Mechanism of the cardioprotective effect of transforming growth factor beta 1 in feline myocardial ischemia and reperfusion.

We studied the effects of transforming growth factor beta 1 (TGF-beta 1) in a feline model of myocardial ischemia (1.5 hr) and reperfusion (4.5 hr). Myocardial ischemia followed by reperfusion resulted in severe myocardial injury, endothelial dysfunction, high cardiac myeloperoxidase activity indicative of neutrophil accumulation in the ischemic myocardium, and significant neutrophil adherence to the ischemic coronary endothelium. In contrast, intravenous administration of TGF-beta 1 (20 micrograms/kg) 30 min prior to reperfusion significantly attenuated myocardial necrosis (13.8% +/- 3.5% vs. 32.2% +/- 2.9% of area-at-risk, P < 0.01) and attenuated endothelial dysfunction (P < 0.01) associated with ischemia-reperfusion. Moreover, myeloperoxidase activity in the ischemic myocardium was significantly lower than vehicle controls (0.2 +/- 0.1 vs. 1.7 +/- 0.3 units/100 mg of tissue, P < 0.01) and neutrophil adherence to ischemic coronary endothelium was significantly (P < 0.01) attenuated in TGF-beta 1-treated cats. These results demonstrate that TGF-beta 1 exerts a significant cardioprotective effect in a feline model of myocardial ischemia and reperfusion. The mechanism of this protective effect appears to relate to endothelial preservation by TGF-beta 1 inhibiting circulating neutrophils from adhering to the endothelium, a critical step in neutrophil-induced reperfusion injury.     

Coronary endothelial and cardiac protective effects of a monoclonal antibody to intercellular adhesion molecule-1 in myocardial ischemia and reperfusion.

BACKGROUND. Intercellular adhesion molecule-1 (ICAM-1) is a major ligand on endothelial cells for adherence of activated polymorphonuclear leukocytes (PMNs). The major purpose of this study was to study the effects of RR1/1, a monoclonal antibody against ICAM-1 (i.e., MAb RR1/1), on myocardial injury and endothelial dysfunction associated with myocardial ischemia and reperfusion. METHODS AND RESULTS. Either MAb RR1/1 (2 mg/kg, n = 7), an antibody that was found to bind selectively to endothelial cells in the cat, or MAb R3.1 (2 mg/kg, n = 7), a nonbinding control antibody, was given as an intravenous bolus 10 minutes before reperfusion. Two hundred eighty minutes later, hearts were excised. The left ventricle area-at-risk (AAR) was similar in MAb RR1/1 (29 +/- 2%) and MAb R3.1 (30 +/- 3%) groups. In MAb R3.1-treated cats, 90 minutes of myocardial ischemia plus 4.5 hours of reperfusion induced a significant myocardial injury (necrotic tissue/AAR, 28 +/- 2%), high myeloperoxidase activity (0.65 +/- 0.16 units/100 mg ischemic tissue), and a marked decrease in endothelium-dependent vasorelaxation in isolated left anterior descending coronary arteries (vasorelaxation to acetylcholine, 29 +/- 3%) with no change in endothelium-independent vasorelaxation (relaxation to NaNO2, 91 +/- 3%). However, cats treated with MAb RR1/1 developed significantly less myocardial necrosis (10 +/- 2% of the AAR, p less than 0.01), lower myeloperoxidase activity in ischemic myocardial tissue (0.2 +/- 0.03 units/100 mg ischemic tissue, p less than 0.01), and enhanced vasorelaxant responses to endothelial-dependent relaxation to acetylcholine (53 +/- 5%) compared with ischemic/reperfused cats treated with Mab R3.1. Furthermore, addition of MAb RR1/1 in vitro significantly inhibited unstimulated PMN adherence to ischemic-reperfused coronary artery endothelium. CONCLUSIONS. These results suggest that ICAM-1-dependent PMN adherence plays an important role in reperfusion injury, and that PMN adherence and infiltration contribute significantly to coronary endothelial dysfunction.     

Protection by superoxide dismutase from myocardial dysfunction and attenuation of vasodilator reserve after coronary occlusion and reperfusion in dog

