Effect of ischemia and reperfusion on sarcoplasmic reticulum calcium uptake.

To investigate the mechanism underlying postischemic cardiac dysfunction (myocardial stunning), contractility and adenine nucleotide metabolism were studied in three groups of isolated perfused rabbit hearts (control, ischemic, and reperfused), whereas Ca2+ uptake by the sarcoplasmic reticulum (SR) was measured in homogenates obtained from them. The hearts were Langendorff-perfused under constant pressure with Krebs-Henseleit solution at 37 degrees C. Global normothermic ischemia was produced by closing the perfusion line. In the reperfused group, after 15 minutes of ischemia, Krebs-Henseleit solution was perfused for 10 minutes. Developed left ventricular pressure (control, 104 +/- 6.3 mm Hg) and left ventricular dP/dt (2,063 +/- 256.6 mm Hg.sec-1) were significantly decreased in reperfused hearts (left ventricular pressure, 78 +/- 5.9 mm Hg; left ventricular dP/dt, 1,339 +/- 216.3 mm Hg.sec-1). Myocardial ATP content (control, 13.6 +/- 0.98 mumol/g dry wt) decreased during ischemia (4.5 +/- 1.23 mumol/g) but was restored to control level on reperfusion (11.8 +/- 0.68 mumol/g). Maximum velocity of Ca2+ uptake by the SR (Vmax) (control, 49.3 +/- 2.54 nmol.min-1 x mg-1) was significantly depressed by ischemia (36.3 +/- 1.94 nmol.min-1 x mg-1) but was restored to the control value after a 10-minute reperfusion (45.3 +/- 0.79 nmol.min-1 x mg-1). Apparent dissociation constant KCa and the Hill coefficient for Ca2+ uptake were not different between control, ischemia, and reperfusion. To test for the possible role of the SR Ca(2+)-release channel in the effect of ischemia and reperfusion, we measured Ca2+ uptake after incubation of homogenates with 610 microM ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular sodium accumulation during ischemia as the substrate for reperfusion injury.

To elucidate the role of intracellular Na+ kinetics during ischemia and reperfusion in postischemic contractile dysfunction, intracellular Na+ concentration ([Na+]i) was measured in isolated perfused rat hearts using 23Na nuclear magnetic resonance spectroscopy. The extension of the ischemic period from 9 minutes to 15, 21, and 27 minutes (at 37 degrees C) increased [Na+]i at the end of ischemia from 270.0+/-10.4% of preischemic level (mean+/-SE, n=5) to 348.4+/-12.0% (n=5), 491.0+/-34.0% (n=7), and 505.3+/-12.1% (n=5), respectively, whereas the recovery of developed pressure worsened with the prolongation of the ischemic period (95.1+/-4.2%, 84.3+/-1. 2%, 52.8+/-13.7%, and 16.9+/-6.4% of preischemic level). The kinetics of [Na+]i recovery during reperfusion was analyzed by the fitting of a monoexponential function. When the hearts were reperfused with low-[Ca]o (0.15 mmol/L) solution, the time constants of the recovery (tau) after 15-minute (8.07+/-0.85 minutes, n=5) and 21-minute ischemia (6.44+/-0.90, n=5) were significantly extended, with better functional recovery (98.5+/-1.4% for 15-minute [P<0.05]; 98.0+/-1.0% for 21-minute [P<0.05]) compared with standard reperfusion ([Ca]o=2.0 mmol/L, tau=3.58+/-0.28 minutes for 15-minute [P<0.0001]; tau=3.02+/-0.20 for 21-minute [P<0.0001]). A selective inhibitor of Na+/Ca2+ exchanger also decelerated the [Na+]i recovery, which suggests that the recovery reflects the Na+/Ca2+ exchange activity. In contrast, high-[Ca]o reperfusion (5 mmol/L) accelerated the [Na+]i recovery after 9-minute ischemia (tau=2.48+/-0.11 minute, n=5 [P<0.0001]) and 15-minute ischemia (tau=2.10+/-0.07, n=6 [P<0. 05]), but functional recovery deteriorated only in the hearts with 15-minute ischemia (29.8+/-9.4% [P<0.05]). [Na+]i recovery after 27-minute ischemia was incomplete and decelerated by low-[Ca]o reperfusion, with limited improvement of functional recovery (42. 5+/-7.9%, n=5 [P<0.05]). These results indicate that intracellular Na+ accumulation during ischemia is the substrate for reperfusion injury and that the [Na+]i kinetics during reperfusion, which is coupled with Ca2+ influx, also determines the degree of injury.     

Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia

Alterations in energy substrate utilization during reperfusion of ischemic hearts can influence the functional recovery of the myocardium. Energy substrate preference by the reperfused myocardium, however, has received limited attention. Therefore, we measured oxidation rates of glucose and palmitate during reperfusion of ischemic hearts. Isolated working rat hearts were perfused with 1.2 mM palmitate and 11 mM [14C]glucose, 1.2 mM [14C]palmitate and 11 mM glucose, or 11 mM [14C]glucose alone, at an 11.5 mm Hg preload and 80 mm Hg afterload. Hearts were subjected to 60-minute aerobic perfusion or 25-minute global ischemia followed by 60-minute aerobic reperfusion. Steady-state oxidative rates of glucose or palmitate were determined by measuring 14CO2 production. In hearts perfused with glucose alone, oxidative rates during reperfusion were not significantly different than nonischemic hearts (1,008 +/- 335 vs. 1,372 +/- 117 nmol [14C]glucose oxidized/min/g dry wt, respectively). In the presence of palmitate, glucose oxidation was markedly reduced in reperfused and nonischemic hearts (81 +/- 11 and 101 +/- 15 nmol [14C]glucose oxidized/min/g dry wt, respectively). Palmitate oxidation rates were not significantly different in reperfused compared with nonischemic hearts (369 +/- 55 and 455 +/- 50 nmol [14C]palmitate oxidized/min/g dry wt, respectively). [14C]Palmitate was incorporated into myocardial triglycerides to a greater extent in reperfused ischemic hearts than in nonischemic hearts (26.0 and 13.8 mumol/g dry wt, respectively). Under the perfusion conditions used, palmitate provided over 90% of the ATP produced from exogenous substrates. Addition of the carnitine palmitoyltransferase I inhibitor, ethyl 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate (Etomoxir, 10(-6) M), during reperfusion stimulated glucose oxidation and improved mechanical recovery of ischemic hearts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heat-shock response is associated with enhanced postischemic ventricular recovery

In cells, hyperthermia induces synthesis of heat-shock proteins and the acquisition of thermotolerance. Thermotolerant cells are resistant to subsequent oxidative stress. In this study, heat-shocked hearts were examined for evidence of protection during ischemia and reperfusion. Rats were exposed to 15 minutes of 42 degrees C hyperthermia. Twenty-four hours later their hearts were isolated and perfused and the contractility examined during and after ischemic perfusion. No protection was observed during ischemic perfusion. However, upon reperfusion heat-shocked hearts had recovery of contractility within 5 minutes of reperfusion, while control hearts showed no contractility at this time. Throughout 30 minutes of reperfusion heat-shocked hearts had significantly improved recovery of contractile force, rate of contraction and rate of relaxation. Creatine kinase release, associated with reperfusion injury, was significantly reduced from a high of 386.8 +/- 78.9 mU/min/g heart wt for controls to 123.7 +/- 82.9 mU/min/g heart wt for heat-shocked hearts at 5 minutes of reperfusion. Following 30 minutes of reperfusion, ultrastructural examination revealed less damage of mitochondrial membranes in the heat-shocked hearts. Further biochemical investigations revealed that the antioxidative enzyme, catalase, was significantly increased to 137 +/- 12.7 U/mg protein in the heat-shocked hearts while the control value was 64.8 +/- 8.3 U/mg protein. Hyperthermic treatment, which induces the heat-shock response, may be therapeutic for salvaging ischemic myocardium during reperfusion, through a mechanism involving increased levels of myocardial catalase.     

Acadesine and myocardial protection. Studies of time of administration and dose-response relations in the rat.

BACKGROUND. Although there are many factors that might contribute to tissue injury during ischemia and reperfusion, the loss of adenine nucleotides has long been considered to be of importance. This has led to the study of interventions designed to limit the loss of nucleotides or to enhance the rate of nucleotide resynthesis during reperfusion. Alternatively, the breakdown of adenosine triphosphate to adenosine might represent a protective response of the ischemic heart because adenosine is considered an anti-injury autocoid. Augmentation of endogenous adenosine levels might be beneficial. For these reasons, the protective properties of acadesine (AICAr: 5-amino-4-imidazole carboxamide riboside) were assessed in a rat model of myocardial ischemia and reperfusion. METHODS AND RESULTS. The protective properties of acadesine were studied in the isolated, perfused rat heart subjected to global hypothermic (20 degrees C) ischemia and reperfusion. When acadesine was given as an in vivo pretreatment (100 mg/kg i.v. 15 minutes before study) followed by being administered as an additive (20 mumol/l) to the St. Thomas' Hospital cardioplegic solution (single dose) and then as an additive (20 mumol/l) to the initial reperfusion (15 minutes) solution, the recovery of aortic flow after 2.5 hours of ischemia was improved from its control value of 16.5 +/- 3.9 ml/min to 28.9 +/- 4.1 ml/min (n = 8 per group; p less than 0.05). Similar protection was seen with other indexes of cardiac function. Analysis of hearts obtained at the end of 2.5 hours of ischemia and 35 minutes of reperfusion revealed no significant differences in metabolite content between control and drug-treated hearts with the exception of inosine monophosphate, which was increased from its drug-free control value of 0.10 +/- 0.01 mumol/g dry wt to 0.86 +/- 0.06 mumol/g dry wt (p less than 0.05). In further studies (n = 8 per group), with multidose (every 30 minutes) cardioplegia and extended periods (6 hours) of hypothermic ischemia, acadesine consistently led to higher mean recoveries of function and lower levels of creatine kinase leakage. Again, the only significant metabolic effect was an increase in tissue inosine monophosphate content. In studies (n = 12 per group) to determine whether acadesine was acting before, during, or after ischemia, the drug was given 1) only as pretreatment (100 mg/kg i.v.), 2) only during single-dose cardioplegia (20 mumol/l), or 3) only during reperfusion (20 mumol/l). Significant protection was observed in the first two groups (recovery of aortic flow increased from 10.6 +/- 2.6 ml/min in the acadesine-free control to 22.6 +/- 2.8 and 23.6 +/- 3.1 ml/min, respectively; p less than 0.05). No significant protection was observed when acadesine was given only during reperfusion. In dose-response studies, acadesine (0, 5, 20, 50, 200, and 1,000 mumol/l; n = 12 per group) was given only as a cardioplegic additive; the postischemic recoveries of aortic flow were 15.4 +/- 2.8, 16.9 +/- 3.6, 29.5 +/- 3.8, 27.4 +/- 3.8, 26.7 +/- 4.2, and 27.1 +/- 2.7 ml/min, respectively. CONCLUSIONS. Acadesine improves the ability of the heart to recover from ischemia and reperfusion when administered before ischemia or with cardioplegia. The mechanism underlying the protection remains to be resolved.     

