Preservation of metabolic reserves and function after storage of myocytes in hypothermic UW solution.

Isolated rat myocytes cold stored anaerobically up to 24 h in University of Wisconsin solution lost 95% of their ATP and 100% of their glycogen. They underwent contracture when rewarmed in a Krebs-Henseleit (KH) medium that contained Ca unless Ca addition was delayed. In the latter case, cell function, measured by stimulation-induced cell shortening, was surprisingly well retained. Aerobically stored cells were resistant to Ca on rewarming, although 96% of glycogen was still lost, along with 46% of ATP. Cells that were incubated for 48 h aerobically with the substrates glucose and pyruvate at pH 6.2 retained 77% of their ATP and 59% of their glycogen, with good cell morphology. At pH 6.2, the demand for ATP was only 55% of that at pH 7.4. However, after rewarming, these cells functioned no better than anaerobically stored cells, although their inotropic response to isoproterenol was improved. We conclude that 1) aerobic conditions with substrates at low pH preserve myocyte metabolic reserves well for 48 h, partly by reducing the demand for ATP; 2) rewarming conditions are critical for anaerobically stored cells with metabolic stores that are severely depleted; and 3) unloaded cell function is surprisingly insensitive to a period of severe metabolic deprivation.

Effects of adenosine and acadesine on interstitial nucleosides and myocardial stunning in the pig.

5-Amino-4-imidazolecarboxamide riboside (AICAr) or acadesine has been proposed to exert cardioprotection by enhancing adenosine production in ischemic myocardium. However, there are conflicting reports on acadesine's effects in ischemic myocardium and few studies in which myocardial adenosine levels have been measured. The purpose of this study was to determine whether acadesine increases interstitial fluid adenosine levels and attenuates myocardial stunning or potentiates the effects of adenosine in the intact pig. In pentobarbital-anesthetized pigs, myocardial stunning was induced by 10 min left anterior descending coronary artery occlusion and 90 min reperfusion. Regional ventricular function was assessed by measuring systolic wall thickening, and interstitial nucleosides were estimated by cardiac microdialysis. Control hearts were compared with hearts treated with acadesine, adenosine, and adenosine plus acadesine. Adenosine pretreatment (100 microg x kg(-1) x min(-1), intracoronary) immediately prior to ischemia increased interstitial adenosine levels 9-fold and improved postischemic functional recovery from a control value of 17.6 +/- 4.1% to 43.6 +/- 3.4% of preischemic systolic wall thickening. In contrast, acadesine (20 mg/kg i.v. bolus 10 min prior to ischemia + 0.5 mg x kg (-1) x min(-1), i.v. infusion through 60 min reperfusion) had no effect on interstitial fluid adenosine levels or the recovery of regional function (21.5 +/- 5.9% recovery), nor were the functional effects of adenosine potentiated by acadesine. These findings indicate that acadesine does not enhance myocardial adenosine levels, attenuate myocardial stunning, or potentiate the cardioprotective effects of adenosine in the pig.

Evidence that cytosolic and ecto 5'-nucleotidases contribute equally to increased interstitial adenosine concentration during porcine myocardial ischemia.