Previous studies indicate impairment of coronary arterial ring relaxation and loss of coronary vasodilator reserve after coronary artery occlusion and reperfusion. These changes are mediated in part through loss of endothelium-derived relaxing factor (EDRF) and/or myocardial neutrophil accumulation. To examine if superoxide dismutase (SOD), a scavenger of superoxide radicals, would modify the diminished coronary vasodilator reserve after temporary coronary occlusion in the intact animal, open-chest mongrel dogs were subjected to 1 hour of circumflex (Cx) coronary artery occlusion followed by 1 hour of reperfusion and treated with saline or SOD. Before Cx occlusion, coronary blood flow increased, and vascular resistance decreased (both p less than 0.01) in response to EDRF-dependent vasodilator acetylcholine as well as EDRF-independent vasodilator nitroglycerin. After Cx reperfusion, resting Cx coronary blood flow and vascular resistance were similar to the preocclusion values. In the saline-treated animals, there was evidence of myocardial dysfunction, which was measured by segmental shortening (-6 +/- 2% vs. 10 +/- 2%). Furthermore, increase in Cx coronary blood flow and reduction in vascular resistance in response to both vasodilators were significantly (p less than 0.01) impaired; these occurrences suggested loss of coronary vasodilator reserve. Myocardial histology showed extensive capillary plugging by neutrophils in the Cx-supplied myocardium. Myocardial myeloperoxidase activity, an index of neutrophil infiltration, was also increased in the Cx compared with the left anterior descending coronary artery region (p less than 0.02). Treatment of dogs with SOD, started at the end of Cx occlusion and continued during reperfusion, exerted significant (p less than 0.01) protective effect against reperfusion-induced attenuation of coronary vasodilator reserve in response to both acetylcholine and nitroglycerin. Loss of myocardial function (segmental shortening 5 +/- 1% vs. 10 +/- 1%) was less than in the saline-treated animals (p less than 0.01). Cx region-myocardial neutrophil accumulation and myeloperoxidase activity were also less (p less than 0.02) in the SOD-treated than in the saline-treated dogs. These observations suggest that coronary reperfusion impairs coronary vasodilator reserve in intact dogs. This impairment can be modified by treatment of animals with SOD before reperfusion. Capillary plugging by neutrophils may contribute to the altered coronary vasodilator reserve observed in the immediate postreperfusion period, and SOD modifies this reperfusion-induced impairment.     

Recombinant soluble P-selectin glycoprotein ligand-1 protects against myocardial ischemic reperfusion injury in cats.

OBJECTIVE: Neutrophils (PMNs) contribute importantly to the tissue injury associated with ischemia and subsequent reperfusion of a vascular bed. The effects of a recombinant soluble human form of P-selectin glycoprotein ligand-1 (rsPSGL.Ig) on PMN-endothelial cell interactions were investigated in a well established model of feline myocardial-ischemia reperfusion injury. METHODS: Cats were subjected to 90 min of myocardial ischemia followed by 270 min of reperfusion. RESULTS: Administration of rsPSGL.Ig (1 mg/kg) just prior to reperfusion resulted in a significant reduction in myocardial necrosis compared to that in cats administered a low affinity mutant form of rsPSGL.Ig (1 mg/kg) (16 +/- 3 vs. 42 +/- 7% of area-at-risk, P < 0.01). Cardioprotective effects were confirmed by significant (P < 0.05) reductions in plasma creatine kinase activity in cats treated with rsPSGL.Ig. Inhibition of PMN-endothelial cell interactions was evidenced by a significant attenuation in cardiac myeloperoxidase activity (P < 0.01) and reduced PMN adherence to ischemic-reperfused coronary endothelium (P < 0.001). In addition, rsPSGL.Ig treatment significantly (P < 0.01) preserved endothelium-dependent vasorelaxation in ischemic-reperfused coronary arteries. CONCLUSION: These results demonstrate that the administration of a recombinant soluble PSGL-1 reduces myocardial reperfusion injury and preserves vascular endothelial function, which is largely the result of reduced PMN-endothelial cell interactions.     

Reduction in coronary vasodilator reserve following coronary occlusion and reperfusion in anesthetized dog: role of endothelium-derived relaxing factor, myocardial neutrophil infiltration and prostaglandins

To examine the effects of acute myocardial ischemia and reperfusion on regional coronary vasodilator (or flow) reserve, peak reactive hyperemic blood flow following a 10 s occlusion was obtained in dogs subjected to circumflex (Cx) coronary artery occlusion for 1 h followed by reperfusion for 1 h. Acute myocardial ischemia resulting from Cx artery occlusion-reperfusion caused an attenuation in peak reactive hyperemic Cx flow (mean +/- S.E., from 215 +/- 29% to 87 +/- 17%, P less than or equal to 0.001). Acetylcholine-induced increase in Cx flow was also significantly (P less than or equal to 0.01) attenuated following Cx occlusion-reperfusion. These alterations were not observed in the left anterior descending (LAD) coronary artery, which was not subjected to occlusion. Pre-treatment of four dogs with indomethacin inhibited prostaglandin release (P less than or equal to 0.01), but did not affect peak reactive hyperemic coronary flow or acetylcholine-induced increase in coronary flow before or after occlusion-reperfusion. Histopathology revealed extensive myocardial neutrophil infiltration in the Cx-supplied region compared to the LAD-supplied region. Myocardial myeloperoxidase activity, an index of neutrophil infiltration, was also increased in the Cx compared to the LAD region (P less than or equal to 0.02). Myocardial neutrophil accumulation and myeloperoxidase activity were similar in the control and indomethacin-treated animals. These observations suggest that acute myocardial ischemia resulting from coronary artery occlusion-reperfusion impairs coronary vasodilator reserve in anesthetized dogs. This impairment, which was not modified by prostaglandin inhibition, may be related to the loss of endothelium-derived relaxing factor and/or decreased microvascular cross-sectional area resulting from capillary plugging by neutrophils.     

