The effects of Trolox, a water-soluble vitamin E analogue, in regionally ischemic, reperfused porcine hearts.

Myocardial protection by the water-soluble vitamin E analogue, Trolox, was investigated in 18 regionally ischemic, reperfused porcine hearts. The left anterior descending coronary artery was distally ligated for 45 min and was reperfused for three days. Five grams of Trolox (n = 9) were infused intravenously before coronary occlusion. Treatment was continued with an intravenous dose of 5 grams Trolox/24 hours until the end of the experiment. Infarct size was determined as the ratio of infarcted (tetrazolium stain) to ischemic myocardium (dye technique). Regional systolic shortening was assessed by sonomicrometry. Generation of free radicals by stimulated neutrophils was evaluated by luminol-enhanced chemiluminescence. Plasma concentrations of Trolox were measured by high-performance liquid chromatography. Aside from heart rate before ischemia, global hemodynamic values including calculated left ventricular oxygen consumption did not differ significantly between the two groups. Plasma concentrations of Trolox measured 1.8 +/- 0.3 mmol/l (before ischemia), 0.96 +/- 0.13 mmol/l (before reperfusion), 0.77 +/- 0.1 mmol/l (40 min of reperfusion), and 0.08 mmol/l (end of the experiment). Generation of free radicals by stimulated neutrophils was reduced by about 30% in the treatment group before ischemia and immediately before reperfusion, but was not reduced at the end of the experiment. Risk regions (control group 19.4 +/- 6 g, treatment group 19.3 +/- 7 g) and infarct sizes (control group 69.3 +/- 8%, treatment group 69.3 +/- 12%) were almost identical. Regional systolic shortening of a control segment and of the risk region were similar in both groups before ischemia, before reperfusion, and after 45 min of reperfusion. After 3 days of reperfusion, regional systolic shortening of the reperfused myocardium of the treated group had recovered to a significantly greater extent (P = 0.027). This parameter amounted to 9 +/- 6% in the treated group and to 3 +/- 3% in the control group. Improved functional recovery was not accompanied by higher tissue concentrations of adenosine triphosphate. It is concluded that the chosen treatment with Trolox does not reduce infarct size but accelerates functional recovery. This finding suggests that the mechanisms resulting in myocardial necrosis during ischemia/reperfusion and in post-ischemic myocardial dysfunction may differ.     

Intracoronary hyperosmotic mannitol during reperfusion does not affect infarct size in ischemic,reperfused porcine hearts

Summary We investigated the effect of reperfusion with hyperosmotic mannitol on the infarct size in porcine hearts. The distal half of the left anterior descending coronary artery was occluded in each of 21 anesthetized pigs for 75 min and was reperfused for 2 h. During reperfusion mannitol (1075 mosmol/kg) was intracoronarily infused at a dose of 0.5 ml/min in 6 pigs (low mannitol group), at a dose of 1.5 ml/min in another 6 pigs (high mannitol group), and at a dose of 5 ml/min in 3 pigs for the first 8 min of reperfusion (very high mannitol group). 6 pigs served as controls. Although mannitol infusion increased plasma osmolality in the ischemic, reperfused myocardium in all experiments, the infarct size expressed as the ratio of the infarcted tissue over the area at risk of necrosis was not significantly influenced. Infarct size amounted to 72±25% in the control group, to 75±14% in the low mannitol group, to 78±18% in the high mannitol group, and to 93±8% in the very high mannitol group. These results clearly indicate that reperfusion with hyperosmotic mannitol after 75 min of ischemia does not exert any beneficial effect on the infarct size.     

