Reduction of myocardial reperfusion injury by intravenous adenosine administered during the early reperfusion period

Adenosine influences the function of several cell types thought to be involved in the pathogenesis of myocardial reperfusion injury. We have previously demonstrated that intracoronary administration of adenosine enhances myocardial salvage 24 hours after reperfusion. To determine if these beneficial effects could be obtained during a prolonged period of reperfusion using an intravenous route of administration, 22 closed-chest dogs were subjected to 90 minutes of proximal left anterior descending coronary artery occlusion and 72 hours of reperfusion. Animals randomly received either intravenous adenosine (0.15 mg/kg/min) or an equal volume of Ringer's lactate during the first 150 minutes of reperfusion. The area at risk was defined in vivo with Monastral blue, and infarct size was measured histologically with Mallory's trichrome stain. Serial global and regional ventricular function were determined with contrast ventriculography and analyzed using a computerized radial shortening method. Biopsies were obtained from the central ischemic zone to assess endothelial ultrastructure and capillary obstruction. No significant effects in heart rate or blood pressure were noted during adenosine infusion. Transmural collateral blood flow during ischemia was similar in the groups. Infarct size expressed as a percentage of the anatomical area at risk was significantly less in the adenosine-treated group (35.3 +/- 4.3% in controls versus 17.1 +/- 4.3% in treated animals, p less than 0.01). A progressive decrease in transmural blood flow was noted in control animals during reperfusion, resulting in a significant reduction at 3 hours compared with the preocclusion value (0.69 +/- 0.11 ml/min/g [at baseline versus 0.45 +/- 0.10 ml/min/g at 3 hours, p less than 0.05]). In contrast, flow in adenosine animals at 3 hours was similar to baseline values (0.91 +/- 0.15 ml/min/g at baseline versus 0.98 +/- 0.14 ml/min/g at 3 hours, p = NS) and was significantly higher (p less than 0.05) than the control group. Radial shortening in the ischemic zone was significantly improved at 3 (-2.6 +/- 2.8% in controls versus 11.6 +/- 3.3% in treated animals, p less than 0.01) and 72 hours (5.5 +/- 2.0% in controls versus 17.3 +/- 3.5% in treated animals, p less than 0.01) after reperfusion in treated animals. Electron microscopy showed reduced neutrophil and erythrocyte plugging of capillaries with relative preservation of endothelial cell structure in the adenosine group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of kappa-opioid receptors at reperfusion affords cardioprotection in both rat and mouse hearts

The temporal properties of kappa-opioid receptor (kappa-OR) mediated cardioprotection are less well characterised than delta-opioid receptor (delta-OR) responses. This study was aimed at delineating the time course of kappa-OR-mediated protection in two experimental models: an in vivo rat model of regional myocardial infarction (30 min of left coronary artery occlusion with 120 min of reperfusion), and an in vitro perfused murine heart model (undergoing 25 min of global ischemia and 45 min of reperfusion). In the rat model, the selective kappa-OR agonist U50, 488 (0.1 mg/kg, IV bolus), administered either 10 min prior to ischemia or 5 min prior to reperfusion, significantly reduced infarct size (38 +/- 3% and 43 +/- 2% infarct size/area-at-risk (IS/AAR), respectively; P < 0.05) compared to untreated rats (56 +/- 1% IS/AAR). Administration of U50, 488 10 s after onset of reperfusion failed to elicit protection. Cardioprotection with U50,448 administered immediately prior to reperfusion was abolished by a kappa-OR antagonist, (0.1 mg/kg nor-BNI), given 10 min prior to reperfusion. In the in vitro murine model, untreated hearts exhibited 28 +/- 2% (IS/AAR) infarct size. Infusion of U50, 488 (at a final 100 nM concentration) significantly limited infarct size in mouse hearts when applied at the onset of reperfusion (15 +/- 2% IS/AAR; P < 0.05), yet failed to afford protection when infused prior to ischemia. Additionally, in both models studied, treatment with either wortmannin or 5-hydroxydecanoate (5-HD) abrogated the protective effects of U50,488 applied just prior to reperfusion. In summary, kappa-ORs afford cardioprotection primarily when activated prior to and not after reperfusion. This protection may involve activation of the PI3 kinase (PI3K) pathway and mitochondrial (mito) K (ATP) channels.     

