TNF-alpha antibodies are as effective as ischemic preconditioning in reducing infarct size in rabbits

Pretreatment with tumor necrosis factor-alpha (TNF-alpha) antibodies abolishes myocardial infarct size reduction by late ischemic preconditioning (IP). Whether or not TNF-alpha is also important for myocardial infarct size reduction by classic IP is unknown. Anesthetized rabbits were untreated (group 1, n = 7), classically preconditioned by 5 min left coronary artery occlusion/10 min reperfusion (group 2, n = 6), or pretreated with TNF-alpha antibodies without (group 3, n = 6) or with IP (group 4, n = 6) before undergoing 30 min of occlusion and 180 min of reperfusion. Infarct size in group 1 was 44 +/- 11 (means +/- SD)% of the area at risk. With a comparable area at risk, infarct size was reduced to 13 +/- 7%, 23 +/- 8%, and 19 +/- 12% (all P < 0.05) in groups 2, 3, and 4, respectively. The circulating TNF-alpha concentration was increased during ischemia in group 1 from 752 +/- 403 to 1,542 +/- 482 U/ml (P < 0.05) but remained unchanged in all other groups. Circulating TNF-alpha concentration during ischemia and infarct size correlated in all groups (r = 0.76). IP, TNF-alpha antibodies, and the combined approach reduced infarct size to a comparable extent. Therefore, the question of whether or not TNF-alpha is causally involved in the infarct size reduction by IP in rabbits could not be answered.     

Postconditioning fails to improve no reflow or alter infarct size in an open-chest rabbit model of myocardial ischemia-reperfusion

Postconditioning (PoC) with brief intermittent ischemia after myocardial reperfusion has been shown to lessen some elements of postischemic injury including arrhythmias and, in some studies, the size of myocardial infarction. We hypothesized that PoC could improve reflow to the risk zone after reperfusion. Anesthetized, open-chest rabbits were subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion. In protocol 1, rabbits were randomly assigned to the control group (n = 10, no further intervention after reperfusion) or to the PoC group, which consisted of four cycles of 30-s reocclusions with 30 s of reperfusion in between starting at 30 s after the initial reperfusion (4 x 30/30, n = 10). In protocol 2, rabbits were assigned to the control group (n = 7) or the PoC group, which received PoC consisting of four cycles of 60-s intervals of ischemia and reperfusion starting at 30 s after the initial reperfusion (4 x 60/60, n = 7). No reflow was determined by injecting thioflavine S (a fluorescent marker of capillary perfusion), risk zone by blue dye, and infarct size by triphenyltetrazolium chloride. In protocol 1, there were no statistical differences in hemodynamics, ischemic risk zone, or infarct size (35 +/- 6% of the risk zone in the PoC group vs. 29 +/- 4% in the control group, P = 0.38) between the groups. Similarly, in protocol 2, PoC failed to reduce infarct size compared with the control group (45 +/- 4% of the risk zone in the PoC group vs. 42 +/- 6% in the control group, P = 0.75). There was a strong correlation in both protocols between the size of the necrotic zone and the portion of the necrotic zone that contained an area of no reflow. However, PoC did not affect this relationship. PoC did not reduce infarct size in this model, nor did it reduce the extent of the anatomic zone of no reflow, suggesting that this intervention may not impact postreperfusion microvascular damage due to ischemia.     

Early treatment with deferoxamine limits myocardial ischemic/reperfusion injury

Oxygen-derived free radicals (the superoxide anion O2- and hydroxyl radical.OH) have been implicated in myocardial injury associated with coronary artery occlusion followed by reperfusion. Transition metals (such as iron or copper) are needed to catalyze the formation of the .OH radical and subsequent .OH-mediated lipid peroxidation, yet the role of these transition metals in the pathogenesis of myocyte necrosis remains undefined. To address this issue, 21 dogs underwent 2 h of coronary artery occlusion and 4 h of reperfusion. Each animal was randomly assigned into 1 of 3 treatment groups: 7 received the iron chelator deferoxamine beginning 30 min preocclusion, 7 received deferoxamine beginning 5 min prior to reperfusion, while 7 dogs served as saline controls. Deferoxamine effectively chelated free iron in both treatment groups (total urine iron content averaged 42 +/- 16, 662 +/- 177 and 803 +/- 2.5 micrograms in control, pretreated, and deferoxamine at reperfusion groups respectively; p less than 0.05), but had no significant effect on in vivo area at risk (AR), hemodynamic parameters, collateral blood flow during occlusion, or myocardial blood flow following reperfusion. Area of necrosis (AN) in dogs pretreated with deferoxamine (34.6 +/- 3.7% of the AR; p less than 0.05) was significantly smaller than that observed in the saline control group (55.4 +/- 4.7% of the AR). Deferoxamine administered at the time of reperfusion, however, had no significant effect on infarct size (AN/AR = 54.3 +/- 8.7%, p = NS vs. controls). Thus, early treatment with the iron chelator deferoxamine acutely reduced the extent of myocyte necrosis produced by 2 h of transient coronary artery occlusion in the canine model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dimethylthiourea does not ameliorate reperfusion lung injury in dogs or rabbits