Recruitment of an inotropic reserve in moderately ischemic myocardium at the expense of metabolic recovery. A model of short-term hibernation.

The regional, functional as well as metabolic consequences of inotropic stimulation on myocardium subjected to prolonged moderate ischemia were investigated. In 35 enflurane-anesthetized swine the left anterior descending coronary artery was cannulated and perfused at constant flow. The vein paralleling the left anterior descending coronary artery was cannulated for measurement of lactate and oxygen content. Transmural biopsies from the anterior myocardium were taken for the measurement of ATP, creatine phosphate, and glycogen. After control measurements, flow was adjusted to reduce regional contractile function (expressed as a work index, determined by sonomicrometry) by approximately 50%. After either 5, 25, 40, or 85 minutes of moderate ischemia, dobutamine was infused for 5 minutes into the ischemic region. In a separate group of five swine also subjected to 85 minutes of ischemia followed by infusion of dobutamine and 2 hours of reperfusion, triphenyltetrazolium chloride staining and light microscopy were used to identify infarcted tissue. Moderate ischemia (regional myocardial blood flow, 0.21 +/- 0.07 ml.min-1.g-1, determined by radiolabeled microspheres) was associated with a reduction of creatine phosphate after 5 minutes (from 9.35 +/- 2.54 to 6.43 +/- 1.06 mumol/g wet wt, p less than 0.05) and a further reduction after 25 minutes (3.18 +/- 0.69 mumol/g wet wt, p less than 0.05). Thereafter, creatine phosphate recovered despite continued ischemia (after 40 minutes, 4.95 +/- 1.37 mumol/g wet wt; after 85 minutes, 5.78 +/- 2.27 mumol/g wet wt). Lactate consumption during control conditions was reversed to production after 5 minutes of ischemia, which moderated during more prolonged ischemia. Without changing regional myocardial blood flow, infusion of dobutamine increased the work index significantly at any time point but also caused worsening of metabolic markers of ischemia. Nevertheless, even after 85 minutes of ischemia followed by the infusion of dobutamine and 2 hours of reperfusion, there was no evidence of necrosis. This experimental model provides a means of characterizing the mechanisms of short-term hibernation.     

Brief ischemia-reperfusion performed after prolonged ischemia (ischemic postconditioning) can terminate reperfusion arrhythmias with no reduction of cardiac function in rats

OBJECTIVE: Recently, it has been reported that ischemic postconditioning, a brief episode of ischemia-reperfusion performed after prolonged ischemia, can reduce ischemic myocardial injury. However, the effects of ischemic postconditioning on ischemia/reperfusion injury remain unclear. We investigated the effects of brief ischemia-reperfusion before (ischemic preconditioning) and after (ischemic postconditioning) prolonged ischemia on myocardial ischemia/reperfusion injury, especially reperfusion arrhythmias. METHODS: Adult male Sprague-Dawley rats weighing about 400-500 g were used. The isolated heart was perfused using a working heart method (Krebs-Henseleit bicarbonate buffer). In the control group, after stabilization, diastolic global ischemia for 15 minutes was produced by a one-way ball valve with electrical pacing (330 bpm, 2.0 V). After ischemia, the heart was reperfused for 20 minutes. In the preconditioning and postconditioning groups, 5-minute global ischemia was produced before and after ischemia for 15 minutes with a 1 minute interval. An electrocardiogram was performed and left ventricular pressure (LVP, +dP/dt, -dP/dt) and CK activity in coronary effluent were measured during the protocol. RESULTS: Ischemic preconditioning did not affect the incidence or duration of reperfusion ventricular arrhythmias. Ischemic postconditioning could terminate reperfusion ventricular arrhythmias completely and reduced the duration of reperfusion ventricular arrhythmias significantly (P < 0.01). Furthermore, the recovery ratio of +dP/dt at 20 minutes after initial reperfusion was significantly higher in the postconditioning group than in the other groups. CONCLUSION: These results suggest that ischemic postconditioning can terminate reperfusion arrhythmias with no reduction of cardiac function, and may be useful for correcting stunned myocardium.     