Adiponectin protects against myocardial ischaemia-reperfusion injury via AMP-activated protein kinase, Akt, and nitric oxide

AIMS: Cardiovascular disease and type 2 diabetes mellitus are associated with low plasma concentration of adiponectin. The aim of this study was to investigate whether adiponectin exerts cardioprotective effects during myocardial ischaemia-reperfusion and whether this effect is related to the production of nitric oxide (NO). METHODS AND RESULTS: Isolated rat hearts were subjected to 30 min of either global or local ischaemia followed by 60 min of reperfusion. The hearts received vehicle, adiponectin (3 microg/mL), the NO-synthase inhibitor nitro-l-arginine (L-NNA) (0.1 mM), or a combination of adiponectin and L-NNA at the onset of ischaemia. Haemodynamics, infarct size, and expression of endothelial NO-synthase (eNOS), AMP-activated protein kinase (AMPK), and Akt were determined. Adiponectin significantly increased left ventricular function and coronary flow during reperfusion in comparison with the vehicle group. Co-administration of L-NNA abrogated the improvement in myocardial function induced by adiponectin. Infarct size following local ischaemia-reperfusion was 40 +/- 6% of the area at risk in the vehicle group. Adiponectin reduced infarct size to 19 +/- 2% (P < 0.01). L-NNA did not affect infarct size per se but abolished the protective effect of adiponectin (infarct size 40 +/- 5%). Phosphorylation of eNOS Ser1177, AMPK Thr172, and Akt Ser 473 was increased in the adiponectin group (P < 0.05). CONCLUSION: Adiponectin protects from myocardial contractile dysfunction and limits infarct size following ischaemia and reperfusion by a mechanism involving activation of AMPK and production of NO.     

Myocardial reperfusion injury in the canine model after 40 minutes of ischemia: effect of intracoronary adenosine

To explore the contribution of reperfusion injury to final infarct size after a short duration of ischemia, closed-chest dogs underwent 40 minutes of proximal left anterior descending artery occlusion followed by 3 days of reperfusion. Animals randomly received intracoronary adenosine (n = 8) at 3.75 mg/min during the first hour of reperfusion or no therapy (control, n = 9). Infarct size was measured histologically. Regional ventricular function was determined with contrast ventriculography. The risk region was similar and collateral blood flow in the inner two thirds of the ischemic zone was markedly reduced in both groups (adenosine: 0.05 +/- 0.07 ml/min/gm; control: 0.02 +/- 0.07 ml/min/gm; p = NS). Infarct size as a percent of the area at risk was significantly reduced in the adenosine group (5.0 +/- 1.3% versus 13.5 +/- 3.2%; p = 0.03), associated with a trend for improved recovery of regional ventricular function. Relative endothelial preservation was seen in the adenosine group. These results suggest that reperfusion injury contributes to final myocardial cell necrosis in the closed-chest canine model subjected to 40 minutes of regional ischemia.     

Myocardial protective effects of adenosine. Infarct size reduction with pretreatment and continued receptor stimulation during ischemia.

BACKGROUND. We hypothesized that 1) endogenous adenosine released during ischemia conferred an inherent cardioprotection, and 2) a pretreatment dose of adenosine before ischemia would provide additional protection independent of hemodynamic effects. METHODS AND RESULTS. Thirty-six anesthetized New Zealand White rabbits underwent 30 minutes of regional ischemia produced by coronary occlusion followed by 2 hours of reperfusion. The adenosine group (ADO, n = 9) received a 5-minute pretreatment infusion of 140 micrograms/kg/min of adenosine before ischemia. A control group (SAL, n = 9) received saline before ischemia. To separate the effects of adenosine used as a pretreatment versus the effects during ischemia, a third group (ADO+SPT, n = 9) received adenosine as pretreatment followed by 10 mg/kg 8-p-sulfophenyl theophylline (8-SPT), an A1/A2-receptor antagonist given before ischemia, thus allowing pretreatment with adenosine but antagonizing its effects during ischemia. To preclude any protection from endogenous adenosine released during ischemia, the fourth group (SAL+SPT, n = 9) received saline as pretreatment and 8-SPT before ischemia. Area of necrosis within the area at risk (infarct size) was determined with tetrazolium and Evans blue stains, and transmural blood flow was measured using radioactive microspheres. Collateral blood flow in the area at risk was similar in all groups, as was the size of the area at risk. Infarct size was reduced by adenosine pretreatment (ADO, 8.4 +/- 7.2%) in contrast to saline vehicle (SAL, 27.8 +/- 6.3%; p less than 0.05 versus ADO). alpha 1/alpha 2-Receptor blockade after adenosine pretreatment abolished the ischemic protection provided by pretreatment adenosine (ADO+SPT, 42.7 +/- 8.3%; p less than 0.05 versus ADO). Finally, receptor blockade of endogenously released adenosine without adenosine pretreatment increased infarct size by 24% over the nonpretreated saline group (SAL+SPT, 51.5 +/- 9.0%; p less than 0.05 versus SAL). CONCLUSIONS. We conclude that 1) endogenous adenosine building up during ischemia is cardioprotective, and 2) pretreatment with adenosine confers cardioprotection independent of hemodynamic effects. Whether pretreatment effects of adenosine subsequently modulate the effects of endogenous adenosine (through alterations in receptor population or sensitivity) or endogenous and exogenous adenosine represent additive compartments is unclear.     