We previously demonstrated that in vivo reperfusion of a dog lung after 48 h of pulmonary arterial (PA) ischemia results in pulmonary edema with a significant infiltrate of polymorphonuclear leukocytes. We hypothesized that the injury resulted from production of hydroxyl radical by activated neutrophils. In the current study, we attempted to prevent the injury in both dogs and rabbits with dimethylthiourea (DMTU), a scavenger of hydroxyl radical. After 48 h of left PA occlusion in 18 dogs, DMTU was administered to 9 animals and 9 were not treated. The occlusion was then released, and the dogs were killed 4 h later. Reperfusion resulted in a drop in leukocyte count and left lung edema, but there was no difference between treated and untreated animals. The wet-to-dry ratios of the lungs in the treated group were 5.76 +/- 0.44 (SE) on the reperfused left side and 4.50 +/- 0.06 (P less than 0.05) on the right side. In the untreated groups the comparable ratios were 5.73 +/- 0.31 and 4.92 +/- 0.10 (P less than 0.05 for right vs. left). Histological examination revealed significant differences between the right and left lungs in the extent of intra-alveolar granulocytes and macrophages but did not reveal differences between the treated and untreated animals. To ensure that neither the model nor the lack of response to DMTU was species specific, we then developed a rabbit model of reperfusion edema.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of thromboxane A2 in reperfusion injury

Thromboxane A2 (TXA2) receptor antagonists can limit infarct size in models of coronary occlusion and reperfusion, but it was unknown if these compounds can mitigate reperfusion injury. Anesthetized open chest dogs were subjected to left circumflex coronary (LCX) occlusion for 90 min. Two minutes before reperfusion, the dogs were given iv saline (0.9% NaCl) or the TXA2 antagonist SQ 29,548 (0.2 mg/kg + 0.2 mg/kg/hr). Reperfusion was instituted for 5 hr at which time infarct size was determined. Regional myocardial blood flow was determined before, during, and after occlusion. SQ 29,548 treatment resulted in a significant reduction in infarct size (57 +/- 7 and 34 +/- 8% of the left ventricular area at risk infarcted in the saline and SQ 29,548 groups, respectively). No differences in collateral flow during occlusion were observed between groups, but SQ 29,548 treatment resulted in a significantly higher subendocardial reperfusion flow (54 +/- 10 and 93 +/- 14 ml/min/100g for the saline and SQ 29,548 groups, respectively). Thus, TXA2 seems to play a role in exacerbating reperfusion injury and TXA2 receptor blockade may have potential as a mode of therapy for ischemia-reperfusion damage.     

Apelin reduces myocardial reperfusion injury independently of PI3K/Akt and P70S6 kinase

Apelin, the endogenous ligand of the G protein-coupled APJ receptor, is a peptide mediator with emerging regulatory actions in the heart. The aim of the present studies was to explore potential roles of the apelin/APJ system in myocardial ischaemia/reperfusion injury. To determine the cardiac expression of apelin/APJ and potential regulation by acute ischaemic insult, Langendorff perfused rat hearts were subjected to regional ischaemia (left coronary artery occlusion, 35 min) or ischaemia followed by reperfusion (30 min). Apelin and APJ mRNA expression were then determined in ventricular myocardium by rt-PCR. Unlike APJ mRNA expression, which remained unchanged, apelin mRNA was upregulated 2.4 fold in ventricular myocardium from isolated rat hearts undergoing ischaemia alone, but returned back to control levels after 30 min reperfusion. We then proceeded to test the hypothesis that treatment with exogenous apelin is protective against ischaemia/reperfusion injury. Perfused hearts were subjected to 35 min left main coronary artery occlusion and 120 min reperfusion, after which infarct size was determined by tetrazolium staining. Exogenous Pyr(1)-apelin-13 (10(-8 )M) was perfused either from 5 min prior to 15 min after coronary occlusion, or from 5 min prior to 15 min after reperfusion. Whilst ineffective when used during ischaemia alone, apelin administered during reperfusion significantly reduced infarct size (47.6+/-2.6% of ischaemic risk zone compared to 62.6+/-2.8% in control, n=10 each, p<0.05) in hearts subject to temporary coronary occlusion followed by reperfusion. This protective effect was not abolished by co-administration of the PI3K inhibitor wortmannin (10(-7 )M, infarct size 49.8+/-4.1%, n=4) or the P70S6 kinase inhibitor rapamycin (10(-9 )M, 41.8+/-8.8%, n=4). In conclusion these results suggest that apelin may be a new and potentially important cardioprotective autacoid, upregulated rapidly after myocardial ischaemia and acting through an unknown pathway.     

