening was assessed by sonomicrometry. Myocardial concentrations of adenosine triphosphate were evaluated at the end of the experiment. Generation of free radicals by stimulated polymorphonuclear neutrophils was determined by luminol-enhanced chemiluminescence. Histologic and immunohistologic techniques were applied to characterize the myocardial inflammatory response. Global hemodynamics did not differ between the two groups. Neither infarct size (control group 68 +/- 18%, treated group 74 +/- 14%), recovery of systolic shortening (control group 3 +/- 6%, treated group 6 +/- 6%), nor myocardial adenosine triphosphate concentrations were improved by iloprost treatment. Myocardial inflammatory response remained unaffected by this treatment. The capacity of coronary venous, stimulated polymorphonuclear neutrophils to generate free radicals was slightly suppressed in the treated group before ischemia, at the end of ischemia and during early reperfusion. In this preparation, iloprost did not exhibit any beneficial effect on infarct size, recovery of systolic shortening and myocardial adenosine triphosphate concentrations.     

Limitation of Infarct Size and No-Reflow by Intracoronary Adenosine Depends Critically on Dose and Duration

ObjectivesIn the absence of effective clinical pharmacotherapy for prevention of reperfusion-mediated injury, this study re-evaluated the effects of intracoronary adenosine on infarct size and no-reflow in a porcine model of acute myocardial infarction using clinical bolus and experimental high-dose infusion regimens.BackgroundDespite the clear cardioprotective effects of adenosine, when administered prior to ischemia, studies on cardioprotection by adenosine when administered at reperfusion have yielded contradictory results in both pre-clinical and clinical settings.MethodsSwine (54 ± 1 kg) were subjected to a 45-min mid–left anterior descending artery occlusion followed by 2 h of reperfusion. In protocol A, an intracoronary bolus of 3 mg adenosine injected over 1 min (n = 5) or saline (n = 10) was administered at reperfusion. In protocol B, an intracoronary infusion of 50 μg/kg/min adenosine (n = 15) or saline (n = 21) was administered starting 5 min prior to reperfusion and continued throughout the 2-h reperfusion period.ResultsIn protocol A, area-at-risk, infarct size, and no-reflow were similar between groups. In protocol B, risk zones were similar, but administration of adenosine resulted in significant reductions in infarct size from 59 ± 3% of the area-at-risk in control swine to 46 ± 4% (p = 0.02), and no-reflow from 49 ± 6% of the infarct area to 26 ± 6% (p = 0.03).ConclusionsDuring reperfusion, intracoronary adenosine can limit infarct size and no-reflow in a porcine model of acute myocardial infarction. However, protection was only observed when adenosine was administered via prolonged high-dose infusion, and not via short-acting bolus injection. These findings warrant reconsideration of adenosine as an adjuvant therapy during early reperfusion.     

Lack of Adenosine Causes Myocardial Refractoriness

Objectives. This study sought to investigate the myocardial mechanisms causing refractoriness to ischemic preconditioning in pigs.Background. Ischemic preconditioning in the pig vanishes after 60 min and cannot be reinstated by a second cycle of brief coronary occlusions at this time point. Ischemic preconditioning has been shown to be mediated by adenosine A₁-receptors. Because myocardial adenosine production during ischemia ceases as the number of repeated brief ischemic episodes increases, we hypothesized that this lack of adenosine may cause this myocardial refractoriness.Methods. In open chest pigs, ischemic preconditioning was achieved by repeated brief coronary occlusions. Myocardial adenosine content was assessed by high performance liquid chromatographic analysis of serial myocardial biopsy samples; infarct size (percent infarcted area of the area at risk) was determined using tetrazolium salts.Results. Ischemic preconditioning by two cycles of occlusion of the left anterior descending coronary artery (10 min) and reperfusion (30 min) decreased infarct size ([mean ± SEM] 40.4 ± 2.9%; control: 76.9 ± 1.8%, p < 0.001). Prolonging the second reperfusion period to 60 min caused ischemic preconditioning to vanish (79.0 ± 0.5%) and caused refractoriness to a second cycle of preconditioning (70.0 ± 2.0%). Myocardial adenosine content increased only during the first coronary occlusion (baseline: 110.9 ± 42.0 nmol/g dry weight; first coronary occlusion: 1,686.2 ± 244.1, p < 0.001) but not during subsequent coronary occlusions. In refractory myocardium, intramyocardial microinfusion of the adenosine A₁-receptor agonist N⁶-cyclohexyladenosine (CHA [0.3 mmol/liter]) again decreased infarct size (27.4 ± 7.0%, p < 0.001 vs. control).Conclusions. Myocardial refractoriness may be caused by the inability to produce adenosine endogenously. In refractory myocardium, application of CHA reinduces cardioprotection.     