Copper loading of hearts increases postischemic reperfusion injury

We studied the role of copper as a potential mediator of postischemic reperfusion injury in the isolated, perfused rat heart. Hearts were equilibrated with Krebs-Henseleit buffer for 10 minutes and then loaded with copper by way of perfusion with buffer containing 20 microM copper(II)-bis-histidial for 30 minutes. Control hearts were perfused with Krebs-Henseleit buffer alone during the loading period. Hearts than were washed with buffer for 10 minutes and subjected to 20 minutes of normothermic global ischemia followed by 30 minutes of reperfusion. Atomic absorption spectroscopy revealed a 67% increase in total copper content in loaded hearts by the end of the wash. By the end of the 30-minute period of reperfusion, control hearts demonstrated a 50-60% recovery of myocardial function as determined by peak systolic pressure, contractility, and heart rate. In contrast, copper-loaded hearts exhibited virtually no functional recovery within the 30-minute time period. Using salicylate as a probe, we determined that peak and duration of .OH formation appears to be increased in copper-loaded hearts during reperfusion. Furthermore, efflux of lactic dehydrogenase was significantly increased in copper-loaded hearts. Our results clearly demonstrate that increasing cardiac content of copper results in enhanced postischemic reperfusion injury associated with increased formation of .OH, thus suggesting an important catalytic role for this transition metal.     

Combination therapy for maximal myocardial infarct size reduction

The aim of this study was to test whether myocardial infarct size reduction would be optimized by combining three known effective therapies: cariporide, regional hypothermia, and ischemic preconditioning (CHIP). Before coronary artery occlusion (CAO), treated rabbits (CHIP, n = 7) received cariporide (0.3 mg/kg), ischemic preconditioning (7 minutes ischemia and 5 minutes reperfusion), then 20 minutes of mild regional hypothermia (34 degrees C). Control rabbits (n = 7) received saline and a 34-minute waiting period. All received 30 minutes of CAO and reperfusion. In another study, rabbits (n = 8 in each group) received 90 minutes of CAO. In the 30-minute protocol, the authors found that hearts in both groups were equally ischemic during CAO. Mean ischemic risk zones were similar in both groups; however, in CHIP hearts, infarct size was 4 +/- 1% of risk zone, a reduction of 91% compared with control rabbits (44 +/- 7% of the risk zone, P = 0.001). In the 90-minute protocol, risk zone size was similar in both groups, but infarct size in control hearts was 76 +/- 3% of the risk zone compared with 34 +/- 7% in CHIP treated hearts (P = 0.0003). In summary, the combined treatment provided extraordinary protection. Infarct comprised only 4% of the risk region after 30-minute ischemia-a far greater reduction than was previously observed in the same laboratory using any single intervention. After 90 minutes of ischemia, infarct was 55% lower in CHIP hearts, suggesting that this therapeutic approach dramatically reduces ischemia/reperfusion cell death, even during long occlusions.     

Coronary cyclic flow variations "precondition" ischemic myocardium.

BACKGROUND. Repeated brief episodes of myocardial ischemia performed by mechanical clamping of a coronary artery "precondition" the heart and reduce infarct size after a subsequent sustained ischemia. It is not known, however, whether spontaneous episodes of transient ischemia caused by formation of platelet thrombi, which may occur in unstable angina, have a similar cardioprotective effect. METHODS AND RESULTS. Therefore, our objective was to determine whether brief spontaneous thrombotic episodes of ischemia/reperfusion could limit infarct size and preserve contractile function following 60 minutes (protocol 1) or 90 minutes (protocol 2) of sustained ischemia and 4-4.5 hours of reperfusion in the canine model. Before the sustained coronary occlusion, dogs underwent a 30-minute "treatment" period consisting of: no intervention (control group), four repeated episodes of 3-minute mechanical occlusion plus 5-minute reperfusion (preconditioned group), or coronary artery stenosis and endothelial injury, resulting in a mean of four spontaneous episodes of cyclic flow variations (CFV group) caused by formation and dislodgment of platelet thrombi. In protocol 1 (60-minute sustained ischemia plus 4.5-hour reperfusion), infarct size was significantly smaller in both the preconditioned and CFV groups compared with controls (3.5 +/- 1.4%,* 3.4 +/- 2.1%,* and 9.9 +/- 2.7% of the myocardium at risk, respectively; *p less than 0.05 versus control). In contrast, neither preconditioning nor CFV preserved contractile function: Segment shortening during sustained occlusion was equally depressed at -15% to -20% of baseline values among the three groups and equally stunned at +12% to +18% of baseline during the 4.5 hours of reflow. In protocol 2 (90-minute sustained ischemia plus 4-hour reperfusion), only CFV continued to exert a cardioprotective effect: Infarct size averaged 15.0 +/- 4.1%, 7.4 +/- 2.5%,* and 16.5 +/- 4.4% of the region at risk in the preconditioned, CFV, and control groups, respectively (*p less than 0.05 versus control). Contractile function, however, was similar among all three groups both during 90 minutes of sustained occlusion and throughout 4 hours of reperfusion. CONCLUSIONS. We therefore conclude that repeated coronary thrombus formation preconditions the ischemic myocardium: In fact, in contrast to mechanical preconditioning, cardioprotection provided by CFV persisted following 90 minutes of sustained coronary occlusion. However, preconditioning by thrombotic or mechanical occlusion neither preserved myocardial contractile function during sustained coronary occlusion nor prevented stunning after reperfusion. These data raise the possibility that clinical episodes of unstable angina prior to acute myocardial infarction may precondition the ischemic myocardium.     

