Na(+)/H(+) exchange inhibitor cariporide attenuates cell injury predominantly during ischemia and not at onset of reperfusion in porcine hearts with low residual blood flow.

BACKGROUND: This study investigated whether myocardial protection by inhibition of Na(+)/H(+) exchange (NHE) occurs during ischemia and/or during reperfusion. METHODS AND RESULTS: The left anterior descending coronary artery was occluded in 32 pigs for 60 minutes and then reperfused for 24 hours. Infarct sizes (nitroblue tetrazolium [NBT] stain, histology) were determined at the end of the experiments. An extracorporeal bypass was used to achieve a constant residual blood flow of 3 mL/min in the myocardium at risk during ischemia. The NHE-1 inhibitor cariporide or distilled water was infused into the extracorporeal bypass system. In group 1, active treatment was administered from the onset of ischemia until 10 minutes of reperfusion (n=8). In group 2, active treatment was infused during the first 30 minutes of ischemia only (n=8). The group 3 animals (n=8) received intracoronary cariporide after 45 minutes of ischemia until 10 minutes of reperfusion. The control animals (group 4, n=7) were treated similarly to group 1 animals, with the cariporide solution being replaced by distilled water. Infarct sizes of group 1 (NBT stain, 41.5+/-20%; histology, 44. 6+/-12%) and group 2 (NBT stain, 33.5+/-14%; histology 34.9+/-15%) differed significantly (at least P:=0.012) from infarct sizes of group 3 (NBT stain, 71.6+/-15%; histology, 69.2+/-12%) and the control group (NBT stain, 76+/-9%; histology 72.4+/-12%). Cariporide treatment in group 1 and group 2 significantly improved functional recovery after 24 hours of reperfusion. CONCLUSIONS: Myocardial protection by cariporide is predominantly achieved by NHE inhibition during ischemia and not during early reperfusion.

Time-dependent protection by Na+/H+ exchange inhibition in a regionally ischemic, reperfused porcine heart preparation with low residual blood flow.

Inhibition of Na+/H+ exchange has been shown to protect the ischemic reperfused myocardium. This study investigated the time-dependent beneficial effect of the Na+/H+ exchange inhibitor HOE642 (4-isopropyl-3-methylsulphonylbenzoyl-guanedine methanesulphonite, cariporide). The left anterior descending coronary artery was ligated in 21 pigs (seven control animals) for 60 min and then reperfused for 24 h. An extracorporeal bypass system was used to achieve a constant residual blood flow of 3 ml/min within the ischemic myocardium. Cariporide (1 mg/kg) was injected intravenously in seven pigs after 15 min of ischemia (group A), and in another seven animals after 45 min of ischemia (group B). Histochemical (tetrazolium stain) and histologic infarct sizes were determined at the end of the experiments. Regional systolic shortening was determined by sonomicrometry. Mean calculated residual blood flows (ml/min/g of ischemic myocardium) amounted to 0.106 (group A), 0.093 (group B), and 0.117 (control group). Histochemical (32.9 +/- 21%) and histologic infarct sizes (36.7 +/- 17.7%) were significantly reduced in group A compared to both the control group (histochemical infarct size, 62.5 +/- 16.1%, P < 0.01; histologic infarct size. 67.8 +/- 16.3%, P = 0.013) and group B (histochemical infarct size 64.8 +/- 12.2%, P < 0.01; histologic infarct size 67.1 +/- 15.6%, P < 0.01). Infarct sizes of group B did not differ from control values. Recovery of regional systolic shortening after 24 h of reperfusion was insignificantly improved in group A compared to both other groups. In conclusion, inhibition of Na+/H+ exchange during early ischemia reduced cell death in an ischemic reperfused preparation with low residual blood flow.

Time delay of cell death by Na+/H+-exchange inhibition in regionally ischemic, reperfused porcine hearts.