RISK and SAFE signaling pathway interactions in remote limb ischemic perconditioning in combination with local ischemic postconditioning

Local ischemic postconditioning (IPost) and remote ischemic perconditioning (RIPer) are promising methods to decrease ischemia–reperfusion (I/R) injury. We tested whether the use of the two procedures in combination led to an improvement in cardioprotection through a higher activation of survival signaling pathways. Rats exposed to myocardial I/R were allocated to one of the following four groups: Control, no intervention at myocardial reperfusion; IPost, three cycles of 10-s coronary artery occlusion followed by 10-s reperfusion applied at the onset of myocardial reperfusion; RIPer, 10-min limb ischemia followed by 10-min reperfusion initiated 20 min after coronary artery occlusion; IPost+RIPer, IPost and RIPer in combination. Infarct size was significantly reduced in both IPost and RIPer (34.25 ± 3.36 and 24.69 ± 6.02%, respectively) groups compared to Control (54.93 ± 6.46%, both p < 0.05). IPost+RIPer (infarct size = 18.04 ± 4.86%) was significantly more cardioprotective than IPost alone (p < 0.05). RISK pathway (Akt, ERK1/2, and GSK-3β) activation was enhanced in IPost, RIPer, and IPost+RIPer groups compared to Control. IPost+RIPer did not enhance RISK pathway activation as compared to IPost alone, but instead increased phospho-STAT-3 levels, highlighting the crucial role of the SAFE pathway. In IPost+RIPer, a SAFE inhibitor (AG490) abolished cardioprotection and blocked both Akt and GSK-3β phosphorylations, whereas RISK inhibitors (wortmannin or U0126) abolished cardioprotection and blocked STAT-3 phosphorylation. In our experimental model, the combination of IPost and RIPer improved cardioprotection through the recruitment of the SAFE pathway. Our findings also indicate that cross talk exists between the RISK and SAFE pathways.     

缺血后适应对活性氧及心肌细胞凋亡的影响

目的观察后适应和远程后适应对大鼠缺血再灌注模型活性氧生成及心肌细胞凋亡的影响，探讨活性氧在后适应和远程后适应中的作用。方法大鼠随机分为6组：①假手术组：只穿线，不结扎；②模型组：前降支缺血30min后再灌注2h：③后适应组：前降支缺血30min后进行3个循环后适应（缺血10s，再灌注10s，然后再灌注2h;④远程后适应组：前降支缺血24min时结扎右股动脉5min，在再灌注前1min开通股动脉，前降支再灌注2h；⑤2-巯基丙酰基甘氨酸组l；⑥2-巯基丙酰基甘氨酸组2：在缺血20min时尾静脉注射2一巯基丙酰基甘氨酸20mg／kg，余处理分别同组③和（或）④。实验结束后检测血丙二醛，心肌标本行HE染色、TUNEL检测和测定Bcl-2、BaxmRNA。结果①血丙二醛活性：后适应组、远程后适应组、2-巯基丙酰基甘氨酸组1和组2分别为（14．6±1．4）、（15．6±1．5）、（14．4±1．6）、（15．0±1．4）μmol／L，低于模型组（18．3±1．9）μmol／L，（P〈0．05）；②后适应组和远程后适应组Bcl-2表达高于模型组，而Bax表达低于模型组（P〈0．05）；2-巯基丙酰基甘氨酸处理有对抗作用；④细胞凋亡率：后适应组（25．3±2．3）％和远程后适应组（26．7±2．2）％低于模型组（50．0±7．9）％（P〈0．05），2-巯基丙酰基甘氨酸处理有对抗作用。结论后适应和远程后适应均能减少活性氧的产生：后适应和远程后适应都有减少细胞凋亡作用，其心肌保护作用与减少活性氧的产生有关：提示活性氧可能在后适应和远程后适应中发挥重要作用。     

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.     