Dimethylthiourea, but not dimethylsulfoxide, reduces canine myocardial infarct size

We studied the effect of treatment with two diffusible, low molecular weight scavengers of toxic oxygen metabolites, dimethylthiourea (DMTU) and dimethylsulfoxide (DMSO), on canine infarcts caused by 90 min of ischemia and 3 h of reperfusion. Infarct size was determined by incubating ventricular slices with triphenyl tetrazolium chloride. Areas at risk were determined by autoradiography of 99Tc microspheres injected in vivo during ischemia and were similar (p greater than 0.05) in DMTU, DMSO, and saline treated dogs. However, the ratio of infarct size to area at risk was reduced (p less than 0.05) in dogs treated 30 min before reperfusion with 500 mg/kg DMTU (31.1 +/- 4.6%, n = 9) compared with saline treated dogs (53.4 +/- 4.6% n = 9). In contrast, the ratio of infarct size to area at risk was not significantly different (p greater than 0.05) in dogs treated with 2000 mg/kg DMSO 30 min before reperfusion (43.7 +/- 4.3%) compared to saline treated dogs. The serum concentration of DMTU (4.5 mM) was one-tenth that of DMSO (48 mM) in early reperfusion. Therefore, DMTU but not DMSO protected against post-ischemic cardiac reperfusion injury.     

不同强度及时间窗骨骼肌缺血后处理对兔心肌的保护作用

目的:通过比较不同强度及时间窗骨骼肌缺血后处理对兔缺血/再灌注心肌的保护效能,试图寻找最佳强度和时间窗的骨骼肌缺血后处理方案。方法:健康新西兰大白兔42只(雄性)随机分为7组(n=6):①假手术组(Sham)、②缺血对照组(CON)、③标准骨骼肌后处理组(SP)、④延迟6min骨骼肌后处理组(6M-DSP)、⑤延迟1 min骨骼肌后处理组(1M-DSP)、⑥骨骼肌后处理加强组(SSP)、⑦骨骼肌后处理减弱组(WSP)。以开胸结扎冠状动脉左室支固定部位方法制作缺血/再灌注模型,以游离并夹闭双侧腹股沟髂外动脉固定位置方法造成骨骼肌缺血,再灌注末以TTC法确定心肌梗死范围,并分别于心肌缺血前、后及再灌注1 h、2 h,以生化法测定血清肌酸激酶(CK)及乳酸脱氢酶(LDH)水平。结果:和CON组相比,1M-DSP组心肌梗死重量比及面积比分别下降了42.32%及42.68%、SP组分别下降了49.97%及43.78%、SSP组分别下降了48.36%及48.86%,(P均<0.05),但三组之间相比,心梗范围未见明显差异;而6M-DSP、WSP组与CON组相比未见心肌保护作用;肌酸激酶(CK)的水平和梗死范围变化趋势一致。结论:兔在心肌缺血/再灌注之前完成骨骼肌5 min缺血/1 min再灌注1次循环的缺血后处理,可以起到明显的心肌保护作用。     

Timing of adenosine 2A receptor stimulation relative to reperfusion has differential effects on infarct size and cardiac function as assessed in mice by MRI