Studies in different preparations have suggested that Na+/H+ exchange is one mechanism causally involved in cell death in myocardial ischemia and reperfusion. The time delay of cell death by pretreatment with the Na+/H+-exchange inhibitor HOE642, cariporide (4-isopropyl-3-methylsulphonylbenzoyl-guanidine methanesulphonate), was investigated in regionally ischemic, reperfused porcine hearts. HOE642 (1 mg/kg) was injected intravenously in 14 thoracotomized pigs 10 min before occlusion of the left anterior descending coronary artery (45 min of ischemia, six pigs; 70 min of ischemia, six pigs; 90 min of ischemia, two pigs). Ischemia was followed by 24 h of reperfusion. Six animals (45 min of ischemia) served as controls. Infarct size was determined as a ratio of infarcted (tetrazolium stain, histology) to ischemic myocardium (dye technique), and regional myocardial function was assessed by sonomicrometry. HOE642 did not affect global hemodynamic parameters. In the pretreated group with 45 min of ischemia, HOE642 significantly decreased histochemical infarct size from 51.2 +/- 12.6% (control group) to 13.2 +/- 12% (p < 0.005) and histologic infarct size from 44.5 +/- 9% to 17.1 +/- 7% (p < 0.005). Recovery of regional systolic shortening after 24 h of reperfusion was improved from 2 +/- 6% (control group) to 12 +/- 7% (p = 0.02). In addition, myocardial contracture and increase in heart rate during early reperfusion were attenuated. When ischemia was prolonged to 70 min after pretreatment with HOE642, infarct size, recovery of systolic shortening, myocardial contracture, and increase in heart rate did not differ from those of the control group. Pretreatment with HOE642 increased the tolerance to ischemia/reperfusion by approximately 20-25 min. Inhibition of Na+/H+ exchange appears to be very promising in the clinical treatment of acute myocardial ischemia and reperfusion.

Ultrastructural Evaluation of Postischemic Cell Death (Lethal Reperfusion Injury) in Porcine Hearts.

This study investigated whether reperfusion results in an increase of ultrastructurally determined myocardial injury in pig hearts. The left anterior descending coronary artery (LAD) was distally occluded in 12 pigs for 35-45 minutes and then reperfused for 3 hours. At the end of ischemia, as well as after 3 hours of reperfusion, one transmural biopsy was removed from the center of the risk region and subdivided into four-specimens, representing the subendocardial (I), subendo-midmyocardial (II), subepi-midmyocardial (III), and subepicardial layers (IV). The degree of injury was assessed by electronmicroscopy and was scored as reversible (1), an almost equal mixture of reversible and irreversible (2), and totally irreversible (3) damage. In addition, infarct size was determined as the ratio of infarcted (tetrazolium stain) to ischemic (dye technique) myocardium. Infarct sizes ranged from 29.3% to 93% (mean 61.2%). The scores of injury of the four tissue layers before and after reperfusion did not differ significantly: layer I, 2.4 +/- 0.8/2.3 +/- 0.9; layer II, 2.2 +/- 0.9/2.0 +/- 0.9; layer III, 1.8 +/- 0.9/2.0 +/- 0.9; and layer IV, 1.6 +/- 0.9/1.3 +/- 0.6. The means of the four layers were almost identical at the end of ischemia (2.1 +/- 0.8) and after 3 hours of reperfusion (2.0 +/- 0.6). A linear regression analysis with 95% confidence limits of the score values before and after reperfusion indicated that maximally 25% of a mean final infarct size of about 50% may be due to lethal reperfusion injury. This study suggests that cell death in regional ischemia and reperfusion occurs predominantly during ischemia and not during reperfusion.

Comparative study on the effects of intracoronary nicorandil and nitroglycerin in ischaemic, reperfused porcine hearts.

The direct cardioprotective properties of nitroglycerin and nicorandil were compared in regionally ischaemic (45 min), reperfused (24 h) porcine hearts. Intracoronary treatments, which were started 15 min prior to occlusion of the distal left anterior descending coronary artery (LAD), were continuously administered for 105 min. The following equi-hypotensive drug dosages were used in nine pigs each; nitroglycerin 6 micrograms.kg-1 x min before ischaemia and during 45 min of reperfusion, 0.6 microgram.kg-1 x min during ischaemia; nicorandil 5 micrograms.kg-1 x min before ischaemia and during 45 min of reperfusion, and 0.5 microgram.kg-1 x min during ischaemia. Nine control animals were treated with isotonic sodium hydrochloride solution (1 ml.min-1). Despite comparable effects on blood pressure, intracoronary nicorandil, in contrast to intracoronary nitroglycerin, did not increase heart rate. Although neither drug affected coronary blood flow significantly, nicorandil substantially reduced regional myocardial oxygen consumption before coronary artery occlusion (-37 +/- 22%, P = 0.003 vs control group, P = 0.01 vs nitroglycerin treatment). Infarct sizes (tetrazolium method) after 45 min of ischaemia and 24 h of reperfusion were significantly decreased by nicorandil (control group 76.9 +/- 19%, nicorandil group 49.3 +/- 24%, P = 0.012) whereas nitroglycerin exhibited a borderline effect (62.5 +/- 15%, P = 0.054). Both treatments resulted in improved regional systolic shortening of the reperfused segment at the end of the experiments but this was not significant. At these drug dosages the direct cardioprotective action of nicorandil is slightly superior to nitroglycerin. This may be ascribed to its K-channel opening property associated with reduced regional myocardial oxygen consumption before the onset of ischaemia.