p38 MAPK inhibition reduces myocardial reperfusion injury via inhibition of endothelial adhesion molecule expression and blockade of PMN accumulation

BACKGROUND: In vitro evidence suggests that the p38 mitogen-activated protein kinase (p38 MAPK) plays a crucial role in PMN activation and inflammatory cytokine production. However, the effect of p38 MAPK on myocardial reperfusion injury, a pathologic condition involving a typical inflammatory response, has not been fully examined. In the present study, we investigated the effect of SB 239063, a specific p38 MAPK inhibitor, on myocardial injury in a murine ischemia/reperfusion (I/R) model and elucidated the mechanism by which p38 MAPK inhibitor may exert its protective effect against I/R injury. METHODS AND RESULTS: I/R resulted in a significant myocardial injury (myocardial infarct 45 +/- 2.9%) and marked PMN accumulation (myeloperoxidase activity 1.03 +/- 0.16 U/100 g tissue). Administration of SB 239063 significantly inhibited the myocardial inflammatory response as evidenced by reduced PMN accumulation in I/R myocardial tissue (0.62 +/- 0.008 U/100 g tissue, P<0.01 vs. vehicle), and markedly attenuated myocardial reperfusion injury (myocardial infarct size: 28 +/- 2.4%, P<0.01 vs. vehicle). Moreover, treatment with SB 239063 significantly attenuated I/R-induced P-selectin and ICAM-1 upregulation (13.8 +/- 2.7 vs. 23.9 +/- 3.1%, and 29.4 +/- 1.6 vs. 56.3 +/- 4.8%, respectively P<0.01). In addition, pre-treatment with R15.7, a monoclonal antibody against CD 18 adhesion molecule on PMN surface that virtually abolished PMN accumulation in ischemic-reperfused myocardial tissue, significantly, but not completely, blocked the cardioprotection exerted by SB 239063. CONCLUSION: These results demonstrated for the first time that p38 MAPK activation plays a significant role in adhesion molecule upregulation on ischemia-reperfused endothelial cells and is an important signaling step in the pathogenesis of PMN-mediated tissue injury.     

Remifentanil limits infarct size but attenuates preconditioning-induced infarct limitation

OBJECTIVES: We investigated the influence of the narcotic anesthetic remifentanil on irreversible myocardial ischemic injury. METHODS: New Zealand White rabbits were anesthetized with propofol (0.7-1.8 mg.kg.min) and then subjected to 30 min regional myocardial ischemia and 3 h reperfusion (CON). Some animals also underwent ischemic preconditioning, elicited by either one (IP1) or two (IP2) cycles of 5 min ischemia and 5 min reperfusion, and/or remifentanil, administered either as a transient infusion mimicking the preconditioning protocol (RP2, 10 microg x kg x min) or as a continuous infusion (R, 3-10 microg x kg x min). Rabbits were randomly assigned to experimental groups. Infarct size was assessed with tetrazolium. Results are reported as mean+/-SD. RESULTS: Non-preconditioned infarct size was approximately 50% of the area-at-risk (49.6+/-20.1% CON). Both one and two cycles of ischemic preconditioning markedly reduced infarct size (49.6+/-20.1% CON versus 18.6+/-8.6% IP and versus 7.5+/-7.6% IP2; both p<0.001). Preconditioning with remifentanil modestly reduced infarct size (49.6+/-20.1% CON versus 29.3+/-8.5% RP2; p<0.01). However, sustained administration of remifentanil did not provide protection (49.6+/-20.1% CON versus 43.9+/-16.2% R), and it attenuated the protection offered by preconditioning (49.6+/-20.1% CON versus 35.6+/-20.7% R+IP1, p=NS; and versus 14.5+/-14.5% R+IP2; p<0.05). CONCLUSION: Transient pre-ischemic administration of remifentanil modestly reduces infarct size in propofol-anesthetized rabbits, but continuous administration of remifentanil increases the threshold for ischemic preconditioning-induced infarct limitation.     