The activation of adenosine 2A receptors before reperfusion following coronary artery occlusion reduces infarct size and improves ejection fraction (EF). In this study, we examined the effects of delaying treatment with the adenosine 2A receptor agonist ATL146e (ATL) until 1 h postreperfusion. The infarct size and EF were serially assessed by gadolinium-diethylenetriaminepentaacetic acid-enhanced MRI in C57BL/6 mice at 1 and 24 h postreperfusion. The infarct size was also assessed by 2,3,5-triphenyltetrazolium chloride staining at 24 h. Mice were treated with ATL (10 microg/kg ip) either 2 min before reperfusion (early ATL) or 1 h postreperfusion (late ATL) following the 45-min coronary occlusion. The two methods used to assess infarct size at 24 h postreperfusion (MRI and 2,3,5-triphenyltetrazolium chloride) showed an excellent correlation (R=0.96). The risk region, determined at 24 h postreperfusion, was comparable between the control and ATL-treated groups. The infarct size by MRI at 1 versus 24 h postreperfusion was 25+/-1 vs. 26+/-1% of left ventricular mass (means+/-SE) in control mice, 16+/-2 versus 17+/-2% in early-ATL mice, and 24+/-2 versus 25+/-2% in late-ATL mice (intragroup, P=not significant; and intergroup, early ATL vs. control or late ATL, P<0.05). EF was reduced in control mice but was largely preserved between 1 and 24 h in both early-ATL and late-ATL mice (P<0.05). In conclusion, after coronary occlusion in mice, the extent of myocellular death due to ischemia-reperfusion injury is 95% complete within 1 h of reperfusion. The infarct size was significantly reduced by ATL when given just before reperfusion, but not 1 h postreperfusion. Either treatment window helped preserve the EF between 1 and 24 h postreperfusion.     

Effect of desflurane-induced preconditioning following ischemia-reperfusion on nitric oxide release in rabbits

Nitric oxide (NO) is the mediator of ischemic preconditioning against myocardial infarction. Desflurane produces anesthetic preconditioning to protect the myocardium against infarction. In the model of myocardial ischemia-reperfusion injury in rabbits, we evaluated desflurane-induced ischemic preconditioning and studied its mechanism of NO synthesis. Thirty-two male adult New Zealand white rabbits were anesthetized with intravenous (IV) 30 mg/kg pentobarbital followed by 5 mg/kg/hr infusion. All rabbits were subjected to 30 minutes (min) long lasting left anterior descending coronary artery (LAD) occlusion and three hours (hr) of subsequent reperfusion. Before LAD occlusion, the rabbits were randomly allocated into four groups for preconditioning treatment (eight for each group). The control group did not receive any preconditioning treatment. The desflurane group received inhaled desflurane 1.0 MAC (minimal end-tidal alveolar concentration) for 30 min that was followed by a 15 min washout period. The L-NAME-desflurane group received L-NAME (NG-nitro-L-arginine methyl ester; non-selective Nitric Oxide Synthetase (NOS) inhibitor) 1 mg/kg IV 15 min before 1.0 MAC inhaled desflurane for 30 min. The L-NAME group received L-NAME 1 mg/kg IV. Infarct volume, ventricular arrhythmia, plasma lactate dehydrogenase (LDH), creatine kinase (CK) activity and myocardial perfusion were recorded simultaneously. We have found that hemodynamic values of the coronary blood flow before, during, and after LAD occlusion were not significantly different among these four groups. For the myocardial ischemia-reperfusion injury animals, the infarction size (mean +/- SEM) in the desflurane group was significantly reduced to 18 +/- 3% in the area at risk as compared with 42 +/- 7% in the control group, 35 +/- 6 in the L-NAME group, and 34 +/- 4% in the L-NAME-desflurane group. The plasma LDH, CK levels, and duration of ventricular arrhythmia were also significantly decreased in the desflurane group during ischemia-reperfusion injury. Our results indicate that desflurane is an anesthetic preconditioning agent, which could protect the myocardium against the ischemia-reperfusion injury. This beneficial effect of desflurane on the ischemic preconditioning is probably through NO release since L-NAME abrogates the desflurane preconditioning effect.     

Differential effects of postconditioning on myocardial stunning and infarction: a study in conscious dogs and anesthetized rabbits