Endothelium and myocardial protecting actions of taprostene, a stable prostacyclin analogue, after acute myocardial ischemia and reperfusion in cats

The effects of taprostene, a synthetic prostacyclin analogue, were investigated in a 6-hour model of myocardial ischemia (MI) with reperfusion in anesthetized cats. Taprostene (100 ng/kg/min) was infused intravenously starting 30 minutes postocclusion of the left anterior descending coronary artery followed by reperfusion 1 hour later, and the cats were observed for an additional 4.5 hours. Taprostene infusion resulted in significantly lower plasma creatine phosphokinase activities at every time from 3 to 6 hours for the MI + taprostene group compared with the MI + vehicle group and were not significantly different when compared with sham MI controls. The areas at risk, expressed as a percentage of the total left ventricular weights, were not significantly different between the MI groups. However, the necrotic area expressed as a percentage of the myocardial area at risk was significantly lower in the taprostene-treated cats compared with the untreated MI group (p less than 0.01). Cardiac myeloperoxidase activities indicated that significantly fewer neutrophils were attracted to the area at risk and to the ischemic zone of the MI + taprostene cats when compared with the MI cats given only the vehicle. Data from isolated left anterior descending coronary artery ring preparations removed from hearts after 6 hours of ischemia indicated that the endothelium was damaged by ischemia-reperfusion injury in the untreated cats. However, endothelial dysfunction was not observed in circumflex coronary arteries of ischemic cats or in coronary rings isolated from MI + taprostene cats. Thus, taprostene exerted a significant cardioprotection in cats subjected to ischemia and reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelium-derived relaxing factor inhibits hypoxic pulmonary vasoconstriction in rats

The hypothesis that endothelium-derived relaxing factor (EDRF) modulates hypoxic pulmonary vasoconstriction (HPV) was tested in isolated, blood-perfused rat lungs ventilated with gas mixtures of 21% O2-5% CO2-74% N2 (normoxia) or of 3% O2-5% CO2-92% N2 (hypoxia); 30 microM NG-monomethyl-L-arginine (L-NMMA), an inhibitor of EDRF production, caused a reduction in the endothelium-dependent relaxant response to acetylcholine (ACh) from 62 +/- 7, 88 +/- 4, and 100 +/- 4% to 26 +/- 8, 49 +/- 12, and 75 +/- 7% at ACh concentrations of 1, 10, and 100 microM, respectively (p less than 0.05 at all concentrations), indicating that L-NMMA acts via the inhibition of EDRF production. L-NMMA induced a concentration-related augmentation in HPV of 20 +/- 5, 32 +/- 8, and 34 +/- 8% at concentrations of 30, 300, and 1,000 microM (p less than 0.05, compared with a vehicle control group at all concentrations). The pressor response to a dose of angiotensin II (A-II), which produced the same increase in pulmonary artery pressure as that induced by hypoxia, was also significantly augmented (2 +/- 0.6%), but to a lesser extent. The augmentation of HPV by 30 microM L-NMMA was completely reversed by 1 mM L-arginine (a precursor of EDRF), but not by D-arginine (an isomer of L-arginine). One and 6 mM L-arginine, but not 6 mM D-arginine caused a significant inhibition of HPV by 20 +/- 2 and 47 +/- 12% (p less than 0.05, compared with the vehicle control group) and a small but not significant reduction in A-II-mediated contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental studies on ischemic injury and reperfusion injury to the cardiac sarcoplasmic reticulum--the myocardial protective effect of nicorandil