Action of aprotinin in myocardial ischemia - an investigation using a plasma-free model.

BACKGROUND: The protease inhibitor aprotinin has been reported to have an anti-ischemic effect on left-ventricular myocardium in patients undergoing cardiopulmonary bypass operation. To examine the anti-ischemic properties beside its antifibrinolytic and inhibitory action on the kallikrein-bradykinin system, we investigated this substance in buffer-perfused rat hearts. METHODS: 24 isolated isovolumically contracting rat hearts received a 10-minute infusion of either 10000 units aprotinin or pure saline followed by 30 minutes of no-flow global ischemia and 45 minutes of reperfusion. Hemodynamics, high-energy phosphates, and troponin T as molecular marker of cardiac injury were studied. RESULTS: During 15 minutes of reperfusion steady state function was identical in both groups, with a recovery of the developed left-ventricular pressure to 81.9+/-1.5% after protease inhibition and 83.0+/-2.6% in the controls. Coronary flow, myocardial oxygen consumption, and contractile reserve after maximum Ca++ stimulation were also identical. High-energy phosphates were comparably reduced in both groups (adenine nucleotides: 3.1+/-0.3 micromol/g ww after aprotinin vs. controls 2.7+/-0.4 micromol/g ww and creatine phosphate: 6.5+/-0.9 micromol/g ww vs. controls 4.7+/-1.1 micromol/g ww). However, release of the cardiac specific marker troponin T was lower after ischemia at several measurements (p<0.05). The total release of troponin T was 44+/-10 ng in the aprotinin treated hearts vs. 90+/-17 ng in the postischemic control hearts (p<0.05). CONCLUSIONS: The findings demonstrate that aprotinin in a moderate dose is effective in reducing postischemic troponin release in a non-blood perfused system. Measurement of myocardial high-energy phosphates after aprotinin use was performed for the first time and indicates that not a reduction in severity of direct myocardial ischemic intensity but a beneficial action on processes causing release of troponin is the mode of action of this effect.     

Intermittent ischemia produces a cumulative depletion of mitochondrial adenine nucleotides in the isolated perfused rat heart

The purpose of the present study was to determine if repetitive myocardial ischemia would result in the cumulative loss of mitochondrial adenine nucleotides. Isolated perfused rat hearts were subjected to continuous or intermittent ischemia. A single 5-minute period of continuous ischemia did not result in a significant decrease in the mitochondrial adenine nucleotide pool; a single 10-minute period of ischemia resulted in a decrease of approximately 17%. Next, the adenine nucleotide content of mitochondria from preischemic and 30-minute continuous ischemic hearts was compared with two groups of hearts undergoing intermittent ischemia (both groups receiving a total of 30 minutes of ischemia). One group received three 10-minute episodes of ischemia interrupted by 5-minute periods of reperfusion (3 x 10-minute intermittent ischemia); the other intermittent ischemic group received six 5-minute episodes of ischemia interrupted by 5-minute periods of perfusion (6 x 5-minute intermittent ischemia). The mitochondrial adenine nucleotide content (expressed as nanomoles per nanomole cytochrome a) for the preischemic and 30-minute continuous ischemic hearts was 14.7 +/- 0.6 and 8.0 +/- 0.4, respectively. The mitochondrial adenine nucleotide content of the 3 x 10-minute intermittent ischemia group (8.5 +/- 0.5) was not significantly different from the 30-minute continuous ischemic group. The mitochondrial adenine nucleotide content of the 6 x 5-minute intermittent ischemia group (11.0 +/- 0.6) was significantly larger than that of the 30-minute continuous and the 3 x 10-minute intermittent ischemia groups (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of preconditioning ischemia on reperfusion arrhythmias after coronary artery occlusion and reperfusion in the rat