Postconditioning, i.e., brief intermittent episodes of myocardial ischemia-reperfusion performed at the onset of reperfusion, reduces infarct size after prolonged ischemia. Our goal was to determine whether postconditioning is protective against myocardial stunning. Accordingly, conscious chronically instrumented dogs (sonomicrometry, coronary balloon occluder) were subjected to a control sequence (10 min coronary artery occlusion, CAO, followed by coronary artery reperfusion, CAR) and a week apart to postconditioning with four cycles of brief CAR and CAO performed at completion of the 10 min CAO. Three postconditioning protocols were investigated, i.e., 15 s CAR/15 s CAO (n=5), 30 s CAR/30 s CAO (n=7), and 1 min CAR/1 min CAO (n=6). Left ventricular wall thickening was abolished during CAO and similarly reduced during subsequent stunning in control and postconditioning sequences (e.g., at 1 h CAR, 33+/-4 vs. 34+/-4%, 30+/-4 vs. 30+/-4%, and 33+/-4 vs. 32+/-4% for 15 s postconditioning, 30 s postconditioning, and 1 min postconditioning vs. corresponding control, respectively). We confirmed this result in anesthetized rabbits by demonstrating that shortening of left ventricular segment length was similarly depressed after 10 min CAO in control and postconditioning sequences (4 cycles of 30 s CAR/30 s CAO). In additional rabbits, the same postconditioning protocol significantly reduced infarct size after 30 min CAO and 3 h CAR (39+/-7%, n=6 vs. 56+/-4%, n=7 of the area at risk in postconditioning vs. control, respectively). Thus, contrasting to its beneficial effects on myocardial infarction, postconditioning does not protect against myocardial stunning in dogs and rabbits. Conversely, additional episodes of ischemia-reperfusion with postconditioning do not worsen myocardial stunning.     

Influence of low-flow infusion and magnesium on tissue necrosis during regional ischemia in the canine myocardium.

Passive intracoronary perfusion of therapeutic agents has been used in the clinical setting to attenuate the effects of brief episodes of myocardial ischemia. The objective of this study was to assess the effects of low-flow coronary infusion with or without Mg2+ on tissue necrosis and cardiac hemodynamics after prolonged regional ischemia. In 33 anesthetized dogs (5 excluded during study), the left anterior descending coronary artery was occluded for 6 h. Dogs were assigned to three groups: the first group (n = 8) was subjected to 6 h coronary occlusion without low-flow perfusion (controls), the second group (n = 10) received a low-flow coronary infusion of Ringer's lactate (Mg(2+)-free), and the third group (n = 10) received a low-flow coronary infusion of Ringer's lactate plus Mg2+ sulfate (15 mM). Tissue necrosis was evaluated using tetrazolium staining and was normalized to the principal baseline predictors of infarct size including anatomic risk zone (microsphere autoradiography) and coronary collateral flow. In control hearts, infarct size comprised 51.1 +/- 4.1% of the risk zone (40.8 +/- 5.1% left ventricular cross-sectional area (LV)). In the Mg(2+)-free and Mg2+ groups, risk zone size was 17.3 +/- 2.2 and 16.8 +/- 1.8% LV (p < 0.05 vs. controls), while infarct size was 23.1 +/- 3.1 and 24.9 +/- 8.1% (p < 0.05 vs. controls), respectively. Coronary collateral flow in the endocardium was similar for all of the experimental groups; however, hearts subjected to ischemia with low-flow perfusion of Ringer's lactate demonstrated significantly higher epicardial coronary collateral flow levels compared with controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

A3 adenosine receptor agonist IB-MECA reduces myocardial ischemia-reperfusion injury in dogs

We examined the effect of the A3 adenosine receptor (AR) agonist IB-MECA on infarct size in an open-chest anesthetized dog model of myocardial ischemia-reperfusion injury. Dogs were subjected to 60 min of left anterior descending (LAD) coronary artery occlusion and 3 h of reperfusion. Infarct size and regional myocardial blood flow were assessed by macrohistochemical staining with triphenyltetrazolium chloride and radioactive microspheres, respectively. Four experimental groups were studied: vehicle control (50% DMSO in normal saline), IB-MECA (100 microg/kg iv bolus) given 10 min before the coronary occlusion, IB-MECA (100 microg/kg iv bolus) given 5 min before initiation of reperfusion, and IB-MECA (100 microg/kg iv bolus) given 10 min before coronary occlusion in dogs pretreated 15 min earlier with the ATP-dependent potassium channel antagonist glibenclamide (0.3 mg/kg iv bolus). Administration of IB-MECA had no effect on any hemodynamic parameter measured including heart rate, first derivative of left ventricular pressure, aortic pressure, LAD coronary blood flow, or coronary collateral blood flow. Nevertheless, pretreatment with IB-MECA before coronary occlusion produced a marked reduction in infarct size ( approximately 40% reduction) compared with the control group (13.0 +/- 3.2% vs. 25.2 +/- 3.7% of the area at risk, respectively). This effect of IB-MECA was blocked completely in dogs pretreated with glibenclamide. An equivalent reduction in infarct size was observed when IB-MECA was administered immediately before reperfusion (13.1 +/- 3.9%). These results are the first to demonstrate efficacy of an A3AR agonist in a large animal model of myocardial infarction by mechanisms that are unrelated to changes in hemodynamic parameters and coronary blood flow. These data also demonstrate in an in vivo model that IB-MECA is effective as a cardioprotective agent when administered at the time of reperfusion.     