Effects of nicardipine, a calcium antagonist, on myocardial salvage and high energy phosphate stores in reperfused myocardial injury

The current study determined the effectiveness of nicardipine, a 1,4-dihydropyridine calcium antagonist, in preserving reperfused myocardium in a cat model of temporary coronary occlusion and ascertained if replenishment of myocardial phosphate stores during reperfusion as defined by phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy was indicative of salvage. Twenty open chest, anesthetized cats were studied with use of a snare ligature around the proximal left anterior descending coronary artery, with a coil sutured to the epicardial surface overlying the distribution of the artery. Peak areas of phosphocreatine, inorganic phosphate and adenosine triphosphate (ATP) NMR signals were measured during 1 h of occlusion followed by 1.5 h of reperfusion. Infarct size and jeopardy area were determined in vitro by simultaneous infusion of phthalocyanine blue dye and triphenyltetrazolium chloride into the aorta and the left anterior descending coronary artery, respectively, after 5 h of myocardial reperfusion. Nicardipine-treated and control groups had similar jeopardy area values (41.2 +/- 1.6% versus 47.4 +/- 3.1% of the left ventricle), but infarct area was significantly reduced in the nicardipine-treated group (3.2 +/- 1.1% versus 24.9 +/- 7.5% of jeopardy area, p less than 0.01). High energy phosphate compounds remained markedly altered during reperfusion in both groups. No significant improvement in phosphocreatine or inorganic phosphate recovery was observed in animals pretreated with nicardipine despite an 87% reduction in infarct size. Myocardial ATP was greater during reperfusion in the nicardipine-treated compared with the control group (average over initial 90 min of reperfusion 58 +/- 6% versus 46 +/- 3% of baseline values, p less than 0.05), suggesting improved recovery of ATP. However, the measured levels of high energy phosphate compounds during reperfusion and their ratios did not correlate with infarct size and thus were not predictive of myocardial salvage.     

中性粒细胞在肺血栓栓塞症犬肺缺血-再灌注损伤中的作用

目的观察中性粒细胞(PMN)在肺血栓栓塞症(PTE)犬肺缺血-再灌注(I/R)损伤中的作用。方法 15只犬按随机数字表法随机分为3组:假手术组、缺血组、再灌注组。对 PTE 犬行取栓术,分别观察肺组织普通病理、组织匀浆髓过氧化物酶(MPO)含量及超微结构;Annexin V-FITC 凋亡检测试剂盒标记后用流式细胞仪检测再灌注前及再灌注后2、4、6 h 外周血 PMN 的凋亡率。结果再灌注6 h 后,再灌注组肺泡腔 PMN 计数明显高于假手术组及缺血组[(31±11)、(1±1)及(8±4)/10个高倍镜视野],肺组织匀浆 MPO 含量分别为(11.7±1.6)、(8.3±2.1)及(9.1±0.5)μg/L;缺血组肺泡腔内少许炎细胞渗出,再灌注组肺泡腔内见大量以 PMN 为主的炎细胞渗出;电镜下,再灌注后PMN 紧紧黏附于肺泡间隔,间隔内含水肿液及空泡样变性等;再灌注6 h 后外周血 PMN 凋亡率为(3.0±2.5)%,明显低于再灌注前的(7.4±4.5)%;术后4 h,再灌注组外周血 PMN 凋亡率为(4.8±2.6)%,明显低于缺血组的(9.3±2.0)%;6 h 组结果类似[(3.0±2.5)%,(8.0±1.6)%]。结论在该 PTE 犬 I/R 模型中,再灌注后 PMN 活性增强、凋亡减少;PMN 的参与是该模型肺 I/R 损伤的重要细胞学机制之一。     

Selective inhibition of the contractile apparatus. A new approach to modification of infarct size, infarct composition, and infarct geometry during coronary artery occlusion and reperfusion.

BACKGROUND. Myocardial reperfusion is associated with calcium overload and cell contracture, mechanisms that may precipitate cell death. In this study, we tested the hypothesis that in vivo inhibition of this contracture could lead to cell preservation in an open-chest large animal model. METHODS AND RESULTS. Regional myocardium function was measured during a selective intracoronary infusion of 2,3-butanedione monoxime (BDM), a specific inhibitor of actin-myosin coupling, in the control state (10 pigs) and in a protocol of a 51-minute coronary occlusion followed by reperfusion (40 pigs). The effects on coronary artery blood flow in the basal state were also studied (seven pigs). Intramyocardial distribution of the infusate during coronary occlusion, myocardial water content after 30 minutes of reperfusion and area at risk, infarct size, type of histological necrosis, and infarct geometry after 24 hours of reperfusion were assessed. Methods used included electromagnetic flowmeter, radiolabeled microspheres, subendocardial sonomicrometers, fluorescein, triphenyl tetrazolium chloride and Masson's trichrome staining, and computer quantification of infarct edges. In the absence of ischemia, BDM infusion inhibited regional shortening in a dose-dependent manner up to full systolic bulging while producing marked regional increase in coronary blood flow. During early reperfusion, BDM reduced end-diastolic length 76% more than the control infusion (p less than 0.05) and increased systolic bulging by 420% compared with no change in control animals. The ratio of infarct size/area at risk was reduced by 31% with BDM (p less than 0.05), with striking modifications of infarct histology and infarct geometry; specifically, the extent of contraction band necrosis was reduced by 63% from 105.5 +/- 18.2 to 39.2 +/- 13.6 mm2 (p less than 0.02), and more patches of necrosis (6.5 +/- 2.1 versus 1.6 +/- 0.4, p less than 0.05) and higher contour (7.7 +/- 1.2 versus 5.03 +/- 0.2, p less than 0.05) and fractal (12.1 +/- 1.3 versus 7.8 +/- 0.2, p less than 0.05) indexes were found. CONCLUSIONS. Selective intracoronary infusion of BDM at doses inhibiting regional wall motion decreased infarct size after reperfusion. The effects of BDM on regional function, the reduction in contraction band necrosis at histology, and the peculiar configuration of these infarcts all suggest that inhibition of contracture can interfere with cell-to-cell progression of myocardial necrosis, supporting a role for contracture in reperfusion-induced cell death.     