Involvement of ATP-sensitive potassium channels in preconditioning protection

Summary Single or multiple brief periods of ischemia (preconditioning, PC) have been shown to protect the myocardium from infarction during a subsequent more prolonged ischemic insult. To test the hypothesis that opening of ATP-sensitive potassium channels (K_ATP) is involved in this mechanism, either bimakalim, a K_ATP channel opener, or glibenclamide, a K_ATP channel blocker, were administered to mimic or to block preconditioning protection in barbital-anesthetized pigs. PC was elicited by a single period of 10 min left anterior descending coronary artery (LADCA) occlusion followed by 15 min of reperfusion before the LADCA was reoccluded for 60 min. Instead of PC, bimakalim infusion was started 15 min before the 60 min LADCA occlusion (TCO) and stopped with the onset of ischemia. Glibenclamide was administered either for 10 min prior to the PC protocol, before bimakakim infusion, or before TCO. Regional wall function was quantified with ultrasonic crystals aligned to measure wall thickening (%WT). At the end of the protocol, infarct size was determined by incubating myocardium with p-nitrobluetetrazolium.In seven preconditioned pigs, infarct size was 9.9±5.1% of the risk region compared with 65.9±6.0% in the seven control pigs subjected to 60 min of ischemia only (p<0.001). In seven pigs treated with bimakalim, infarct size was reduced to 35.3±6.6 (p<0.05 vs. controls). Blocking ATP-sensitive potassium channels with glibenclamide prior to PC abolished its protective effect (infarct size, 62.2±4.5%;p<0.001 vs. PC alone). Glibenclamide also antagonized the protective effect of bimakalim (infarct size, 55.2±4.0%), but did not affect infarct size, when solely administered prior to the prolonged ischemic period (62.2±4.3%). We conclude that in swine myocardium K_ATP channels are involved in the protective effect of ischemic preconditioning, since glibenclamide completely abolished the protective effect of preconditioning, while bimakalim could — at least in part — mimic it.Dedicated to Prof. Dr. H.-J. Langmann on the occassion of his 70th birthday.     

Pathophysiology of Myocardial Infarction

One or several short cycles of ischemia/reperfusion before (preconditioning) or after (postconditioning) a sustained coronary occlusion with subsequent reperfusion reduce the ultimate infarct size. The protection is potent, but limited to a narrow time frame. In animal experiments, a complex signal transduction cascade was identified which results specifically in a reduction of reperfusion injury. There is evidence for both ischemic pre- and postconditioning in patients with coronary artery disease.     

Long-term inhibition of myocardial infarction by postconditioning during reperfusion

Cardioprotection with postconditioning has been well demonstrated after a short period of reperfusion. This study tested the hypothesis that postconditioning reduces infarct size, vascular dysfunction, and neutrophil accumulation after a long-term reperfusion. Canines undergoing 60 min left anterior descending artery (LAD) occlusion were divided into two control groups of either 3 h or 24 h of full reperfusion and two postconditioning groups with three 30 s cycles of reperfusion and re-occlusion applied at the onset of either 3 h or 24 h of reperfusion. Size of the area at risk (AAR) and collateral blood flow during ischemia were similar among groups. In controls, infarct size as percentage of the AAR (30 ± 3 vs. 39 ± 2* %) by TTC staining, superoxide anion generation from the post-ischemic coronary arteries by lucigenin-enhanced chemiluminescence [(89 ± 5 vs. 236 ± 27* relative light units (RLU/mg)], and neutrophil (PMN) accumulation by immunohistochemical staining in the AAR (52 ± 11 vs. 84 ± 14* cells/mm² myocardium) significantly increased between 3 and 24 h of reperfusion. Postconditioning reduced infarct size (15 ± 4† and 27 ± 3.6† %), superoxide anion generation (24 ± 4† and 43 ± 11† RLU/mg), and PMN accumulation (19 ± 6† and 45 ± 8† cells/mm² myocardium) in the 3 and 24 h reperfusion groups relative to time-matched controls. These data suggest that myocardial injury increases with duration of reperfusion; reduction in infarct size and attenuation in inflammatory responses with postconditioning persist after a prolonged reperfusion. * p < 0.05 24 vs. 3 h control; † p < 0.05 postconditioning vs. time-matched control.     