Is microvascular protection by cariporide and ischemic preconditioning causally linked to myocardial salvage?

Two independent cardioprotective interventions, Na(+)/H(+) exchange inhibition and ischemic preconditioning (PC), were investigated with respect to differential effects on microvascular and myocardial salvage in anesthetized rabbits (30 min of ischemia, 180 min of reperfusion). Cariporide (Car, 300 microg/kg) administered before occlusion and PC reduced infarct size (IS) as measured by triphenyltetrazolium staining [control, 46.0 +/- 4.2% of risk area (RA); Car, 17.6 +/- 3.7% (P < 0.01); PC, 27.5 +/- 4.1% (P < 0.01)] and concomitantly decreased the area of anatomic no reflow (ANR) as measured by thioflavin S staining [control, 40.4 +/- 3.7%; Car, 19.0 +/- 2.9% (P < 0.01); PC, 26.9 +/- 3.4% (P < 0.05)]. Regional myocardial blood flow (RMBF, measured by radioactive microspheres) in the RA, which deteriorated between 30 and 180 min of reperfusion (control, from 79 +/- 6 to 26 +/- 2% of nonischemic flow), was shifted to higher values with both treatments [Car, from 110 +/- 12 to 49 +/- 7% (P < 0.05); PC, from 109 +/- 8 to 38 +/- 6% (P < 0.05)]. However, neither intervention uncoupled the close relationship between IS and ANR (r = 0.92-0.95) or RMBF. Car given at reperfusion did not alter IS, ANR, RMBF, or the close interrelationships. Because size and spatial distribution of no reflow and myocardial necrosis remained closely coupled with independent cardioprotective interventions, a potential causal connection between microvascular and myocardial salvage is discussed.     

No ischemic preconditioning in heterozygous connexin43-deficient mice

Protein kinase Cepsilon (PKCepsilon) plays a central role in ischemic preconditioning (IP) in mice and rabbits, and activated PKCepsilon colocalizes with and phosphorylates connexin43 (Cx43) in rats and humans. Whether or not Cx43 contributes to the mechanism(s) of IP in vivo is yet unknown. Therefore, wild-type (n = 8) and heterozygous Cx43-deficient mice (n = 8) were subjected to 30 min occlusion and 120 min reperfusion of the left anterior descending coronary artery. IP was induced by one cycle of 5 min occlusion and 10 min reperfusion (n = 8/8 mice) before the sustained occlusion. Infarct size was reduced by IP in wild-type mice [11.3 +/- 3.4% vs. 23.7 +/- 7.2% of the left ventricle (LV), P < 0.05] but not in Cx43-deficient mice (26.0 +/- 6.0% vs. 25.1 +/- 3.8% of LV). Also, three cycles of 5 min occlusion and 10 min reperfusion (n = 5) did not induce protection in Cx43-deficient mice (27.6 +/- 5.5 % of LV). Thus Cx43 contributes to the protection of IP in mice in vivo.     

Attenuation of ischemic preconditioning in pigs by scavenging of free oxyradicals with ascorbic acid

Free oxyradicals are involved in the signal transduction of ischemic preconditioning in rats and rabbits. Data from larger mammals in which the infarct development is closer to that in humans are lacking. We have therefore investigated the impact of the radical scavenger ascorbic acid on ischemic preconditioning in pigs. In 33 anesthetized pigs, the left anterior descending coronary artery was perfused from an extracorporeal circuit. Infarct size (measured as percent area at risk) was determined by triphenyltetrazolium chloride staining. In placebo-treated animals undergoing 90 min of severe ischemia and 120 min of reperfusion, infarct size averaged 26.9 +/- 3.9% (mean +/- SE; n = 9). Ischemic preconditioning by 10 min of ischemia and 15 min of reperfusion reduced infarct size to 6.4 +/- 2.4% (P < 0.05 vs. placebo; n = 9). Intravenous infusion of ascorbic acid (30 min before ischemic preconditioning or ischemia; 2-g bolus followed by 25 mg/min until the end of ischemia) had no effect on infarct size per se (22.6 +/- 6.5%; n = 6), but largely abolished the infarct size reduction by ischemic preconditioning (19.1 +/- 5.4%; n = 9). Scavenging of free oxyradicals with ascorbic acid largely attenuates the beneficial effect of ischemic preconditioning in pigs.     