The purpose of this study was to determine the roles of cytosolic and ecto 5'-nucleotidase in myocardial ischemia-induced increases in interstitial fluid (ISF) adenosine. Pentobarbital anesthetized, open chest pigs were instrumented with two microdialysis fibers in the distally perfused bed of the left anterior descending (LAD) coronary artery to estimate ISF metabolites. Fibers in control hearts were perfused with standard Krebs buffer. In two additional groups, after collecting one dialysate sample with normal Krebs, fibers were perfused with buffer supplemented with either L-homocysteine thiolactone (5 mM) or the ecto 5'-nucleotidase inhibitor alpha, beta-methylene adenosine 5'-diphosphate (AOPCP, 5 mM). Hearts were then submitted to 60 minutes LAD occlusion and two hours reperfusion. Dialysate nucleosides and AMP were measured by high performance liquid chromatography. The local delivery of homocysteine did not alter preischemic dialysate adenosine concentration (0.30 +/- 0.04 microM) compared to pre-homocysteine infusion (0.39 +/- 0.04 microM) or control hearts (0.36 +/- 0.04 microM), but AOPCP significantly decreased preischemic dialysate adenosine levels (from 0.36 +/- 0.02 to 0.14 +/- 0.03 microM). During LAD occlusion both homocysteine and AOPCP reduced dialysate levels by approximately 50%. At 30 minutes ischemia dialysate adenosine concentrations were 19.47 +/- 2.72, 11.41 +/- 2.44, and 7.93 +/- 1.01 microM in control, homocysteine, and AOPCP hearts, respectively. AOPCP significantly increased dialysate AMP levels; at 60 minutes ischemia AMP levels were 6.22 +/- 2.97 microM in control hearts and 38.60 +/- 5.69 microM in AOPCP treated hearts. These results suggest that both cytosolic and ecto 5'-nucleotidase contribute to ischemia-induced increases in ISF adenosine in porcine myocardium.

Adenosine attenuates in vivo myocardial stunning with minimal effects on cardiac energetics.

Adenosine has been shown to modulate myocardial intermediary metabolism. The purpose of this study was to determine whether adenosine-mediated attenuation of in vivo myocardial stunning is associated with improved myocardial phosphorylation potential. Adult, open chest pigs were subjected to 10 minutes of regional myocardial ischemia and 90 minutes reperfusion. Regional ventricular function was assessed by measuring systolic wall thickening. Myocardial phosphorylation potential was estimated from the tissue (CrP/CrxPi) ratio determined in rapid-frozen tissue biopsy samples from normal and stunned myocardium. Control pigs were compared to animals treated prior to ischemia with intracoronary adenosine (50 micrograms/kg/min). Postischemic regional systolic wall thickening in adenosine treated pigs was significantly improved (40 +/- 3% of preischemic values) compared to control untreated pigs (26 +/- 3%). Myocardial stunning was associated with decreased ATP levels, but neither the total creatine pool (CrP + Cr) nor the (CrP/CrxPi) ratio was reduced. Adenosine pretreatment was associated with decreased Pi and Cr contents resulting in improved postischemic (CrP/CrxPi) ratio in the stunned bed compared to controls, but this effect occurred only after postischemic function had attained maximal improvement. These results suggest that adenosine attenuation of in vivo myocardial stunning is independent of elevated myocardial phosphorylation potential.

Comparison of interstitial fluid and coronary venous adenosine levels in in vivo porcine myocardium.

There are numerous reports of interstitial fluid (ISF) and coronary venous adenosine measurements in isolated perfused hearts. This study was designed to simultaneously compare ISF and coronary venous adenosine concentrations during various interventions in in vivo porcine myocardium. In anesthetized, open-chest pigs, ISF adenosine, inosine, and hypoxanthine were sampled with cardiac microdialysis. Coronary sinus or venous purines were sampled with a metabolism-stop solution. During basal conditions, ISF adenosine was greater than coronary venous adenosine, but vascular inosine and hypoxanthine were greater than corresponding ISF levels. Dobutamine (20 micrograms/kg/min, i.v.) and systemic hypoxia produced three- and two-fold increases in ISF adenosine, but had no significant effect on coronary sinus adenosine concentration. Hypoxia, but not dobutamine, increased coronary sinus total purines 50%. In contrast to these interventions, intracoronary adenosine infusion (0.5-50 micrograms/kg/min) was associated with significantly greater coronary venous adenosine concentrations than ISF levels. Only during a coronary artery occlusion/reperfusion protocol were ISF and coronary venous adenosine concentrations comparable. The results of this study thus provide in vivo evidence of the powerful endothelial and red blood cell metabolic barriers to both exogenous and endogenous adenosine. These results also illustrate the differences in adenosine concentrations in the ISF and vascular spaces.