Severe arrhythmias occur predictably on reperfusion after 5 minutes of coronary occlusion in the rat. There is little data available on whether ischemic preconditioning (PC) of hearts can reduce the incidence of such arrhythmias. The effect of PC (three cycles of 2 minutes of coronary occlusion and 5 minutes of reperfusion) on development of arrhythmias after a subsequent 5-minute coronary artery occlusion and reperfusion was studied. Rats (n = 16 each group) underwent 5-minute occlusion and reperfusion alone or preceded by PC; arrhythmias were monitored during ischemia and for 10 minutes of reperfusion, and biopsies were taken for creatine phosphate and adenosine triphosphate in ischemic and nonischemic zones of the left ventricle. PC reduced the incidence of ventricular tachycardia (VT) during occlusion (81% control versus 13% PC, p less than 0.001). On subsequent reperfusion, ventricular fibrillation (VF) developed in zero PC animals versus 13 (81%) of controls (p less than 0.001), and irreversible VF in zero of PC versus seven (44%) of controls (p = 0.007). VT occurred in four (25%) of PC versus all (100%) of controls (p less than 0.001). PC reduced mean duration of VT plus VF from 320 +/- 54 to 5 +/- 1 seconds (p less than 0.001) and delayed arrhythmia onset from 8 +/- 2 to 85 +/- 35 seconds after reperfusion. There was no difference in creatine phosphate levels in the ischemic zone at the end of reperfusion in PC animals compared with controls without irreversible VF (16.2 +/- 4.1 versus 15.5 +/- 3.9 nmol/mg protein, p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)     

The significance of underlying coronary stenosis for recovery of myocardial function after transient ischemia in the dog

The rate of recovery of myocardial function after transient coronary occlusion (CO) has been considered to depend on the duration and frequency of CO. However, underlying coronary stenosis has not been previously demonstrated to be a determinant of the rate of myocardial functional recovery. Thus, 12 open-chest dogs were studied to examine the influence of critical coronary stenosis (CCS) on functional recovery after transient CO. Regional functional recovery following 2-minute CO was examined under two different conditions in eight dogs: patent coronary artery stenosis and fixed CSS that exhausted coronary reserve but did not cause a deficit in resting coronary flow or regional function. Following reperfusion with the coronary artery patent, regional function in the ischemic zone was fully recovered (100 +/- 18.0% of pre-CO value) at 30 seconds and was significantly increased (postischemic hypercontraction) compared to pre-CO value at 1 and at 2 minutes after reperfusion. Following CO and reperfusion in the setting of CCS, regional functional recovery was delayed and regional function remained depressed until 2 minutes after reperfusion. No cumulative effect on functional recovery following repeated 2-minute CO was demonstrated in a control group of four dogs. We conclude that coronary artery patency is a determinant of the rate of myocardial function recovery after a transient ischemic episode, and postischemic hypercontractility was suppressed by the underlying CCS.     

Effect of verapamil on postischemic "stunned" myocardium: importance of the timing of treatment

Timely administration of verapamil has been shown to reduce indexes of ischemic injury in experimental models of prolonged coronary artery occlusion, yet its effect on contractile function of reversibly injured (that is, "stunned") myocardium remains unknown. The objective of the present study was to determine whether verapamil--administered either 30 min before coronary artery occlusion, at the time of reperfusion or 30 min after reperfusion--could attenuate the regional contractile dysfunction and alterations in high energy phosphate metabolism produced by 15 min of transient coronary artery occlusion in anesthetized, open chest dogs. All treatment groups exhibited passive systolic bulging during occlusion. In the control dogs receiving saline solution, segment shortening in the previously ischemic tissue averaged only 31 +/- 8% of normal baseline values after 3 h of reperfusion. In addition, endocardial adenosine triphosphate (ATP) stores were depleted by -8.7 +/- 0.8 nmol/mg cardiac protein to 26.5 +/- 1.1 nmol/mg protein, and endocardial creatine phosphate content increased by 9.6 +/- 4.3 nmol/mg cardiac protein over normal values. Pretreatment with verapamil essentially ablated the phenomenon of postischemic stunning: segment shortening was restored to 115 +/- 8% of normal after 3 h of reflow (p less than 0.01 versus control), endocardial ATP stores were partially preserved (30.6 +/- 1.2 nmol/mg protein; p less than 0.05 versus control) and creatine phosphate overshoot was blunted (endocardial creatine phosphate content decreased by -5.6 +/- 2.9 nmol/mg protein; p less than 0.05 versus control). Verapamil administered at or after reperfusion also attenuated postischemic contractile dysfunction: segment shortening for both groups recovered to 65 +/- 9% of baseline at 3 h after reperfusion (p less than 0.05 versus control). Verapamil given at or after reperfusion had no beneficial effect, however, on high energy phosphate stores. Thus, even when treatment was "delayed," that is, initiated at or after reperfusion, administration of verapamil significantly increased contractile function of the postischemic stunned myocardium.     