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

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

Acute treatment with vitamin E does not protect the regionally ischemic,reperfused porcine heart

Summary Thirty pigs were randomly assigned to a blind treatment with vitamin E or placebo. Ten animals each received 0.5g d-alpha tocopherol intravenously before ischemia (group 1) or before reperfusion (group 2). Ten control pigs were treated with a lipid emulsion as placebo. The left anterior descending coronary artery was distally ligated for 45 min followed by 3 days of reperfusion. Infarct size was determined as ratio of infarcted (tetrazolium stain) to ischemic myocardium (dye technique). Regional systolic shortening was assessed by sonomicrometry. Myocardial and plasma concentrations of vitamin E were determined by high-performance liquid chromatography. Global hemodynamic parameters and estimated left ventricular oxygen consumption did not differ among the three groups. Intravenous treatment with vitamin E raised the plasma levels of this vitamin from 1 ± 0.3 mg/l (control group) to 21 ± 6 mg/l before ischemia, to 4 ± 2 mg/l before reperfusion and to 2 ± 0.6 mg/l at the end of the experiments in group 1. In group 2, vitamin E plasma levels increased from 1 ± 0.3 mg/l to 24 ± 13 mg/l before reperfusion and to 2 ± 0.6 mg/l after 3 days of reperfusion. At the end of the experiments, myocardial vitamin E concentrations amounted to 4.2 ± 0.7 ng/mg fresh weight (control group), 9.7 ± 2.1 ng/mg (group 1), and to 8.7 ± 1.4 ng/mg (group 2). The increase in vitamin E plasma concentration was not associated with a cardioprotective effect. Infarct sizes of the three groups (group 1: 68 ± 12%, group 2: 66 ± 15%, control group: 69 ± 8%) were almost identical. Furthermore, recovery of systolic shortening was not improved by the acute vitamin E treatment. Mean systolic shortening of the reperfused segment amounted to 4% in the two treatment groups and 3% in the control group after 3 days of reperfusion. These results suggest that an acute increase in vitamin E plasma concentration before ischemia or during the early phase of reperfusion does not protect the ischemic, reperfused porcine heart.This study was supported by a grant from the Deutsche Forschungsgemeinschaft, KL 724     

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

The direct cardioprotective properties of nitroglycerin andnicorandil were compared in regionally ischaemic (45 min), reperfused(24 h) porcine hearts. Intracoronary treatments, which werestarted 15 min prior to occlusion of the distal left anteriordescending coronary artery (LAD), were continuously administeredfor 105min. The following equi-hypotensive drug dosages wereused in nine pigs each; nitroglycerin 6 fig. kg^–1 x minbefore ischaemia and during 45 min of reperfusion, 0.6 µg.kg^–1x min during ischaemia; nicorandil 5 fig. kg^–1x min before ischaemia and during 45 min of reperfusion, and0.5 fig. kg^–1 x min during ischaemia. Nine control animalswere treated with isotonic sodium hydrochloride solution (1ml. min^–1). Despite comparable effects on blood pressure, intracoronarynicorandil, in contrast to intracoronary nitroglycerin, didnot increase heart rate. Although neither drug affected coronaryblood flow significantly, nicorandil substantially reduced regionalmyocardial oxygen consumption before coronary artery occlusion( – 37±22%, P=0003 vs control group, P=0.01 vsnitroglycerin treatment). Infarct sizes (tetrazolium method)after 45 min of ischaemia and 24 h of reperfusion were significantlydecreased by nicorandil (control group 76.9 ± 19%, nicorandilgroup 49.3 ± 24%, P=0.012)whereas nitroglycerin exhibiteda borderline effect (62.5 ± 15%, P=0.054). Both treatmentsresulted in improved regional systolic shortening of the reperfusedsegment at the end of the experiments but this was not significant.At these drug dosages the direct cardioprotective action ofnicorandil is slightly superior to nitroglycerin. This may beascribed to its K-channel opening property associated with reducedregional myocardial oxygen consumption before the onset of ischaemia.     