Ryanodine receptor dysfunction in porcine stunned myocardium.

We investigated the effects of myocardial stunning on the function of the two main Ca2+ transport proteins of the sarcoplasmic reticulum (SR), the Ca(2+)-adenosinetriphosphatase and the Ca(2+)-release channel or ryanodine receptor. Regional myocardial stunning was induced in open-chest pigs (n = 6) by a 10-min occlusion of the left anterior descending coronary artery (LAD) and 2 h reperfusion. SR vesicles isolated from the LAD-perfused region (stunned) and the normal left circumflex coronary artery (LC)-perfused region were used to assess the oxalate-supported 45Ca2+ uptake, [3H]ryanodine binding, and single-channel recordings of ryanodine-sensitive Ca(2+)-release channels in planar lipid bilayers. Myocardial stunning decreased LAD systolic wall thickening to 20% of preischemic values. The rate of SR 45Ca2+ uptake in the stunned LAD bed was reduced by 37% compared with that of the normal LC bed (P < 0.05). Stunning was also associated with a 38% reduction in the maximal density of high-affinity [3H]ryanodine binding sites (P < 0.05 vs. normal LC) but had no effect on the dissociation constant. The open probability of ryanodine-sensitive Ca(2+)-release channels determined by single channel recordings in planar lipid bilayers was 26 +/- 2% for control SR (n = 33 channels from 3 animals) and 14 +/- 2% for stunned SR (n = 21 channels; P < 0.05). This depressed activity of SR function observed in postischemic myocardium could be one of the mechanisms underlying myocardial stunning.

Safety, tolerance, and efficacy of adenosine as an additive to blood cardioplegia in humans during coronary artery bypass surgery.

Myocardial stunning after heart surgery is associated with increased morbidity and mortality in patients with severe multivessel disease and reduced myocardial function. The purpose of this study was to evaluate the safety, tolerance, and efficacy of adenosine as a cardioprotective agent when added to blood cardioplegia in patients undergoing coronary artery bypass surgery. Sixty-one patients were randomized to standard cold-blood cardioplegia, or cold-blood cardioplegia containing 1 of 5 adenosine doses (100 microM, 500 microM, 1 mM, 2 mM, and 2 mM with a preischemic infusion of 140 microg/kg/min of adenosine). Invasive and noninvasive measurements of ventricular performance and rhythm were obtained preoperatively, prebypass, and then at 1, 2, 4, 8, 16, and 24 hours postbypass. Use of inotropic agents and vasoactive drugs pastoperatively was recorded; blood samples were collected for measurement of nucleoside levels. High-dose adenosine treatment was associated with a 249-fold increase in the plasma adenosine concentration and a 69-fold increase in the combined levels of adenosine, inosine, and hypoxanthine (p <0.05). Increasing doses of the adenosine additive were also associated with lower requirements of dopamine (p = 0.003) and nitroglycerine (p = 0.001). The 24-hour average doses for dopamine and nitroglycerine in the placebo group were 28-fold and 2.6-fold greater than their respective high-dose adenosine treatment cohorts. Finally, the placebo- and 100 microM-adenosine group was associated with a lower ejection fraction when compared to patients receiving the intermediate dose or high-dose treatment. These findings are consistent with the hypothesis that adenosine is effective in attenuating myocardial stunning in humans.

Myocardial stunning: a therapeutic conundrum.