Effect of intracoronary adenosine infusion during coronary intervention on myocardial reperfusion injury in patients with acute myocardial infarction

Despite early recanalization of an occluded infarct artery, up to 33% of patients with acute myocardial infarction do not obtain complete myocardial reperfusion due to a process of reperfusion injury. This study assessed whether adjunctive therapy with adenosine might prevent or attenuate the phenomenon of myocardial reperfusion injury. Myocardial reperfusion was assessed in 79 consecutive patients receiving a 20-minute intracoronary infusion of adenosine during percutaneous coronary intervention (PCI) and in a historical cohort of 200 patients with acute myocardial infarction who were treated with PCI (controls). Myocardial reperfusion injury was defined as persistent (> or =50% of initial value) ST-segment elevation after successful recanalization. Its effect on infarct size was evaluated by calculating the Selvester QRS score before intervention and at follow-up. Myocardial reperfusion injury was present in 19% of patients receiving adenosine versus 35% of control patients (p = 0.004). Evaluation of infarct expansion over time showed almost no change in the QRS score in patients receiving adenosine (3.4 +/- 3.0 before PCI; 3.5 +/- 3.1 at follow-up). In contrast, infarct QRS score in the control group worsened from 3.1 +/- 2.7 before PCI to 4.5 +/- 3.2 at follow-up (p = 0.003 treatment with adenosine vs control). Multivariate analysis identified adjunctive therapy with adenosine as an independent protective determinant of myocardial reperfusion injury and of infarct expansion. The rate of major adverse cardiac events (death and myocardial infarction) at 1 month tended to be lower in patients receiving adenosine (4% vs 6.5%, p = 0.7) and was mainly observed in patients with evidence of myocardial reperfusion injury (cardiac event rate 2% in patients with ST-segment elevation of <50% vs 14% in patients with ST-segment elevation > or =50%, p = 0.003). Thus, impaired myocardial reperfusion is the most important determinant of clinical outcome in patients with acute myocardial infarction treated with PCI. Adjunctive therapy with intracoronary infusion of adenosine during PCI prevents the occurrence of severe myocardial reperfusion injury and is associated with less infarct expansion.     

Pharmacologic perturbation of neutrophils by Fluosol results in a sustained reduction in infarct size in the canine model of reperfusion.

Previous studies have demonstrated that intravenous administration of large doses of Fluosol, a perfluorochemical preparation, reduced infarct size 24 h after reperfusion, an effect that was associated with reduced neutrophil infiltration. The effect of a clinically tolerable dose of Fluosol on infarct size after a prolonged period of reperfusion and its mechanism of action on neutrophils remain unknown. Twenty-one anesthesized closed chest dogs were subjected to 90 min of proximal left anterior descending coronary artery occlusion and 72 h of reperfusion. An additional five dogs that did not undergo regional myocardial ischemia were utilized to explore the mechanism of action of Fluosol on neutrophil function. In the infarct study, animals were randomized to receive either intravenous Fluosol (n = 10) or an equivalent volume of Ringer's lactate solution (control; n = 11) at 15 ml/kg body weight during the last 30 min of occlusion and for the 1st 30 min of reperfusion. Fluosol significantly reduced infarct size when expressed as percent area at risk 72 h after reperfusion (13.7 +/- 2.7% vs. 38.3 +/- 4.5%, respectively, p less than 0.001). This reduction was associated with significant improvement in regional wall motion (18.4 +/- 2.3% vs. 5.5 +/- 2%, p less than 0.001). Endocardial blood flow in the ischemic bed was significantly higher 3 h after reperfusion in Fluosol-treated dogs (0.63 +/- 0.08 vs. 0.34 +/- 0.07 ml/min per g, p = 0.01). Reduced capillary plugging by neutrophils with relative preservation of endothelial cell structure was observed in Fluosol-treated animals. Infusion of Fluosol produced a marked transient decrease in peripheral neutrophil and platelet counts in both ischemic and nonischemic dogs and was associated with a significant reduction in total hemolytic complement levels. Studies of neutrophil function ex vivo revealed a reduction in chemotaxis and lysozyme degranulation after infusion of Fluosol. In vitro experiments showed that Fluosol produced a rapid and sustained activation of neutrophils determined by superoxide anion production. These data demonstrate that low dose intravenous Fluosol produces a sustained reduction in infarct size in the canine model. The beneficial effect may be in part due to the suppression of various neutrophil functions in the reperfused myocardium subsequent to peripheral activation by Fluosol. Such interventions may offer a novel therapy to enhance myocardial salvage by sequestration of circulating neutrophils during the critical early reperfusion period.     