Cell death in ischemic,reperfused porcine hearts: A histochemical and functional study

Summary The temporal development of infarcts was histochemically and functionally determined in porcine hearts. In one series of experiments (22 pigs), the distal third of the left anterior descending coronary artery (LAD) was transiently occluded for periods between 20 and 90 min and was reperfused for another 24h. At the end of the experiments, the infarcted myocardium of four tissue slices was determined with a tetrazolium stain and related to the risk region which was delineated by a fluorescent dye. Infarcts started to develop in the ischemic septum and the subendocardial layer of the free anterior wall between 20 and 35 min of ischemia. Thereafter, infarctions progressed rapidly from the inner towards the outer layer at risk. The jeopardized anterior left ventricular wall became almost completely infarcted within 60 min of ischemia. In a second series of experiments (10 pigs) recovery of systolic shortening was studied with implanted ultrasonic crystals over 3 weeks of reperfusion. At the end of the experiments, systolic shortening was about 75% of baseline level when ischemia had lasted between 20 and 35 min. Almost no recovery was observed when the occlusion time lasted 45 to 60 min. This study suggests that the assessment of myocardial infarction with a tetrazolium stain after 24 h of reperfusion corresponds very well with functional recovery after 3 weeks of reperfusion. Furthermore, determination of regional myocardial function of the ischemic, reperfused segment in the chronic stage may be considered an additional tool to evaluate therapeutic effects on infarct size in this model.The study was supported by a grant of Deutsche Forschungsgemeinschaft (DFG) Sonderforschungsbereich 330 Organprotektion Göttingen.This paper contains parts of the Habilitationsschrift of Dr. H. H. Klein.     

Comparative study on the enhancement of ischemic tolerance by intracoronary pretreatment with three calcium antagonists in pig hearts

Summary The effect of intracoronary (IC) pretreatment with different calcium antagonists (diltiazem, nifedipine, verapamil) on the development of infarcts was investigated in two experimental series including 35 open-chest pigs. The left anterior descending coronary artery (LAD) was distally ligated for 75 minutes (series A) or for 45 minutes (series B) and was reperfused for 24 hours. Infarct size was determined as the ratio of infarcted myocardium (tetrazolium stain) to the risk region (dye technique). In series A, 20 pigs were pretreated immediately before occlusion with either IC diltiazem (n=5, 4 mg/2 min), IC nifedipine (n=5, 0.4 mg/2 min), IC verapamil (n=5, 1 mg/2 min), or isotonic sodium chloride solution (n=5). In series B, IC diltiazem (n=5, 4 mg/2 min), IC verapamil (n=5, 1 mg/2 min), or isotonic saline solution (n=5) were administered 8 minutes prior to ischemia. The IC infusion of all calcium antagonists (series A) depressed left ventricular peak pressure, diastolic blood pressure, and dp/dt max and increased heart rate and coronary venous oxygen saturation. The development of infarcts was significantly delayed by IC diltiazem and IC verapamil. Mean infarct sizes (series A) amounted to 62% in the diltiazem group, 88% in the nifedipine group, 40% in the verapamil group, and 94% in the control group. In series B, where a time period of 8 minutes elapsed between pretreatment and induction of ischemia, mean infarct sizes after 45 minutes of ischemia and 24 hours of reperfusion amounted to 47% in the diltiazem group, 4% in the verapamil group, and 76% in control experiments. The better protection of verapamil in series B can mainly be ascribed to its longer lasting regional depression compared to diltiazem.In conclusion, at the given drug concentrations, IC verapamil and IC diltiazem enhanced the ischemic tolerance to a greater extent than IC nifedipine (series A). When a time period of 8 minutes had elapsed between IC treatment and the onset of ischemia (series B), verapamil still exhibited a protective effect on the treated myocardium, which can mainly be ascribed to its long-lasting negative inotropic action.This study was supported by a grant from the Deutsche Forschungs-gemeinschaft, SFB 330 Organprotektion. The paper contains parts of the Habilitationschrift of Dr. H. H. Klein.     