Ischemic and reperfusion injuries to the myocardium were evaluated by measuring cardiac function and the calcium binding capacity of the sarcoplasmic reticulum (SR) and by studying the myocardial protective effect of nicorandil. While undergoing cardiopulmonary bypass, dogs were subjected to 120-min global myocardial ischemia and then to 120-min reperfusion. Group I hearts were arrested with untreated potassium cardioplegic solution; Group II with the same solution containing 2 mg/L and Group III with the same plus 10 mg/L of nicorandil. Group III exhibited better recovery from ischemic and reperfusion injuries than Group I with recovery rates of LV dp/dt max (95.0 +/- 28.9% vs 61.1 +/- 30.4, p less than 0.05) and LV negative dp/dt max (69.0 +/- 12.5% vs 46.8 +/- 21.7, p less than 0.05). The 3 groups showed a marked decrease in the max calcium binding capacity during ischemia compared with the Control Group but exhibited no further decrease after reperfusion. After ischemia and reperfusion, Group III (30.4 +/- 9.13 nmol Ca/mg protein, 30.0 +/- 8.50) demonstrated a significantly higher binding capacity than Group I (17.0 +/- 2.41 and 18.3 +/- 1.01, p less than 0.05), while Group II did not. These results suggest that ischemia is more injurious to the calcium binding capacity of SR than reperfusion and that 10 mg/L of nicorandil added to the cardioplegic solution preserves SR function and enhances the recovery of cardiac function.     

Effects of transient coronary ischemia and reperfusion on myocardial edema formation and in vitro magnetic relaxation times

The effects of transient ischemia and reperfusion on regional myocardial function, salvage and swelling have been systematically analyzed in experimental canine preparations. The results of these interventions on myocardial in vitro measurements of magnetic relaxation times (T1 = magnetization recovery, T2 = spin echo) are of significant importance with respect to future nuclear magnetic resonance tomographic imaging. Thus, using a pulsed magnetic resonance spectrometer (10.7 MHz), myocardial tissue samples from two groups of dogs were evaluated. In group 1 (n = six dogs), the left anterior descending artery was occluded for 3 hours before sacrifice; in group 2 (six dogs), 3 hours of occlusion was followed by 1 hour of reperfusion. Multiple tissue samples from normal and ischemic (or ischemic and reperfused) myocardium were obtained for measurement of T1, T2 and % water content (wet weight--dry weight/wet weight). Water content increased with ischemia (78 +/- 4%) and reperfusion (81 +/- 4%) (both p less than 0.01 versus control values). Values for T1 increased with ischemia (598 +/- 39 versus 487 +/- 23 ms in normal tissue from the same heart, p less than 0.01). Even greater T1 changes occurred in the animals with reperfusion (654 +/- 52 ms, p less than 0.01 versus the intra-animal control values). Changes in T2 were similar but less marked (ischemic zone 43.9 +/- 1.0 versus 41.2 +/- 1.0 ms in nonischemic tissue in the corresponding heart, p less than 0.05; reperfusion zone 48.3 +/- 3.5 versus 41.9 +/- 2.3 ms in the normal zone, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationships of high energy phosphates, tissue pH, and regional blood flow to diastolic distensibility in the ischemic dog myocardium

Myocardial ischemia due to increased oxygen demand (pacing tachycardia plus critical coronary stenoses) alters diastolic distensibility and relaxation more than ischemia of comparable duration due to coronary occlusion. To investigate the relationship between myocardial diastolic function and metabolism, we compared myocardial high energy phosphate content, tissue pH, and regional blood flow for these two types of ischemia in anesthetized open-chest dogs. Myocardial biopsies were done with a high-speed air-turbine biopsy drill, permitting rapid (less than 1-second) freezing of tissue samples from both nonischemic and ischemic areas, while myocardial pH was measured with a hydrogen ion-selective polymer membrane implanted in the subendocardium. After 3 minutes of pacing tachycardia in dogs with critical coronary stenoses (demand-type ischemia, n = 14), regional systolic function (% segment shortening by ultrasonic crystals) was mildly depressed (from 19 +/- 2% control to 13 +/- 2% post-pacing, P less than 0.01), while left ventricular diastolic pressure-segment length relations shifted upward, indicating decreased distensibility of the ischemic myocardial segment. Associated with these changes in function, subendocardial adenosine triphosphate decreased (from 31.3 +/- 1.5 to 27.9 +/- 1.0 nmol/mg protein, P less than 0.01), as did creatine phosphate (53.8 +/- 2.1 to 39.6 +/- 2.5 nmol/mg protein, P less than 0.01), while myocardial pH declined slightly (delta pH = -0.14 +/- 0.02, P less than 0.01). In contrast, at 3 minutes of coronary artery occlusion (primary ischemia, n = 14), regional segment shortening was replaced by systolic bulging (% shortening decreased from 17 +/- 2% to -2 +/- 1% during occlusion, P less than 0.01), while left ventricular pressure-segment length relations were not shifted upward, and there was no decrease in diastolic distensibility of the ischemic segment. With coronary artery occlusion, subendocardial adenosine triphosphate declined slightly (33.2 +/- 0.5 to 29.2 +/- 2.0 nmol/mg, P less than 0.05), while creatine phosphate decreased substantially (51.1 +/- 2.3 to 7.8 +/- 1.4 nmol/mg protein, P less than 0.01). Myocardial pH fell strikingly (delta pH = -0.33 +/- 0.03, P less than 0.01), and the decline was 236% of that seen with demand-type ischemia. Regional myocardial blood flow (microsphere technique) showed a decreased endocardial:epicardial (endo:epi) ratio (1.04 +/- 0.04 control vs. 0.40 +/- 0.05 during pacing, P less than 0.01) and absolute subendocardial flow (1.02 +/- 0.47 to 0.47 +/- 0.05 ml/min per g, P less than 0.01) with demand-type ischemia.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assessment of myocardial salvage after ischemia and reperfusion using magnetic resonance imaging and spectroscopy