Aspirin before reperfusion blunts the infarct size limiting effect of atorvastatin

We assessed whether aspirin (acetylsalicylic acid, ASA), administered before reperfusion, abrogates the infarct size (IS)-limiting effect of atorvastatin (ATV). Statins reduce IS. This dose-dependent effect is mediated by upregulation of cycloxygenase-2 (COX2) and PGI(2) production. Administration of selective COX2-inhibitors either with ATV for 3 days or immediately before coronary occlusion blocks the IS-limiting effect of ATV. Sprague-Dawley rats received 3-day ATV (10 mg x kg(-1) x day(-1)) or water alone. Rats underwent 30 min coronary artery occlusion and 4 h reperfusion (IS protocol, n=8 in each group), or rats underwent 30 min coronary artery occlusion and 10 min reperfusion (enzyme expression and activity protocol, n=4 in each group). Immediately before reperfusion rats received intravenous ASA (5, 10, or 20 mg/kg) or saline. Area-at-risk (AR) was assessed by blue dye and IS by triphenyltetrazolium chloride. ATV reduced IS (10.1 +/- 1.4% of the AR) compared with controls (31.0 +/- 2.2%). Intravenous ASA alone did not affect IS (29.0 +/- 2.6%); however, ASA dose dependently (5, 10, and 20 mg/kg) attenuated the protective effect of ATV on IS (15.8 +/- 0.9%, 22.0 +/- 1.6%, and 23.7 +/- 3.8%, respectively). ASA dose dependently blocked the upregulation of COX2 by ATV. COX2 activity was as follows: control, 8.93 +/- 0.90 pg/mg; ATV, 75.85 +/- 1.08 pg/mg; ATV + ASA5, 34.39 +/- 1.48 pg/mg; ATV + ASA10, 19.87 +/- 1.10 pg/mg; and ATV + ASA20, 9.36 +/- 0.94 pg/mg. ASA, administered before reperfusion in doses comparable to those used in the clinical setting, abrogates the IS-limiting effect of ATV in a model with mechanical occlusion of the coronary artery. This potential adverse interaction should be further investigated in the clinical setting of acute coronary syndromes.     

Nicorandil induces late preconditioning against myocardial infarction in conscious rabbits

Nicorandil has been shown to induce an infarct-limiting effect similar to that induced by the early phase of ischemic preconditioning (PC). The goals of this study were to determine whether nicorandil induces a delayed cardioprotection that is analogous to the late phase of ischemic PC and, if so, whether nicorandil-induced late PC is associated with upregulation of cardioprotective proteins. Chronically instrumented, conscious rabbits received vehicle (intravenous normal saline; control group, n = 10), nicorandil (100 microg/kg bolus + 30 microg x kg(-1) x min(-1) i.v. for 60 min; nicorandil group, n = 10), or ischemic PC (6 cycles of 4-min coronary occlusion/4-min reperfusion; PC group, n = 8). Twenty-four hours later, rabbits underwent a 30-min coronary occlusion, followed by 3 days of reperfusion. Myocardial infarct size was significantly reduced in rabbits pretreated with nicorandil (27.5 +/- 5.3% of the risk region) or with ischemia (30.3 +/- 4.2%) versus controls (59.1 +/- 4.7%, P < 0.05 vs. both). Furthermore, the expression of cyclooxygenase-2 (COX-2) and Bcl-2 was significantly elevated (+38% and +126%, respectively; P < 0.05) in myocardium of rabbits given nicorandil 24 h earlier versus controls. We conclude that nicorandil induces delayed cardioprotection against myocardial infarction similar to that afforded by the late phase of ischemic PC, possibly by upregulating COX-2 and Bcl-2.     

Is the sarcolemmal or mitochondrial KATP channel activation important in the antiarrhythmic and cardioprotective effects during acute ischemia/reperfusion in the intact anesthetized rabbit model?