Regional differences in postischemic recovery in the stunned canine myocardium

To determine if differences exist in the degree of ischemic damage and in postischemic recovery when different coronary arteries are occluded and reperfused, 40 barbital-anesthetized dogs were subjected to brief 15-minute periods of coronary artery occlusion followed by 3 hours of reperfusion ("stunned" myocardium) of the left anterior descending (LAD) or the left circumflex (LCX) coronary arteries. Myocardial segment shortening (%SS) in the subendocardium of nonischemic and ischemic reperfused areas was measured by sonomicrometry, and regional myocardial blood flow was measured by radioactive microspheres. Transmural tissue biopsies were taken at the end of reperfusion for the measurement of adenine nucleotides and total tissue water content. Arterial and local coronary venous blood samples were collected during preocclusion, during occlusion, and at 30 and 180 minutes of reperfusion for determination of blood oxygen content and oxygen consumption in the ischemic area. During occlusion, subendocardial blood flow (LAD flow = 0.11 +/- 0.02; LCX flow = 0.15 +/- 0.04 ml/min/gm), myocardial oxygen consumption (LAD = 2.4 +/- 0.7; LCX = 2.7 +/- 0.7 ml/min/100 gm), and areas of the left ventricle at risk (LAD = 27.4 +/- 2.3%; LCX = 32.4 +/- 2.4) were similar in both groups, thus indicating equivalent degrees of ischemia. There were no differences between groups in hemodynamics throughout the experiment or in the loss of myocardial high-energy phosphates or increase in total tissue water in the ischemic reperfused area at 3 hours of reperfusion. There was a significantly greater loss (p less than 0.05) of systolic wall function during LAD versus LCX occlusion and a greater recovery of segment function from 5 minutes throughout 1 hour of reperfusion after LCX occlusion (p less than 0.05), with no difference in %SS at 2 and 3 hours following reperfusion. Thus, although similar changes occurred in blood flow, metabolite parameters, tissue edema, wall function, and overall hemodynamics when either the LAD or LCX perfusion territories were occluded and reperfused, the loss of systolic wall function and recovery of segment shortening were more variable after regional stunning of the LCX perfusion bed. These data suggest that evaluation of pharmacologic or surgical interventions to improve postischemic functional recovery may be more reliably performed when the LAD coronary artery is the vessel occluded.     

Effects of inosine on glycolysis and contracture during myocardial ischemia

The effects of inosine (INO) on substrate metabolism and rigor formation in ischemic myocardium were examined in isolated rabbit hearts. Metabolite content was assessed in tissue extracts by chemical analysis and in the whole heart by 13C and 31P nuclear magnetic resonance spectroscopy. In ischemic hearts metabolizing either [3-13C]pyruvate or [1-13C]glucose, 1 mM INO increased both total and 13C-labeled alanine content; lactate content was unaffected. At 3 minutes of ischemia, tissue alanine was 1.81 +/- 0.11 microM/g wet wt (mean +/- SEM) in hearts perfused with pyruvate+INO versus 1.23 +/- 0.15 microM/g wet wt in hearts perfused with pyruvate alone (p less than 0.05). INO reduced tissue glycogen during ischemia in pyruvate-perfused hearts. Tissue alanine content in ischemic hearts that were supplied glucose+INO (1.29 +/- 0.13 microM/g wet wt) was greater than in ischemic hearts supplied glucose alone (0.65 +/- 0.14 microM/g wet wt). Alanine was found to originate from pyruvate and was a glycolytic end product in glucose-perfused hearts. INO raised the [3-13C]alanine/[3-13C]lactate ratio in ischemic, intact hearts (glucose = 0.24 +/- 0.07 versus glucose+INO = 0.60 +/- 0.09; pyruvate = 0.49 +/- 0.08 versus pyruvate+INO = 0.89 +/- 0.08). At 7 minutes of ischemia, ATP content fell to 70 +/- 3% with glucose+INO versus 58 +/- 5% with glucose alone. Rigor (stone heart) was delayed from 14.7 +/- 1.3 to 23.2 +/- 1.6 minutes with INO. INO did not change ATP content in ischemic hearts that were supplied pyruvate but delayed rigor (pyruvate = 9.9 +/- 1.2 minutes; pyruvate+INO = 15.6 +/- 1.0 minutes), possibly at the expense of glycogen. Supplemental glucose improved the effectiveness of INO with pyruvate to preserve ATP (pyruvate+glucose = 42 +/- 6%; pyruvate+glucose+INO = 72 +/- 6%) and further delayed rigor (pyruvate+glucose = 13.3 +/- 1.5 minutes; pyruvate+glucose+INO = 20.3 +/- 1.8 minutes). Glucose metabolism supported improved energetic and contractile states in ischemic hearts treated with INO. Thus, cardioprotection of the ischemic heart by INO was associated with preservation of functional integrity and improved energy production due to increased glycolytic activity. Activation of glycolysis in the presence of INO was accommodated by augmented alanine production without the additional accumulation of lactate.     