Effects of R56865, an Na(+)- and Ca(2+)-overload inhibitor, on myocardial injury in ischemic, reperfused porcine hearts.

The cardioprotective effect of R56865, an Na(+)- and Ca(2+)-overload inhibitor, was studied in 20 regionally ischemic reperfused (I/R) porcine hearts. Ten pigs were treated with a single intravenous (i.v.) injection of 0.4 mg/kg 15 min before occlusion of the distal left anterior descending coronary artery (LAD) for 45 min. Ten other animals served as controls. Infarct size (IS) was determined as percentage of infarcted (tetrazolium method) to ischemic (dye technique) myocardium after 24-h reperfusion. Regional systolic shortening (SS) was determined by sonomicrometry. Fifteen minutes after i.v. administration of R56865, a significant decrease in heart rate (HR) (from 83 +/- 15 to 74 +/- 16 beats/min), dP/dtmax (from 2,033 +/- 604 to 1,822 +/- 524 mm Hg/s), LAD blood flow (BF, from 17 +/- 7 to 14 +/- 7 ml/min), and calculated global myocardial O2 consumption (MVO2) (from 5.9 +/- 0.9 to 5.4 +/- 0.9 ml O2/min x 100 g) was observed. Although this investigational drug attenuated the increase in HR during early reperfusion, the incidence of ventricular fibrillation (VF) was not affected during either ischemia or reperfusion. R56865 reduced IS by 24% from 67.1 +/- 16% (control group) to 50.8 +/- 13%. In addition, this treatment improved systolic shortening after 24-h reperfusion from 6 +/- 8% (control group) to 15 +/- 9%. Our results support the concept that inhibition of intracellular Na(+)- and Ca(2+)-overload is a promising new principle in treatment of myocardial I/R.

Effect of epinephrine treatment during late ischemia and early reperfusion on regional myocardial function and infarct size in partially infarcted reperfused porcine hearts.

This study investigated whether epinephrine treatment during late ischemia and early reperfusion improves systolic shortening after 45 minutes of reperfusion at the cost of increased infarct size. A model consisting of both stunned and dead myocytes was used. The left anterior descending coronary arteries of 10 control and 10 treated pigs were occluded distally for 40 minutes and then reperfused for 3 days. Regional systolic shortening was determined by sonomicrometry, and infarct size was assessed as the percentage of infarcted (tetrazolium stain) to ischemic (dye technique) myocardium. Intravenous administration of epinephrine was started 10 minutes before the onset of reperfusion (5 micrograms/min) and continued until 45 minutes of reperfusion (mean 18 micrograms/min). Immediately before and during 45 minutes of reperfusion, left ventricular peak pressure, dp/dtmax, and heart rate were significantly increased in the treated animals. After 45 minutes of reperfusion, epinephrine treatment improved systolic shortening of the reperfused myocardium (treated group 9% +/- 8%; control group -1% +/- 6%; p < 0.01). Transient beta-adrenergic stimulation of the reperfused myocardium did not increase infarct size (treated group 57.2% +/- 19%; control group 55.4% +/- 17%). In conclusion, epinephrine treatment during late ischemia and early reperfusion improved systolic shortening after 45 minutes of reperfusion without affecting infarct size.

Postischemic cell death in reperfused porcine hearts is not attenuated by the spin trap agent PBN during early reperfusion.