Dobutamine and pyruvate are two inotropic agents with different mechanisms of action. Although both agents alter postischemic myocardial dysfunction, their potential metabolic effects in the setting of in vivo myocardial stunning have not been addressed. In this study, the effects of dobutamine and pyruvate on systolic wall thickening, myocardial phosphorylation potential index, interstitial fluid adenosine level, and myocardial oxygen consumption in in vivo stunned porcine myocardium were assessed. Stunning was induced with a 10-minute occlusion of the left anterior descending coronary artery. After 30 minutes of reperfusion, pigs were treated with either intravenous dobutamine (10 micrograms/kg per minute) or intracoronary pyruvate (1 ml/min, 150 mmol/L solution, pH 7.4). Infusion of both agents resulted in a marked improvement in regional systolic wall thickening. The dobutamine effect, however, produced a marked increase in myocardial oxygen consumption and was associated with an increase in interstitial adenosine caused by myocardial de-energization, because the myocardial phosphorylation potential index ratio decreased from 0.17 +/- 0.02 to 0.09 +/- 0.02 (p < 0.05). In contrast, pyruvate enhanced myocardial energy status, because the myocardial phosphorylation potential index ratio increased from 0.20 +/- 0.03 to 0.55 +/- 0.08 (p < 0.01). These experimental findings suggest that under certain circumstances the use of beta-receptor agonists to treat myocardial stunning may be suboptimal, if not undesirable. Further investigation is warranted to determine the optimum therapy for the stunned heart.

Effects of ischemic and adenosine preconditioning on interstitial fluid adenosine and myocardial infarct size.

The accumulation of adenosine during a brief coronary occlusion has been proposed to mediate the infarct size-limiting effect of ischemic preconditioning. The purpose of this study was to compare the effects of ischemic preconditioning and a transient adenosine infusion on myocardial interstitial fluid (ISF) adenosine levels and infarct size. Microdialysis fibers (10.0 mm length) were placed in the left ventricular myocardium of pentobarbital sodium-anesthetized rabbits to estimate ISF adenosine. Ischemic preconditioning was induced by 5 min of coronary artery occlusion and 10 min of reperfusion before 45 min of occlusion. Adenosine preconditioning was induced with 5 min of intravenous adenosine infusion (140 micrograms.kg-1.min-1) followed by a 10-min washout before the prolonged occlusion. Myocardial infarct size was determined by triphenyltetrazolium chloride staining after 3 h of reperfusion. Five minutes of ischemia and 5 min of adenosine infusion produced comparable increases in dialysate adenosine levels (from 0.19 +/- 0.02 to 0.69 +/0- 0.11 and 0.28 +/- 0.10 to 0.71 +/- 0.18 microM, respectively) that decreased to baseline before the prolonged ischemia; however, ischemic-preconditioned hearts exhibited elevated dialysate adenosine levels for the first 5 min of reperfusion. Ischemic-preconditioned hearts exhibited significantly reduced dialysate adenosine concentrations for the first 20 min of the prolonged occlusion (P < 0.05 vs. control), and infarct size was reduced from 41 +/- 6 to 10 +/- 4% of risk area. Adenosine preconditioning had no effect on dialysate adenosine levels during prolonged ischemia but did reduce infarct size to 25 +/- 5% of risk area. These results indicate that a transient increase in ISF adenosine can reduce myocardial infarct size, but adenosine alone does not fully replicate the protective effects of ischemic preconditioning.

Improved heart preservation with UW preservation solution.

Despite the good clinical results obtained with the current heart preservation techniques, these methods need to be improved. The UW solution has provided excellent preservation for the pancreas, kidney, and liver after extended cold ischemic storage times. We have tested the ability of the UW solution to store hearts for 5 and 12 hours and compared the results with those obtained from hearts preserved by either Stanford or modified Collins' solutions. Three groups of five canine hearts each underwent 5 hours, and three groups of five canine hearts underwent 12 hours of ischemia at 4 degrees C. Then the hearts were reperfused in an isolated working canine heart preparation. Those hearts preserved for 5 hours had nearly normal ventricular function and adenosine triphosphate contents and were able to maintain normal tissue electrolyte concentration and water contents. After 12 hours of storage time only adenosine triphosphate contents were similar among the groups. Hearts preserved with the UW solution rapidly recovered, reaching nearly normal left ventricular function by 60 minutes of reperfusion; hearts preserved by the modified Collins' solution recovered more slowly, but function was good after 120 minutes of reperfusion. Hearts preserved by the Stanford solution never attained adequate function. The three groups of hearts preserved for 12 hours did not differ in their ability to utilize lactate or in their rates of oxygen utilization. Tissue water and sodium contents were considerably lower in the hearts preserved with the UW solution after 150 minutes of reperfusion compared with hearts stored in the modified Collins' or Stanford solutions. Hearts stored 12 hours in the UW solution under cold ischemic conditions recovered left ventricular function rapidly after reperfusion with normal blood, utilized lactate and oxygen at normal rates, and were able to regulate tissue water and sodium contents to nearly normal levels. Because of the superior preservation obtained by the UW solution, the solution deserves further evaluation for possible future use in clinical heart transplant programs.