To test the hypothesis that contrast-enhanced magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) can differentiate reversible from irreversible myocardial injury, these modalities were used to study ischemia and reperfusion in a rat model. The presence of ischemia and reperfusion were confirmed with radiolabeled microspheres (n = 6). Groups of animals were subjected to either 16 (n = 17), 30 (n = 14), 60 (n = 11), or 90 (n = 14) minutes of left coronary artery (LCA) occlusion and 60 minutes reperfusion. After albumin-gadolinium (Gd)-DTPA injection, contrast-enhanced, T1-weighted, spin-echo proton images were acquired at baseline and every 16 minutes during LCA occlusion and reperfusion. In separate experiments, 31phosphorus (31P) spectra were acquired at similar time points during ischemia and reperfusion. After 16 minutes occlusion, normally perfused myocardium enhanced significantly compared with ischemic myocardium on MRI (104 +/- 7.9% vs. 61 +/- 11.0%, p less than 0.05, n = 5, mean +/- SEM, % of baseline value). MRS showed reduced phosphocreatine (PCr) and adenosine triphosphate (ATP) (58.8 +/- 2.4%, p less than or equal to 0.01; 81.4 +/- 2.4, p less than or equal to 0.01, n = 12). After 16 or 30 minutes ischemia, reflow resulted in uniform MRI signal intensity of the ischemic zone compared with normal myocardium (93.5 +/- 11.3 vs. 80.9 +/- 7.0, p = NS, n = 11, % of baseline value at 30 minutes reperfusion) and PCr recovery on MRS (94.3 +/- 4.0%, p = NS, n = 20, % baseline value at 30 minutes reflow). After 60 and 90 minutes ischemia, reflow resulted in marked enhancement of reperfused compared with normal myocardium on MRI (254.0 +/- 30.0 vs. 78.3 +/- 9.2, p less than or equal to 0.01, n = 10) and no recovery of PCr on MRS (64.1 +/- 3.0, p = NS, n = 14). Triphenyltetrazolium chloride (TTC) staining revealed transmural myocardial infarction (MI) in all hearts subjected to 60 or 90 minutes ischemia and reflow, and small nontransmural MIs in only 2/11 hearts subjected to 16 or 30 minutes ischemia and reperfusion. Thus, 1) MRI with albumin-Gd-DTPA is useful for identifying myocardial ischemia by enhancing the contrast between normally perfused and ischemic myocardia; 2) MRI with albumin-Gd-DTPA is useful for identifying reperfusion after myocardial ischemia; and 3) after reperfusion, reversible can be distinguished from irreversible myocardial injury by characteristic findings on MRI and MRS.     

Iloprost inhibits neutrophil function in vitro and in vivo and limits experimental infarct size in canine heart

The prostacyclin analogue iloprost (ZK 36374) inhibits neutrophil activation in vitro, reduces neutrophil accumulation in inflammatory skin lesions, and reduces ultimate infarct size in an anesthetized open-chest canine model of regional ischemia and reperfusion. Iloprost (0.1-100 microM) inhibited the in vitro production of superoxide anion by canine neutrophils in a concentration-dependent manner. Iloprost (100 ng/kg/min i.v.) inhibited C5a-induced neutrophil migration into inflammatory skin lesions as assessed by the neutrophil-specific enzyme marker, myeloperoxidase. The myeloperoxidase activity determined 2 hours after the intradermal administration of C5a in each of the groups was control 13.3 +/- 1.8 units/g tissue (n = 12) and iloprost 6.5 +/- 0.9 units/g (n = 12), p less than 0.01. Iloprost was administered to anesthetized open-chest dogs (100 ng/kg/min) 10 minutes after left circumflex coronary artery (LCCA) occlusion and continued during the 90-minute occlusion period and the first 2 hours of reperfusion. Regional myocardial blood flow was similar between treatment groups at baseline, 5 minutes and 80 minutes after LCCA occlusion, and after 1 hour of reperfusion. Infarct size, assessed 6 hours after reperfusion, was reduced by iloprost treatment: 22.4 +/- 3.1% of the area at risk (n = 15) compared with 42.4 +/- 3.3% of control (n = 13), p less than 0.01. Iloprost treatment reduced the accumulation of neutrophils (measured by myeloperoxidase activity) in the ischemic myocardium at the interface between infarcted and noninfarcted tissue: control (n = 9) 9.0 +/- 1.8 units/g tissue, iloprost (n = 6) 2.0 +/- 0.4 units/g, p less than 0.01.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypothermia during reperfusion does not reduce myocardial infarct size in pigs