Ischemic, reperfused porcine hearts were used to investigate whether the spin trap agent PBN (N-tert-butyl-alpha-phenylnitrone) attenuates postischemic cell death by scavenging of free radicals. The left anterior descending coronary artery (LAD) was ligated distally in 16 pigs for 45 min and then reperfused for 3 h. PBN (coronary concentration approximately 1 mM) was infused into the LAD of eight pigs during the first 45 min of reperfusion. Electron spin resonance spectroscopy (ESR) was performed to identify free radical adducts in the reperfused coronary venous blood. Regional systolic shortening (SS%) was determined by sonomicrometry. Infarct size was evaluated as the percentage of infarcted (tetrazolium stain) to ischemic (dye technique) myocardium. The transmural ultrastructural degree of myocardial injury as well as myocardial ATP levels were assessed at the end of the experiment. Intracoronary treatment with PBN during early reperfusion did not attenuate myocardial damage. Infarct sizes (control group 59 +/- 19%, treated group 55 +/- 14%), transmural ultrastructural alterations, myocardial ATP concentrations (control group 1.8 +/- 0.3 mumol/mg frozen weight, treated group 1.7 +/- 0.4 mumol/mg) and regional systolic shortening at the end of the experiments (control group -1 +/- 5%, treated group -2 +/- 6% did not differ significantly. Furthermore, under various experimental conditions of spin trapping, free radical adducts could not be identified in coronary venous blood during early reperfusion. The results suggest that the spin trap agent PBN (1 mM) does not affect postischemic cell death in porcine hearts.

Interstitial myocardial neutrophil accumulation between 3 and 72 h of reperfusion does not significantly affect infarct size in porcine hearts.

The inflammatory response--mediated by activated neutrophils--is assumed to play an important role in the pathogenesis of reperfusion injury. This study investigated whether extravascular myocardial neutrophil accumulation between 3 and 72 h of reperfusion affects infarct size in porcine hearts. The left anterior descending coronary artery was ligated distally in 24 pigs for 45 min and reperfused for 3 h (n = 8), 24 h (n = 8), or 72 h (n = 8). Infarct size and extravascular myocardial accumulation of neutrophils was evaluated at the end of the experiments. Global hemodynamic characteristics were comparable for the three groups both before and during ischemia. Neutrophil count/mm2 reperfused myocardium increased substantially from 4 +/- 2 (3 h of reperfusion) to 129 +/- 70 (24 h of reperfusion, p < 0.001). After 3 d of reperfusion, the neutrophil count had decreased to 10 +/- 7. The invasion of neutrophils between 3 and 24 h of reperfusion did not significantly affect the extent of myocardial necrosis. Infarct sizes after 3 h (62.6 +/- 20%), 24 h (71.8 +/- 13%), and 72 h of reperfusion (67.6 +/- 18%) did not differ significantly. This finding does not suggest that a significant amount of myocytes are destroyed by extravasated neutrophils between 3 and 72 h of reperfusion.

Failure of chronic, high-dose, oral vitamin E treatment to protect the ischemic, reperfused porcine heart.

Lipid peroxidation, presumably the result of free radical-mediated injury, has been shown to occur during myocardial ischemia and reperfusion. Since vitamin E is a very effective, naturally occurring, chain-breaking antioxidant, it was investigated whether a vitamin E-supplemented diet increased myocardial tolerance towards ischemia and reperfusion in pigs. In addition to a standard diet which contained 30 mg vitamin E/kg (approximately daily vitamin E intake 30 mg), ten pigs were fed with 10 g vitamin E (all-rac-alpha-tocopherol acetate, Merck AG, Darmstadt, Germany) daily for at least 4 weeks. Ten control pigs remained on the standard diet. In an open chest preparation, the left anterior descending coronary artery was distally ligated for 45 min followed by 3 d of reperfusion. Infarct size was determined as the ratio of infarcted (tetrazolium stain) to ischemic myocardium (dye technique). Regional systolic shortening was evaluated by sonomicrometry. Vitamin E concentrations in plasma and myocardium were measured by high-performance liquid chromatography. Global hemodynamic characteristics did not differ between the two groups. Oral pretreatment with vitamin E raised the plasma concentration of this vitamin from 1.1 +/- 0.3 to 5.0 +/- 1.0 mg/l and the myocardial content from 4.2 +/- 0.7 to 18.6 +/- 2.7 ng/mg fresh weight. Vitamin E treatment did not reduce infarct size, which amounted to 71.3 +/- 5% in the control group and to 71.7 +/- 8.2% in the treated animals. Furthermore, recovery of regional systolic shortening of the reperfused segment did not significantly differ in the two groups after 3 d of reperfusion; it measured 2 +/- 4% in the controls and 6 +/- 6% (p = 0.16) in the treated animals. Therefore, chronic, oral treatment with vitamin E which raised myocardial and plasma concentrations of this vitamin 4- to 5-fold did not increase myocardial tolerance towards ischemia and reperfusion in this animal model.