Opening of Ca-activated K channels is involved in ischemic preconditioning in canine hearts

Brief periods of ischemia that precede sustained ischemia can markedly reduce infarct size (IS), a phenomenon that is known as ischemic preconditioning (IP). Several investigators have shown that elevation of the intracellular Ca(2+) level ([Ca(2+)](i)) during the antecedent brief periods of ischemia triggers the cardioprotective mechanism of IP. Since opening of Ca(2+) activated K(+) (K(Ca)) channels is reported to be cardioprotective, we hypothesized that these channels may be involved in the cardioprotective mechanism of IP. In anesthetized open-chest dogs, myocardial ischemia/reperfusion injury was created by occlusion of the left anterior descending coronary artery (LAD) for 90 min followed by 6 h of reperfusion. First, we showed that the treatment with NS1619, a K(Ca) channel opener, reduced IS (IS in NS1619 group and control group, 19.8 +/- 5.5% vs. 45.4 +/- 3.5% of the area at risk, P < 0.05). Next, four cycles coronary occlusion for 5 min and reperfusion (IP) were performed before the 90-min occlusion with or without the infusion of potent K(Ca) channel inhibitors, iberiotoxin (IbTX) and charybdotoxin (ChTX). IP markedly reduced IS (IS in the IP group was 8.2 +/- 1.8%, P < 0.01 vs. control group). Infusion of either of K(Ca) channel blockers during IP blunted the IS-limiting effect of IP (IS in the IP + IbTX and IP + ChTX groups was 30.7 +/- 7.0% and 35.5 +/- 3.7%, respectively, P < 0.05, vs. IP group). However, the cardioprotective effect of IP was not blunted by the treatment with ChTX when treated only during reperfusion (14.0 +/- 4.1%). Thus, we conclude that the opening of K(Ca) channel is involved in early trigger phase of the molecular mechanism of IP.     

Opening of Ca2+-activated K+ channels is involved in ischemic preconditioning in canine hearts

Brief periods of ischemia that precede sustained ischemia can markedly reduce infarct size (IS), a phenomenon that is known as ischemic preconditioning (IP). Several investigators have shown that elevation of the intracellular Ca(2+) level ([Ca(2+)](i)) during the antecedent brief periods of ischemia triggers the cardioprotective mechanism of IP. Since opening of Ca(2+) activated K(+) (K(Ca)) channels is reported to be cardioprotective, we hypothesized that these channels may be involved in the cardioprotective mechanism of IP. In anesthetized open-chest dogs, myocardial ischemia/reperfusion injury was created by occlusion of the left anterior descending coronary artery (LAD) for 90 min followed by 6 h of reperfusion. First, we showed that the treatment with NS1619, a K(Ca) channel opener, reduced IS (IS in NS1619 group and control group, 19.8 +/- 5.5% vs. 45.4 +/- 3.5% of the area at risk, P < 0.05). Next, four cycles coronary occlusion for 5 min and reperfusion (IP) were performed before the 90-min occlusion with or without the infusion of potent K(Ca) channel inhibitors, iberiotoxin (IbTX) and charybdotoxin (ChTX). IP markedly reduced IS (IS in the IP group was 8.2 +/- 1.8%, P < 0.01 vs. control group). Infusion of either of K(Ca) channel blockers during IP blunted the IS-limiting effect of IP (IS in the IP + IbTX and IP + ChTX groups was 30.7 +/- 7.0% and 35.5 +/- 3.7%, respectively, P < 0.05, vs. IP group). However, the cardioprotective effect of IP was not blunted by the treatment with ChTX when treated only during reperfusion (14.0 +/- 4.1%). Thus, we conclude that the opening of K(Ca) channel is involved in early trigger phase of the molecular mechanism of IP.     

The role of L-arginine in ameliorating reperfusion injury after myocardial ischemia in the cat.