Diltiazem and progression of myocardial ischemic damage during coronary artery occlusion and reperfusion in porcine hearts

This study was designed to investigate whether a cardioprotective intervention could delay the completion of necrosis so that subsequent reperfusion would be more useful. Thirty-six pigs were randomly allocated to treatment with diltiazem (15 micrograms/kg per min) or saline solution and to a 60 or 120 minute coronary occlusion period followed by reperfusion. The treatment was begun 15 minutes before coronary occlusion and terminated 75 minutes after reperfusion. Twenty-four hours after the procedure, the heart was sliced and incubated in triphenyltetrazolium chloride. The infarct area and the maximal transmural area of extension of the infarct were calculated by planimetry. The total number of red blood cells in a transmural section was also counted. In the pigs with a 60 minute coronary occlusion, diltiazem (compared with saline solution) significantly reduced infarct size from 9.7 +/- 1.5% of left ventricular mass to 5.9 +/- 0.6% (p less than 0.05) and the percent transmural extension from 0.72 +/- 0.05 to 0.61 +/- 0.05% (p less than 0.05). Red blood cell extravasation in the infarcted area was reduced from 161,934 +/- 59,905 to 78,525 +/- 46,484 cells/mm3 (p less than 0.05) with diltiazem and the percent transmural extension of the hemorrhagic necrosis from 70 +/- 10 to 36 +/- 15% (p less than 0.05). No such differences were observed in the 120 minute coronary occlusion groups. Mean red blood cell counts and the extent of hemorrhagic necrosis did not correlate with either infarct size or transmural extension.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of increased cytosolic free calcium concentration in myocardial ischemic injury

Summary Increases in cytosolic free calcium concentration ([Ca²⁺]_I) may play an important role in myocardial ischemic injury. An early effect of the rise in [Ca²⁺]_I may be impaired postischemic contractile function if the ischemic myocardium is reperfused during the reversible phase of ischemic injury; furthermore, if the rise in [Ca²⁺]_I is prolonged, a cascade of events may be initiated which ultimately results in lethal injury. With the development of methods for measuring [Ca²⁺]_I, it has become possible to evaluate directly the role of increased [Ca²⁺]_I in myocardial ischemic injury. Although it has been possible to show that inhibition of the transport processes which contribute to the early rise in [Ca²⁺]_I attenuates stunning and the rise in [Ca²⁺]_I concurrently, if increased [Ca²⁺]_I plays an important role in ischemic injury, then it should be possible to show that interventions which alter the timecourse of ischemic injury also alter the timecourse of the rise in [Ca²⁺]_I in a parallel manner. Recently, considerable effort has been expended to investigate the mechanisms underlying the preconditioning phenomenon, whereby repetitive brief periods of ischemia prior to a sustained period of ischemia protects the myocardium from injury during the sustained period of ischemia, and this has stimulated additional work to understand the possible involvement of adenosine as a mediator of preconditioning as well as to understand the protective effects of adenosine. Measurements of [Ca²⁺]_I using 19F NMR of 5FBAPTA-loaded hearts have shown that preconditioning attenuates the rise in [Ca²⁺]_I during 30 min of ischemia and reduces stunning during reflow. Adenosine pretreatment mimics the effects of preconditioning on the rise in [Ca²⁺]_I and on stunning, but adenosine receptor antagonists do not eliminate the protective effects of preconditioning, although some adenosine antagonists also block hexose transport and under these conditions, the ability of preconditioning to attenuate the rise in [Ca²⁺]_I is abolished and there is a corresponding loss of the protective effect of preconditioning on stunning. Although it has been suggested that the beneficial effect of preconditioning on infarct size can be eliminated by pretreatment with glibenclamide, in the isolated rat heart glibenclamide does not affect the attenuation of the rise in [Ca²⁺]_I induced by preconditioning and does not affect stunning. All of these studies show a consistent relationship between the magnitude of the rise in [Ca²⁺]_I during ischemia and the degree of stunning during reperfusion. The data suggest that increased [Ca²⁺]_I plays a very important role in myocardial ischemic injury.     