Proarrhythmic effects of ischemic preconditioning in anesthetized pigs

Conflicting data exist on how ischemic preconditioning affects the incidence of arrhythmias during ischemia. The present study was, therefore, performed to clarify if ischemic preconditioning alters the incidence of arrhythmias during the early phase of ischemia in pigs.Twenty-four pigs (23–36.5 kg) in pentobarbital anesthesia were preconditioned by a 10 min LAD-occlusion and 30 min reperfusion prior to a second 10 min occlusion. The first occlusion served as the preconditioning ischemic period, and the second occlusion as the test ischemic period. To verify that the preconditioning protocol induced protection, infarct size was assessed in an additional nine pigs.The arrhythmic index (arrhythmic beats/min) increased from 12±3 during the first occlusion to 38±8 during the second occlusion (P<0.05). Four pigs (17%) fibrillated during the first ischemic period, while 10 pigs (42%) fibrillated during the second ischemic period (P<0.05). All pigs that fibrillated during the first occlusion also fibrillated during the second occlusion. Mean heart rate was 108±4 beats/min before the first occlusion and increased to 113±4 beats/min before the second occlusion (P<0.001). There was a positive correlation between heart rate before the second occlusion and occurrence of fibrillation during this occlusion (r_s=0.42; P<0.05). The preconditioning protocol reduced infarct size, after a subsequent 45 min ischemic period followed by two hours of reperfusion, from 58±3% of area at risk to 40±5% (P<0.05).In conclusion, our data show that ischemic preconditioning increases both the arrhythmic index and the incidence of ventricular fibrillation during the early phase of a subsequent ischemic period.     

Therapeutic Hypothermia in STEMI

In this review article we tried to find an answer to the question, should local coronary hypothermia be a part of the early reperfusion strategy in patients with STEMI to prevent reperfusion injury, no-reflow phenomenon, and to reduce the infarct size and mortality.Hypothermia can save cardiomyocytes if achieved in a timely fashion before reperfusion. Intracoronary hypothermia can be adjunct to PCI by lessening ischemia/reperfusion injury on cardiomyocytes and reduction in infarct size. Reperfusion induced Calcium overload, generation of ROS and subsequent activation of Mitochondrial permeability transition pore (MPT) are major contributors to reperfusion injury. Hypothermia reduces calcium loading of the cell and maintains cellular energy and tissue level glucose which can scavenger ROS. Hypothermia reduces MPT activation and thus reduces infarct size. Systemic cooling trials failed to reduce infarct size, perhaps because the target temperature was not reached fast enough, and it was associated with systemic side effects. The need for rapid induction of hypothermia to <35 °C with the ethical concern of delaying reperfusion while cooling the patient and the inconsistency of endovascular cooling results lead to a belief that endovascular cooling may exceed the acceptable level of invasiveness in the context of other novels cardioprotective, regenerative and reperfusion therapies. Clinical trials showed the safety and feasibility of novel intracoronary hypothermia with rapid induction and maintenance of hypothermia using routine PCI equipment ahead of reperfusion. Two phases of cooling were applied without significant delay in the door to balloon time. Cooling of the coronary artery leads to cooling of its dependant myocardium without affecting adjacent myocardium. Heat transfer occurred by heat conduction during the occlusion phase and heat convention during the reperfusion phase. Fine-tuning of saline temperature and infusion rate helped to improve the protocol. The best duration of hypothermia before and after reperfusion is not known and needs further investigation. A balance between the undoubted cardioprotective effects of hypothermia with iatrogenic prolongation of ischemia time needs to be established. A reduction in infarct size was observed but needs to be validated with large randomized trials. Furthermore, it might be possible to augment the cardioprotective effects of intracoronary hypothermia by combination with other cardioprotective approaches such as antioxidant drugs and afterload reducing agents.     