BACKGROUND. Myocardial ischemia followed by reperfusion results in endothelial dysfunction characterized by a reduced release of endothelium-derived relaxing factor (EDRF). Because EDRF has been characterized as nitric oxide, we examined the ability of L-arginine, the substrate for nitric oxide synthesis, to protect in a feline model of myocardial ischemia plus reperfusion. METHODS AND RESULTS. The effects of L-arginine were investigated in a 6-hour model of myocardial ischemia and reperfusion in pentobarbital-anesthetized cats. A bolus administration (30 mg/kg) of L-arginine, or its enantiomer D-arginine, was given followed by a continuous infusion of 10 mg/kg/min for 1 hour starting 10 minutes before reperfusion. Myocardial ischemia plus reperfusion in cats receiving D-arginine resulted in severe myocardial injury and endothelial dysfunction characterized by marked myocardial necrosis, high cardiac myeloperoxidase activity in ischemic cardiac tissue, and loss of acetylcholine- and A-23187-induced endothelium-dependent relaxation in coronary artery rings. In contrast, myocardial ischemia plus reperfusion cats treated with L-arginine exhibited a reduced area of cardiac necrosis (16 +/- 2% versus 41 +/- 5% of area at risk, p less than 0.01), lower myeloperoxidase activity in the ischemic region (0.3 +/- 0.08 versus 0.8 +/- 0.10 units/100 mg tissue, p less than 0.05), and significant preservation of acetylcholine- (p less than 0.01) and A-23187- (p less than 0.01) induced endothelial-dependent relaxation. CONCLUSIONS. These results demonstrate the ability of L-arginine to reduce necrotic injury in a cat model of myocardial ischemia plus reperfusion, and this reduction in infarct size is associated with the preservation of endothelial function and attenuation of neutrophil accumulation in ischemic cardiac tissue.     

Caffeine attenuates early post-exercise hypotension in middle-aged subjects

BACKGROUND: Sustained hypotension after an acute dynamic exercise bout is due primarily to peripheral vasodilation. We tested the hypothesis that adenosine-mediated vasodilation contributes to hypotension after exercise, by determining the effect of blocking its actions with caffeine. METHODS: Fourteen healthy middle-aged subjects (mean age = 51 +/- 3 years), cycled to peak effort on 2 study days, after a randomized double-blind intravenous infusion of caffeine (4 mg/kg) selective for adenosine receptor blockade, or vehicle. Both studies were performed after 72 h of caffeine abstinence. RESULTS: Infusion achieved 52.0 +/- 6.1 mumol/L caffeine in plasma. Significant reductions in mean and diastolic blood pressure (BP) were elicited by prior exercise on the vehicle day (from 93 +/- 2 to 85 +/- 2 mm Hg v from 79 +/- 2 to 73 +/- 3 mm Hg, respectively; both P < .05), but not after caffeine infusion. Systolic and mean BP, 10 min after exercise, were higher on the caffeine than on the vehicle day (by 9 +/- 3 and 6 +/- 2 mm Hg, respectively; P < .05), as was heart rate (HR) (100 +/- 5 v 93 +/- 4 beats/min; P < .05). CONCLUSIONS: These data suggest that endogenous adenosine contributes to early hypotension after exercise in healthy middle-aged subjects and underscore the importance of caffeine abstinence if BP or HR immediately after exercise is used to infer cardiovascular risk.     

Rapid effect of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibition on coronary endothelial function

Treatment with 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) decreases cardiovascular event rates in hypercholesterolemic patients. Whether statins exert effects within 24 hours on the coronary vasculature in patients with endothelial dysfunction has not been elucidated. Twenty-seven patients with stable angina pectoris and average low-density lipoprotein cholesterol concentrations of 138+/-9 mg/dL at baseline were allocated to treatment with placebo (14 patients) or 40 mg/d pravastatin (13 patients) in a randomized, double-blind, prospective trial. Coronary endothelial function was assessed before and 24 hours after single treatment by quantitative coronary angiography during intracoronary infusion of nitroglycerin or increasing concentrations of acetylcholine (0.01, 0.1, and 1 micromol/L). Coronary blood flow reserve was measured by Doppler velocimetry during adenosine infusion. Intracoronary acetylcholine infusion induced abnormal vasoconstriction in both groups before treatment, indicating coronary endothelial dysfunction. Treatment with a single oral 40-mg dose of pravastatin significantly attenuated acetylcholine-mediated vasoconstriction after 24 hours (mean+/-SE decrease in luminal diameter before and after treatment: 0.01 micromol/L, 6.1+/-2.2% versus 3.0+/-1.2%; 0.1 micromol/L, 15.6+/-2.6% versus 7.4+/-1.8%; P<0.05; 1 micromol/L, 22.9+/-2.9% versus 13.2+/-2.6%; P<0.05). There was no significant difference in the response to acetylcholine in the placebo group (8.1+/-2.4% versus 9.7+/-2.4%, 16.1+/-2.9% versus 16.8+/-3.2%, and 21.4+/-3.9% versus 23.3+/-4.2%). The response to nitroglycerin infusion was not altered in both groups. Increase in coronary blood flow in response to adenosine and coronary flow reserve remained unchanged during placebo and statin treatment. Serum concentrations of blood lipids and high-sensitive C-reactive protein were not significantly altered after 24 hours in response to placebo or pravastatin therapy. Statin treatment improves endothelium-dependent coronary vasomotion within 24 hours in the absence of significant cholesterol reduction. The full text of this article is available online at http://www.circresaha.org.     