R56865 is antifibrillatory in reperfused ischemic guinea-pig hearts,even when given only during reperfusion

Summary R56865 was previously characterized as an inhibitor of Na and Ca overload that has beneficial effects during ischemia and reperfusion. An isolated guinea-pig heart preparation was subjected to 60 minutes of ischemia and 60 minutes of reperfusion. R56865 was given before ischemia and with the onset of reperfusion, applying different dosing schedules, including an initial loading dose. R56865 below 0.1 µmol/l had no cardiodepressant effects in normoxic hearts and at 0.1 µmol/l reduced left ventricular pressure marginally. The onset of ischemic contracture was delayed only at this concentration. R56865 given before ischemia potently inhibits delayed sustained fibrillation occurring during reperfusion in the concentration range between 0.01 µmol/l and 0.1 µmol/l. Analysis of cellular Na⁺, K⁺, and Ca²⁺ concentrations revealed that R56865 substantially improves the ionic homeostasis of myocardial cells. Most importantly, the compound also reduced the incidence of delayed sustained fibrillation when given at the onset of reperfusion. R56865 was most effective when fast equilibration of drug with tissue was achieved by giving an initial loading dose. In particular, the cellular Na⁺ and Ca²⁺ contents were improved using this dosing scheme. The results are compatible with the classification of R56865 as an inhibitor of Na⁺ and Ca²⁺ overload.     

A calcium channel blocker amlodipine increases coronary blood flow via both adenosine- and NO-dependent mechanisms in ischemic hearts

Amlodipine reduces oxidative stress that decreases NO and adenosine release. This study was undertaken to examine whether amlodipine mediates coronary vasodilation and improves myocardial metabolism and contractility in ischemic hearts via either adenosine- or NO-dependent mechanisms. In open-chest dogs, amlodipine (2 mug kg per min) was infused at the minimum dose that caused maximal coronary vasodilation. The perfusion pressure was reduced in the left anterior descending coronary artery so that coronary blood flow (CBF) decreased by 50%. Amlodipine increased the difference of the adenosine level (VAD (Ado): 119+/-14 to 281+/-46 nM) and the nitrate+nitrite level (VAD (NOx): 7.8+/-1.3 to 16.1+/-1.1 muM) between coronary venous and coronary arterial blood, and also increased CBF (50+/-3 to 69+/-6 ml/100 g/min). These changes were partially reversed by either 8-sulfophenyeltheophylline (8SPT) or l(omega)-nitro arginine methyl ester (l-NAME), and were completely blocked by both 8SPT and l-NAME. The reduction of CBF increased VAD (8-iso-prostaglandin F(2alpha)), and this increase was reduced by amlodipine (10.8+/-1.1 to 5.0+/-0.5 pg/ml). In addition, pretreatment with superoxide dismutase mimicked the coronary effects of amlodipine and blunted the response to amlodipine administration. Amlodipine-induced coronary vasodilation via both adenosine- and NO-dependent mechanisms. Adenosine and NO may interact in ischemic hearts to mediate coronary vasodilation by amlodipine.     

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.     

Protective role of intracoronary fatty acid binding protein in ischemic and reperfused myocardium

In this study, fatty acid binding protein was used to protect an ischemic heart from reperfusion injury. Isolated rat heart was preperfused in the presence of 1.4 microM liposome-bound fatty acid binding protein for 15 minutes, followed by 30 minutes of ischemia and 30 minutes of reperfusion. Our results indicated better preservation of myocardial high-energy phosphate compounds (including ATP and creatine phosphate), reduced creatine kinase and lactate dehydrogenase release from the heart, and improved coronary flow in hearts treated with fatty acid binding protein compared with untreated controls. Fatty acid binding protein enhanced reacylation of arachidonic acid into phospholipids, thereby preserving membrane phospholipids and reducing free fatty acid contents during ischemia and reperfusion. In addition, fatty acid binding protein-bound long-chain free fatty acids and their thioesters as well as carnitine esters were increased in the cytosolic compartment of the heart. These results suggest that fatty acid binding protein may be used as a possible therapeutic agent to improve myocardial function during reperfusion of ischemic heart.     