We previously described a method for regional myocardial cooling that reaches the target temperature within 4 min. The present study evaluated whether this method for regional myocardial cooling during reperfusion reduces myocardial infarct size (IS) in 75-kg pigs. Myocardial infarction was induced by inflation of an angioplasty balloon in the left anterior descendent artery for 45 min followed by 3 h reperfusion. First, 15 pigs were randomized to regional myocardial cooling during reperfusion (n = 8) or control (n = 7). As further control experiments, systemic hypothermia was induced prior to ischemia (n = 3) and during reperfusion (n = 3). IS and area at risk (AAR) were evaluated in vivo by single photon emission cardiac tomography (SPECT) and by standard histochemical staining. Regional cooling during reperfusion did not reduce IS/AAR as assessed by histochemistry (cooling: 0.71 +/- 0.8; control: 0.68 +/- 0.10; p = ns) and SPECT (cooling: 0.90 +/- 0.20; control: 0.88 +/- 0.32; p = ns). Systemic hypothermia during ischemia reduced IS/AAR (histochemistry: 0.09 +/- 0.11; SPECT: 0.25 +/- 0.22; p < 0.001 and p = 0.01 vs control, respectively). Induction of systemic hypothermia during reperfusion had no significant effect on IS/AAR (histochemistry: 0.63 +/- 0.07; SPECT: 0.74 +/- 0.09; p = ns vs control for both comparisons). In conclusion, hypothermia during ischemia is strongly myocardioprotective while hypothermia during reperfusion does not reduce myocardial infarct size in human-sized pigs.     

Intramyocardial variability in integrated backscatter: effects of coronary occlusion and reperfusion

The present study was undertaken to characterize regional myocardial alterations of reflected ultrasound during the cardiac cycle in normal, ischemic, and postischemic reperfused myocardium. Time-averaged integrated backscatter (IB) and cardiac cycle-dependent amplitude modulation were measured from subepicardial, midmyocardial, and subendocardial regions of the left ventricular apex and the midportion of the right ventricular free wall under normal conditions (n = 5), after 1 hr of 100% acute left anterior descending (LAD) occlusion (n = 8), and after 15 min LAD occlusion plus 120 min reperfusion (n = 5) in anesthetized, ventilated open-chest dogs. A significant increase in time-averaged IB was observed in the subepicardium, the midmyocardium, and the subendocardium during ischemia and reperfusion, but there was no intramyocardial variability. Cardiac cycle-dependent amplitude modulation of IB was significantly higher in the normal subendocardium than in the subepicardium (4.3 +/- 0.6 vs 2.9 +/- 0.8 dB, p less than .01) and midmyocardium (2.8 +/- .05 dB, p less than .01). This transmural gradient in amplitude modulation was abolished during ischemia and reperfusion. We conclude that cardiac cycle-dependent amplitude modulation in IB has a transmural dependence in the normal myocardium and this is abolished during acute myocardial ischemia.     

Ethyl pyruvate enhances ATP levels, reduces oxidative stress and preserves cardiac function in a rat model of off-pump coronary bypass

BACKGROUND: Off-pump coronary artery bypass grafting is associated with transient periods of myocardial ischemia during revascularization resulting in myocardial contractile dysfunction and oxidative injury. The purpose of this study was to investigate the efficacy of ethyl pyruvate as a myocardial protective agent in a rat model of off-pump coronary artery bypass grafting associated with transient myocardial dysfunction without infarction. METHODS: Wistar rats were subjected to transient ischemia via 10 min occlusion of the LAD coronary artery followed by 10 min of reperfusion. Animals received an IV bolus of Ringer's solution as a control (n=10) or Ringer's ethyl pyruvate (n=10) immediately before the initiation of ischemia and reperfusion. Myocardial ATP and lipid peroxidation levels were quantified for an estimation of energetics and oxidative stress, respectively. In vivo cardiac function was assessed throughout the ischemia and reperfusion periods. RESULTS: Ethyl pyruvate significantly increased myocardial ATP levels compared to controls (2650+/-759 nmol/g versus 892+/-276 nmol/g, p=0.04). Myocardial oxidative stress was significantly reduced in animals treated with ethyl pyruvate compared to controls (70.4+/-2.6 nmol/g versus 81.8+/-2.4 nmol/g, p=0.04). dP/dt max and cardiac output were significantly greater in the ethyl pyruvate group compared to controls during ischemia and reperfusion. CONCLUSIONS: Ethyl pyruvate enhances myocardial ATP levels, reduces oxidative stress, and preserves myocardial function in a model of transient ischemia/reperfusion injury not subject to myocardial infarction.     