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.     

Adenosine A(1) receptor mediates delayed cardioprotective effect of sildenafil in mouse

Sildenafil induces powerful cardioprotection against ischemia/reperfusion (I/R) injury. Since adenosine is known to be a major trigger of ischemic preconditioning, we hypothesized that A(1) adenosine receptor (A(1)AR) activation plays a role in sildenafil-induced cardioprotective signaling. Adult male C57BL wild-type (WT) mice or their corresponding A(1)AR knockout (A(1)AR-KO) mice were treated intraperitoneally (i.p.) with either sildenafil (0.71 mg/kg, equivalent to 50 mg dose for a 70 kg patient) or volume-matched saline. The selective A(1)AR antagonist 8-cyclopentyl-1,3-dipropyxanthine (DPCPX; 0.1 mg/kg, i.p.) was administered 30 min before sildenafil. The hearts were isolated 24 h later and subjected to 30 min of global ischemia and 1 h of reperfusion in Langendorff mode. Post-ischemic myocardial infarct size (mean+/-SEM; % of risk area) was reduced in C57BL-WT mice treated with sildenafil (5.6+/-0.9) versus saline control group (27.3+/-2.1; p<0.05; n=6/each). However, sildenafil failed to protect the A(1)AR-KO hearts (31.6+/-1.9 vs. 32.3+/-1.5 with saline, p>0.05). Additionally, DPCPX treatment abolished the infarct limiting effect of sildenafil (27.3+/-3.2, p<0.05). DPCPX alone had no effect on infarct size as compared with the control group. No significant changes in post-ischemic recovery of left ventricular pressure and heart rate were observed in the sildenafil-treated group. We further examined the effect of sildenafil in protection against simulated ischemia and reoxygenation injury in adult cardiomyocytes derived from WT and A(1)AR-KO mice. WT myocytes treated with sildenafil (1 microM) demonstrated significantly lower trypan blue-positive necrotic cells. However, cardiomyocytes derived from A(1)AR-KO mice or DPCPX-treated WT cells failed to show protection against necrosis with sildenafil. These results suggest that A(1)AR activation following treatment with sildenafil plays an integral role in the signaling cascade responsible for delayed protection against global I/R injury.     

Effect of Buckberg cardioplegia and peripheral cardiopulmonary bypass on infarct size in the closed chest dog.

OBJECTIVES. To simulate a human catheterization laboratory setting of controlled reperfusion during myocardial infarction, regional infusion of commercially available Buckberg cardioplegic solution and peripheral vented bypass were administered in the closed chest dog. BACKGROUND. Studies in open-chest dogs have demonstrated a significant reduction in infarct size and improvement in regional wall motion with a similar controlled reperfusion method using infusion of substrate-enriched (Buckberg) cardioplegic solution during cardiopulmonary bypass coupled with left ventricular venting. METHODS. After 100 or 180 min of balloon occlusion of the proximal left anterior descending artery, controlled reperfusion was performed with cardioplegic infusion and vented bypass. Dogs matched for occlusion time underwent balloon deflation without bypass or cardioplegia (uncontrolled reperfusion groups). Microspheres were used to quantify coronary ischemia during balloon inflation. All four groups (n = 8 to 9 per group) were followed up at 1 week to determine regional wall motion and infarct size. RESULTS. Qualitative echocardiographic analysis demonstrated no significant difference among groups in recovery of regional wall motion at 1 week; however, wall motion improved significantly in all groups between the ischemia and 1-week recovery periods. The histologic infarct size compared with the area at risk for dogs with uncontrolled versus controlled reperfusion, respectively, was 17.9 +/- 10.5% versus 31.9 +/- 8.3% (p < 0.05) for dogs with 100 min of occlusion and 40.1 +/- 11.7% versus 46.2 +/- 8.4% (p = NS) for dogs with 180 min of occlusion. A greater rate-pressure product in the dogs with controlled reperfusion after 100 min of occlusion (p < 0.05) may explain the larger infarct size observed for that group. CONCLUSIONS. These results demonstrate that regional infusion of substrate-enriched cardioplegic solution in combination with peripheral vented bypass does not further reduce infarct size after prolonged ischemia in the closed chest dog (compared with uncontrolled reperfusion).