F(ab')2 fragments of anti-Mo1 (904) monoclonal antibodies do not prevent myocardial stunning

To determine if inhibition of leukocyte adhesion and aggregation could improve postischemic ventricular dysfunction ("stunning"), a monoclonal antibody (904) that binds to the adhesion-promoting Mo1 glycoprotein on the cell surface of leukocytes was administered intravenously (0.5 mg/kg) to open-chest dogs before a 15-minute coronary occlusion. Ultrasonic crystals placed in ischemic and control myocardium were used to measure systolic wall thickening during a 15-minute occlusion of the left anterior descending artery and for 3 hours after reperfusion. Myocardial blood flow was measured with tracer-labeled microspheres before occlusion, after 10 minutes of occlusion, 3 minutes of reperfusion, and at 1 and 3 hours after reperfusion. Six animals receiving anti-Mo1 antibody had antibody excess demonstrated with immunofluorescence techniques at 5 minutes and 3 hours of reperfusion; this finding indicated saturation of binding sites. Five animals served as controls and received an antibody (murine immunoglobulin G) that does not influence neutrophils. The two groups did not differ hemodynamically during ischemia and reperfusion. Risk areas and myocardial blood flow were also not significantly different between the two groups. The main parameter used to define regional myocardial stunning, percentage systolic wall thickening in the ischemic/reperfused area, did not differ significantly between the two groups. Specimens from nonischemic myocardium were compared with ischemic specimens for myeloperoxidase content. There were no significant differences within or between groups. These data indicate that the anti-Mo1 monoclonal antibody (904) is not effective in improving the profound myocardial dysfunction that persists for 3 hours of reperfusion after 15 minutes of ischemia.     

Alleviation of myocardial stunning by leukocyte and platelet depletion

Neutrophils accumulate in myocardium rendered ischemic and reperfused. Activated neutrophils release mediators such as metabolites of oxygen that can compromise myocellular integrity and provoke cardiac dysfunction. Although it is established that leukopenia reduces infarct size, the role of leukocytes and the source of free radicals in postischemic contractile dysfunction is unresolved. A carotid left anterior descending coronary-artery extracorporeal circuit without (n = 8) or with a Leukopak filter (n = 6) to deplete the leukocytes and platelets from blood entering the left anterior descending artery was established in the anesthetized, open-chest dog 30 minutes before ischemia. Subendocardial segmental function was monitored by sonomicrometry, and ischemia was produced by stopping flow for 15 minutes followed by 3 hours of reperfusion. Depleting leukocytes by 90 +/- 3.2% and platelets by 100% improved segmental function (from 30.5 +/- 7% to 74.1 +/- 12.7% for control versus leukocyte-depleted dogs, respectively) at 15 minutes of reperfusion. In the leukopenic group, however, there was a progressive decline in contractility to 32.5 +/- 13.8% by 3 hours of reperfusion that was associated with a return of leukocytes and, to a lesser extent, a return of platelets in the extracorporeal blood to 70.2 +/- 21.9% and 15.5 +/- 4.3% of systemic values, respectively. Removal of leukocytes and platelets from blood perfusing the coronary vascular bed only at reperfusion improved contractile function to 67.7 +/- 6.9% at 15 minutes and 54.7 +/- 12.1% at 3 hours (n = 6). Scanning electron microscopy revealed adherent leukocytes in the epicardial coronary arteries of control animals after 3 hours of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

The independent effects of oxygen radical scavengers on canine infarct size. Reduction by superoxide dismutase but not catalase

Previous studies demonstrated a significant reduction of ultimate infarct size in the canine heart by the combined administration of superoxide dismutase plus catalase. This study was performed to assess the independent effects of each enzyme on ultimate infarct size due to ischemia/reperfusion. Dogs received 2-hour infusions of superoxide dismutase, catalase, or albumin (controls) via the left atrium beginning 15 minutes before and ending 15 minutes after a 90-minute occlusion of the left circumflex coronary artery. The dogs were killed 6 hours after reperfusion. After histochemical staining, infarct and risk area masses were calculated by gravimetric and planimetric analysis. Infarct size expressed as a percentage of the area at risk was: superoxide dismutase, 19 +/- 5; catalase, 30 +/- 5; and controls, 40 +/- 3. Infarct size in the superoxide dismutase group, but not the catalase group, was significantly less than in controls (P less than 0.05). No significant differences in hemodynamics or area at risk were observed that could explain the differences in infarct size. The results indicate that superoxide dismutase alone protects reperfused ischemic myocardium as well as does the combination of superoxide dismutase and catalase. The beneficial effect of superoxide dismutase and insignificant effect of catalase suggest that tissue damage during ischemia and reperfusion may be mediated largely by superoxide anion but not by hydrogen peroxide.