Role of leukocytes in coronary vascular endothelial injury due to ischemia and reperfusion

A possible cause of the coronary endothelial injury that occurs with ischemia and reperfusion is the local accumulation of leukocytes during these events. To investigate the role of leukocytes in coronary endothelial injury, we tested the effect of leukocyte removal by filtering on coronary endothelial function in a canine model of regional myocardial ischemia and reperfusion. Blood was supplied to the left anterior descending and circumflex arteries of anesthetized dogs via an extracorporeal circulation. A 60-minute left anterior descending occlusion was followed by 120 minutes of reperfusion either with (n = 6) or without (n = 6) leukocyte filters in the extracorporeal circuit. Regional myocardial blood flow was measured with radiolabeled microspheres. Radiolabeled autologous transferrin (113mIn) and erythrocytes (99mTc) were given intravenously during reperfusion for assessment of microvascular permeability. Left anterior descending and circumflex coronary artery rings were assessed in vitro for endothelium-dependent dilation to acetylcholine, ADP, and thrombin. In unfiltered dogs, ischemia and reperfusion increased the protein leak index of ischemic myocardium 2.3-fold compared with that of nonischemic myocardium (2.3 +/- 0.5 to 5.2 +/- 1.6, p less than 0.05). In filtered dogs, there was no difference in the protein leak index of nonischemic versus ischemic myocardium (1.5 +/- 0.4 versus 1.9 +/- 0.5, p = NS). There was impaired left anterior descending coronary artery relaxation (versus circumflex) in response to endothelium-dependent vasodilators in vitro. However, relaxation was not consistently improved by leukocyte filtering. We conclude that leukocytes are responsible for the endothelial injury secondary to ischemia and reperfusion in the coronary microvasculature but have little or no effect on the endothelial injury in epicardial coronary arteries.     

Protective effects of N-2-mercaptopropionyl glycine against myocardial reperfusion injury after neutrophil depletion in the dog: evidence for the role of intracellular-derived free radicals

Reperfusion of the previously ischemic myocardium is associated with the production of oxygen free radicals and their metabolites, which contribute to the ultimate extent of irreversible myocardial injury. The relative importance of polymorphonuclear leukocytes vs intracellular-derived oxygen metabolites has remained uncertain. We evaluated the effectiveness of a free-radical scavenger, N-2-mercaptopropionyl glycine (MPG), in limiting infarct size after ischemia/reperfusion in dogs that were depleted of neutrophils with specific antisera. Twenty-four urethane-anesthetized open-chest dogs were subjected to 90 min of ischemia by occlusion of the left circumflex coronary artery followed by 6 hr of reperfusion. Dogs were randomly assigned to receive nonimmune serum, neutrophil antiserum, or neutrophil antiserum plus MPG (20 mg/kg intra-atrially 15 min before reperfusion was initiated and for 45 min after reperfusion). Infarct size, as a percent of the area at risk, was reduced by 33% in the neutrophil antiserum group as compared with the nonimmune group (30.7 +/- 2.7% vs 45.6 +/- 3.7%, p less than .01). The combined administration of neutrophil antiserum plus MPG reduced the size of infarction by 63% of the area at risk compared with that in the nonimmune group (17.0 +/- 2.7% vs 45.6 +/- 3.7%, p less than .01). The reduction in infarct size with neutrophil antiserum plus MPG was significantly greater than that with the neutrophil antiserum alone (p less than .01). The areas at risk did not differ among the groups. Myocardial protection could not be explained on the basis of hemodynamic differences. The observation that MPG enhances the protective effects of neutrophil depletion suggests that both extramyocardial- and intramyocardial-derived oxygen free radicals contribute significantly to reperfusion-induced myocardial injury.     

Activated neutrophils aggravate endothelial dysfunction after reperfusion of the ischemic feline myocardium.