The relative contributions of cardiomyocyte sarcolemmal ATP-sensitive K(+) (K(ATP)) and mitochondrial K(ATP) channels in the cardioprotection and antiarrhythmic activity induced by K(ATP) channel openers remain obscure, though the mitochondrial K(ATP) channels have been proposed to be involved as a subcellular mediator in cardioprotection afforded by ischemic preconditioning. In the present study, we sought to investigate the effects of administration of ATP-sensitive K(+) channel (K(ATP)) openers (nicorandil and minoxidil), a specific mitochondrial K(ATP) channel blocker (5-hydroxydecanoate (5-HD)) and a specific sarcolemmal K(ATP) channel blocker (HMR 1883; (1-[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl-3-methylthiourea) prior to coronary occlusion as well as prior to post-ischemic reperfusion on survival rate, ischemia-induced and reperfusion-induced arrhythmias and myocardial infarct size in anesthetized albino rabbits. The thorax was opened in the left 4th intercostal space and after pericardiotomy the heart was exposed. In Group I (n=88), occlusion of the left main coronary artery and hence, myocardial ischemia-induced arrhythmias was achieved by tightening a previously placed loose silk ligature for 30 min. In Group II (n=206), arrhythmias were induced by reperfusion following a 20-min ligation of the left main coronary artery. Both in Group I and Group II, intravenous (i.v.) administration of nicorandil (0.47 mg/kg), minoxidil (0.5 mg/kg), HMR 1883 (3 mg/kg)/nicorandil and HMR 1883 (3 mg/kg)/minoxidil before coronary artery occlusion increased survival rate (86%, 75%, 75% and 86% vs. 55% in the control subgroup in Group I; 75%, 67%, 67% and 75% vs. 46% in the control subgroup in Group II), significantly decreased the incidence and severity of life-threatening arrhythmias. In Group II, i.v. administration of nicorandil and minoxidil before coronary artery occlusion significantly decreased myocardial infarct size. However, i.v. administration of nicorandil or minoxidil before reperfusion did neither increase survival rate nor confer any antiarrhythmic or cardioprotective effects. The antiarrhythmic and cardioprotective effects of both nicorandil and minoxidil were abolished by pretreating the rabbits with 5-HD (5 mg/kg, i.v. bolus), a selective mitochondrial K(ATP) channel blocker but not by HMR 1883 (3 mg/kg). In the present study, higher levels of malondialdehyde (MDA) and lower levels of reduced glutathione (GSH) and superoxide dismutase (SOD) in necrotic zone of myocardium in all the 16 subgroups in Group II suggest little anti-free radical property of nicorandil and minoxidil. We conclude that intervention by intravenous administration of nicorandil and minoxidil (through the selective activation of mitochondrial K(ATP) channels) increased survival rate and exhibited antiarrhythmic and cardioprotective effects during coronary occlusion and reperfusion in anesthetized rabbits when administered prior to coronary occlusion. The cardiomyocyte mitochondrial K(ATP) channel may be a pharmacologically modulable target of cardioprotection and antiarrhythmic activity.     

Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs

Transient episodes of ischemic preconditioning (PC) render myocardium protected against subsequent lethal injury after ischemia and reperfusion. Recent studies indicate that application of short, repetitive ischemia only during the onset of reperfusion after the lethal ischemic event may obtain equivalent protection. We assessed whether such ischemic postconditioning (Postcon) is cardioprotective in pigs by limiting lethal injury. Pentobarbital sodium-anesthetized, open-chest pigs underwent 30 min of complete occlusion of the left anterior descending coronary artery and 3-h reflow. PC was elicited by two cycles of 5-min occlusion plus 10-min reperfusion before the 30-min occlusion period. Postcon was elicited by three cycles of 30-s reperfusion, followed by 30-s reocclusion, after the 30-min occlusion period and before the 3-h reflow. Infarct size (%area-at-risk using triphenyltetrazolium chloride macrochemistry; means +/- SE) after 30 min of ischemia was 26.5 +/- 5.2% (n = 7 hearts/treatment group). PC markedly limited myocardial infarct size (2.8 +/- 1.2%, n = 7 hearts/treatment group, P < 0.05 vs. controls). However, Postcon had no effect on infarct size (37.8 +/- 5.1%, n = 7 hearts/treatment group). Within the subendocardium, Postcon increased phosphorylation of Akt (74 +/- 12%) and ERK1/2 (56 +/- 10%) compared with control hearts subjected only to 30-min occlusion and 15-min reperfusion (P < or = 0.05), and these changes were not different from the response triggered by PC (n = 5 hearts/treatment group). Phosphorylation of downstream p70S6K was also equivalent in PC and Postcon groups. These data do not support the hypothesis that application of 30-s cycles of repetitive ischemia during reperfusion exerts a protective effect on pig hearts subjected to lethal ischemia, but this is not due to a failure to phosphorylate ERK and Akt during early reperfusion.