Effect of intracoronary verapamil on infarct size in the ischemic,reperfused canine heart: Critical importance of the timing of treatment

In an effort to determine whether the beneficial effect of calcium blocking drugs occurs only during ischemia or during reperfusion as well, anesthetized dogs were subjected to 3 hours of occlusion of the left anterior descending coronary artery followed by 3 hours of reperfusion. In protocol A, intracoronary verapamil (0.01 mg/kg/min) was begun 90 minutes after coronary occlusion and continued for 1 hour into the reperfusion phase (n = 6) while a control group received an infusion of saline solution (n = 6). In vivo area at risk determined by dye injection was 29 ± 3% of the left ventricle (± standard error of the mean) in the control group and 30 ± 3% in the verapamil group (difference not significant [NS]), whereas the area of necrosis determined by triphenyltetrazolium staining and expressed as a percent of area at risk was smaller in the verapamil group (29 ± 8%) than in the control group (57 ± 8%, p <0.05). In protocol B, verapamil infusion into the left anterior descending coronary was begun 5 minutes before blood reperfusion and continued throughout the 3-hour reperfusion phase. Area at risk was similar in both groups (control, 25 ± 1%, n = 8; verapamil, 28 ± 2%, n = 8, NS); area of necrosis expressed as a percentage of area at risk was 49 ± 6% in the control group and 45 ± 10% in the verapamil group (NS). Therefore, calcium blockade of ischemic myocytes delays death and enhances salvage produced by reperfusion. However, calcium blockade begun after prolonged coronary occlusion does not enhance reperfusion-induced myocardial salvage.     

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.     

Dilazep dihydrochloride prolongs the infarct size-limiting effect of ischemic preconditioning in rabbits

Recent studies have indicated the key role of adenosine receptor activation as a trigger for ischemic preconditioning (PC). Hence, the augmentation of endogenous adenosine may potentiate the cardioprotective effects of PC. In this study, we aimed to test the hypothesis that dilazep dihydrochloride, an adenosine transport inhibitor, potentiates the PC effect. Protocol 1: Infarcts were produced in open-chest anesthetized rabbits by 30-min occlusion of a coronary artery and 2 days' reperfusion. PC was elicited by a preceding 5-min occlusion and either 5, 40, or 120 min of reperfusion. PC with the 5-min reperfusion markedly limited the infarct size after the 30-min ischemia (infarct size to area at risk (IS): 10% ± 3% vs 41% ± 3%, P < 0.05). PC was not protective when the reperfusion periods were 40 or 120 min (IS: 47% ± 5% and 44% ± 3%, P = not significant (NS) vs control, respectively). However, concomitant treatment with dilazep (0.2 mg/kg) preserved the PC effect in the 40-min reperfusion group (18% ± 5%, P < 0.05 vs control) but not in the 120-min reperfusion group (43% ± 4%, P = NS vs control). Protocol 2: Infarct was produced in a similar rabbit model by either a 45- or 50-min occlusion of a coronary artery and 2 days of reperfusion. PC was elicited by a preceding 5-min occlusion and a 5-min reperfusion. PC was protective in the 45-min occlusion group (30% ± 7% vs 67% ± 3%, P < 0.05) but not in the 50-min occlusion group (74% ± 4% vs 79% ± 5%, P = NS). Treatment with dilazep (0.2 mg/kg) failed to retrieve protection in this preconditioned group (77% ± 6%, P = NS vs 50-min occlusion group without PC). Thus, dilazep prolonged the infarct size-limiting effect of PC, but failed to retrieve protection in the group with a longer sustained ischemia. Received: September 6, 2000 / Accepted: March 9, 2001