Endothelial dysfunction, as evidenced by decreased stimulated release of endothelium-derived relaxing factor (EDRF), occurs after reperfusion of the ischemic myocardium. To better understand this endothelial dysfunction, isolated cat hearts were perfused under constant flow by the Langendorff procedure with Krebs-Henseleit solution devoid of blood cells. Following global ischemia (90 minutes) and reperfusion (20 minutes), coronary vasorelaxation to the endothelium-dependent vasodilator acetylcholine (ACh) was 70 +/- 3% of initial values (p less than 0.01) compared with 90 +/- 4% in nonischemic control perfused hearts. No decrement occurred in response to the endothelium-independent vasodilator nitroglycerin (NTG). Coronary artery rings isolated from the ischemic left circumflex coronary artery showed a similar degree of endothelial dysfunction to ACh, with normal relaxation in response to NaNO2. Autologous cat neutrophils (100 million cells), activated with 100 nmol/L f-met-leu-phe infused into the heart directly before and throughout reperfusion, resulted in a further decrement in ACh-induced vasodilation, to 55 +/- 5% of initial response, with no effect on NTG-induced vasodilation. Similar results were obtained with coronary artery rings isolated from perfused cat hearts and exposed to neutrophils. This neutrophil-enhanced endothelial dysfunction was inhibited by human superoxide dismutase as well as by an antibody to the adherence glycoprotein complex CD-18 (i.e., MAbR 15.7). Therefore endothelial dysfunction occurs initially upon reperfusion of the previously ischemic heart and is aggravated by superoxide radicals produced by activated neutrophils.     

The hypertrophied myocardium and coronary disease. Structural changes in patients submitted to aortocoronary bypass surgery.

Seventeen patients with coronary disease submitted to myocardial revascularization were studied. Ten patients had a hypertrophied ventricle, and 7 had normal ventricular mass. Myocardial biopsies were obtained before ischemia and at the time of reperfusion and were assessed for: volume fraction of fibrous tissue, myocyte diameter, morphometric mitochondrial studies and ultrastructural changes. The volume fraction of fibrous tissue in patients with hypertrophied ventricle was 1.9 +/- 0.04, and in patients with normal ventricular mass was 0.9 +/- 0.01 (p less than 0.05). The diameter of the myocyte was 23 +/- 0.3 microns and 18 +/- 1.2 microns for patients with hypertrophied and normal ventricular mass, respectively (p less than 0.01). The value of volumetric density for pre-ischemia samples in patients with a hypertrophied ventricle was 23 +/- 2.2 and in patients with normal ventricular mass was 35 +/- 2.7 (p less than 0.02). Grades 3 and 4 of damaged mitochondria were significantly increased in reperfusion samples from patients with a hypertrophied ventricle compared to pre-ischemia samples. Collagen growth was increased in hypertrophied hearts which were also more sensitive to the ischemia/reperfusion mechanism.     

Lysoplasmenylethanolamine accumulation in ischemic/reperfused isolated fatty acid-perfused hearts.

Lysophospholipid accumulation has been implicated in the pathogenesis of irreversible injury during myocardial ischemia and reperfusion. Plasmalogens (phospholipids with a vinyl-ether bond in the sn-1 position) account for more than 50% of total myocardial sarcolemmal and sarcoplasmic reticulum phospholipids. Accumulation of plasmalogen choline and ethanolamine lysophospholipids (lysoplasmenylcholine and lysoplasmenylethanolamine) or the effects of exogenous fatty acids on lysoplasmalogen accumulation during ischemia and reperfusion have not been examined. Isolated working rat hearts perfused with buffer containing either 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate were subjected to aerobic, ischemic, or ischemia/reperfusion protocols. Levels of lysoplasmenylcholine and lysoplasmenylethanolamine were quantified using a two-stage high-performance liquid chromatographic technique. In hearts perfused with glucose alone, no significant differences in levels of lysoplasmenylcholine or lysoplasmenylethanolamine were seen during ischemia or reperfusion. In fatty acid-perfused hearts, however, significant accumulation of lysoplasmenylethanolamine occurred during reperfusion but not during ischemia (723 +/- 112, 734 +/- 83, and 1,394 +/- 193 nmol/g dry wt for aerobic, ischemic, and ischemic/reperfused hearts, respectively; p less than 0.05 for ischemic/reperfused hearts versus aerobic or ischemic hearts). Lysoplasmenylcholine levels after ischemia and reperfusion did not differ significantly from aerobic values, regardless of whether fatty acids were present or absent from the perfusate. Aerobic and ischemic/reperfused rabbit hearts, in the presence of fatty acid, showed a similar profile in their lysoplasmalogen content. We conclude that differential lysoplasmenylethanolamine accumulation occurs during myocardial reperfusion when exogenous fatty acid concentrations are high. This may reflect the selective action of fatty acid intermediates on the metabolism of lysoplasmenylethanolamines.(ABSTRACT TRUNCATED AT 250 WORDS)