Intracellular free calcium concentration measured with 19F NMR spectroscopy in intact ferret hearts.

Changes in the intracellular free Ca2+ concentration, [Ca2+]i, mediate excitation-contraction coupling in the heart and contribute to cellular injury during ischemia and reperfusion. To study these processes directly, we measured [Ca2+]i in perfused ferret (Mustela putorius furo) hearts using 19F NMR spectroscopy to detect the 5,5'-difluoro derivative of the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). To load cells, hearts were perfused with the acetoxymethyl ester derivative of 5,5'-F2-BAPTA. We measured 19F NMR spectra and left ventricular pressure simultaneously, at rest and during pacing at various external Ca concentrations [( Ca]o). Although contractile force was attenuated by the Ca2+ buffering properties of 5,5'-F2-BAPTA, the decrease in pressure could be overcome by raising [Ca]o. Our mean value of 104 nM for [Ca2+]i at rest in the perfused heart agrees well with previous measurements in isolated ventricular muscle. During pacing at 0.6-4 Hz, time-averaged [Ca2+]i increased; the effect of pacing was augmented by increasing [Ca]o. [Ca2+]i more than tripled during 10-20 min of global ischemia, and returned toward control levels upon reperfusion. This approach promises to be particularly useful in investigating the physiology of intact hearts and the pathophysiology of alterations in the coronary circulation.     

Effects of calcium antagonists and free radical scavengers on myocardial ischemia and reperfusion injury: evaluation by 31P-NMR spectroscopy

The Langendorff perfused rat heart was used to investigate whether myocardial damage during ischemia and reperfusion could be protected by free radical scavengers, calcium antagonist and adenosine. Myocardial high energy phosphates were measured by phosphorus-31 NMR spectroscopy during normal perfusion, 20 min of ischemia and 20 min of reperfusion. In hearts, which were treated both with free radical scavengers (FRS) (Superoxide dismutase): 24 IU/ml and catalase 22 IU/ml) and verapamil (10(-7) M), beta-ATP was significantly higher than that of FRS at the end of ischemia. However, beta-ATP recovered only to 83% of baseline value at the end of reperfusion. In view of myocardial metabolism, verapamil treated hearts were good for recovery of creatine phosphate (PCr) but not ATP at the end of reperfusion. Hearts which were treated with only adenosine did not differ from control hearts. However, when hearts were treated with both verapamil and adenosine (10(-4) M), recovery of both ATP and PCr content was significantly greater than that of control hearts. These results suggested that pretreatment with both verapamil and adenosine before and after global ischemia could protect ischemic myocardium, but, further studies are necessary to clarify the precise mechanism of protection.     

Hypercontractile female hearts exhibit increased S-nitrosylation of the L-type Ca2+ channel alpha1 subunit and reduced ischemia/reperfusion injury

Mechanisms underlying gender differences in cardiovascular disease are poorly understood. We found previously that, under hypercontractile conditions, female hearts exhibit significantly less ischemia/reperfusion injury than males. Here we show that male wild-type (WT) mouse hearts pretreated with 10 nmol/L isoproterenol before ischemia exhibited increased injury versus female hearts, but this relative protection in females was absent in eNOS(-/-) and nNOS(-/-) hearts. In isoproterenol-treated female versus male hearts, there was also more endothelial NO synthase (eNOS) associated with cardiomyocyte caveolin-3, and more neuronal NOS (nNOS) translocation to caveolin-3 during ischemia/reperfusion. S-nitrosothiol (SNO) formation was increased in isoproterenol-treated ischemic/reperfused hearts in all mouse genotypes, but only in WT mice was SNO content significantly higher in females than males. Using the biotin switch method, we identified the L-type Ca2+ channel alpha1 subunit as the predominant S-nitrosylated protein in membrane fractions, and following isoproterenol and ischemia/reperfusion male/female differences in SNO were seen only in WT hearts, but not in constitutive NOS(-/-) genotypes. The isoproterenol-induced increase in L-type Ca2+ current (ICa) was smaller in females versus in males, but NOS blockade increased ICa in females. This gender difference in ICa in isoproterenol-treated myocytes (and abolition on NOS inhibition) was mirrored exactly in Ca2+ transients and SR Ca2+ contents. In conclusion, these data suggest that eNOS and nNOS both play roles in the gender differences observed in ischemia/reperfusion injury under adrenergic stimulation, and also demonstrate increased S-nitrosylation of the L-type Ca2+ channels in female cardiomyocytes.     

Effects of intracoronary administration of contrast media on coronary hemodynamics in a canine post ischemic reperfusion model

Hemodynamic changes due to intracoronary injections of nonionic contrast medium Omnipaque-350 (OM), ionic dimer Hexabrix (HB), and ionic contrast medium Renografin-76 (R76) were compared at baseline and during reperfusion after a 30-minute left anterior descending coronary artery (LAD) occlusion. In 11 open chest, anesthetized, and atrially paced dogs, 4 ml of either OM, HB, R76, or 0.9% NaCl were injected into the carotid-LAD bypass system. Coronary blood flow (CBF) and coronary vascular resistance (CVR) were measured before, during and after the intracoronary injection. The maximal hyperemic change (in percentage) from the preinjection value of CBF and CVR were calculated. The results at baseline and during reperfusion for CBF were: 104 +/- 14% vs. 85 +/- 10% for OM (NS); 76 +/- 11% vs. 39 +/- 9% for R76 (p less than 0.05); 57 +/- 8% vs. 33 +/- 5% for HB (P less than 0.05); and 30 +/- 7% vs. 9 +/- 4% for 0.9% NaCl (p less than 0.05). Consequently, the hyperemic changes of CVR at baseline and during reperfusion were: -49 +/- 3 vs. -42 +/- 4% for OM (NS); -44 +/- 3% vs. -24 +/- 6% for R76 (p less than 0.01); -36 +/- 3% vs. -24 +/- 4% for HB (p less than 0.05); and -18 +/- 4% vs. -7 +/- 3% for 0.9% NaCl (p less than 0.05). Thus, ischemia and reperfusion significantly dampened the coronary hemodynamic and vascular response to R76, HB, and 0.9% NaCl but not to OM. The preserved coronary vascular reserve with high flow in this canine post-ischemic reperfusion model may explain the advantage of nonionic over ionic contrast media used in emergency coronary angiography following thrombolysis.     

Intracellular free calcium concentration, Ca++ channel and calmodulin level in experimental hypertension in rats

Using fluorescent calcium indicator quin2, we studied intracellular free calcium concentration in platelets that have a number of features similar to vascular smooth muscle cells. Intracellular free calcium concentration in platelets of male SHR was significantly higher at 4, 11 and 28 weeks old compared with age-matched male WKY. However, no significant difference was observed in platelets cytosolic free calcium level of DOCA-salt hypertensive and two-kidney, one clip hypertensive rats in the chronic stage. Cardiac Ca++ channels were estimated by means of radioligand binding method with [3H]-nimodipine. No significant changes were observed in the concentration and affinity of cardiac Ca++ channel in SHR, DOCA-salt hypertensive and two-kidney, one clip hypertensive rats. Calmodulin levels in mesenteric arteries of SHR were significantly decreased in comparison with those of WKY. However no significant differences were observed in DOCA-salt hypertensive rats in the chronic stage. These results indicate that the increase in intracellular free calcium concentration of SHR is not the secondary change caused by high blood pressure. It is impossible to detect the ratio of the three states (open, resting and inactivated) of Ca++ channel. Therefore, there remains a possibility of the changes in the ratio of the states of Ca++ channel. The observed abnormalities of Ca++ regulation may contribute to the pathogenesis of hypertension.