Quantitation of absolute flow in coronary artery bypass grafts using digital subtraction angiography.

Coronary angiography defines the location and size of obstructive lesions, but does not assess their physiological significance. To assess a new method to measure the blood-flow waveform, reversed saphenous vein grafts from the left subclavian artery to the left anterior descending coronary artery were placed in five mongrel dogs. Contrast material was injected selectively into the vein graft while obtaining fluoroscopic images from AP and 45 degrees LAO projections. Blood flow was measured under baseline, low-flow, and hyperemic conditions using an electromagnetic flow probe (EM). Seventeen radiographic determinations of mean blood flow (range 18-130 ml/min) were linearly correlated to simultaneous EM measurements (r = 0.91 and 0.88, respectively). Contrast material injections changed EM flow measurements by an average of 35%, which though large, is less than with other radiographic methods. The computed blood-flow waveforms had a time resolution of 1/30 sec and were in good agreement with EM waveforms measured simultaneously. Clinical application of this radiographic method for determining the blood-flow waveform may allow early prediction of coronary artery bypass graft closure.

Pump-induced hemolysis in a rabbit model of neonatal ECMO.

There is no significant difference in hemolysis between pediatric centrifugal pumps and roller pumps during ECMO of 8 hours duration in rabbits or in comparable 24 hour isolated pump circuits. There is also no significant difference in heat production by either pump. Reports of severe hemolysis using the centrifugal pump in neonatal ECMO are likely due to other factors. Proper cannula selection and positioning are important measures to reduce hemolysis by minimizing circuit pressure and maximizing venous return. Avoidance of inadvertently high rotor speed in the centrifugal pump is also recommended.

A comparison of blood and crystalloid cardioplegia during heart transplantation after 5 hours of cold storage.

Four methods of protecting the heart during implantation were compared. All hearts were arrested in situ by perfusing 4 degrees C cardioplegic solution into the aortic root and were stored by a nonperfused cold storage technique for 5 hours at 4 degrees C. The hearts were then transplanted orthotopically with the use of topical iced slush alone or with infusions of either blood cardioplegic solution or one of two crystalloid cardioplegic solutions after each atrial anastomosis. Five dog hearts were included in each group. Biopsy samples to test for adenylates were taken before the arrest, at the end of storage, before cross-clamp removal, and 3.5 hours after cross-clamp removal. The dogs were removed from cardiopulmonary bypass, and with the chest open, left ventricular function curves were measured at 1, 2, and 3 hours after cross-clamp removal. At 3.5 hours of reperfusion time, a full-width section was obtained from the left ventricle for measurement of tissue sodium and water content. No differences in tissue water, sodium, or potassium content were found among the groups. Left ventricular function was significantly better in the blood cardioplegia group than in any other groups. Adenosine triphosphate levels were significantly reduced 3.5 hours after reperfusion in the crystalloid cardioplegia groups but were not significantly depressed at any other measurement time. Excellent early graft function was observed after crystalloid cardioplegic arrest and blood cardioplegic reperfusion during graft implantation.

Inhibition of calcium influx in isolated adult rat heart cells by ATP depletion.

Using 45Ca, indo1, and quin2, calcium uptake was measured in isolated quiescent adult rat heart cells under different metabolic conditions. Exposure of cells in a medium containing 1 mM CaCl2 to rotenone and uncoupler resulted in adenosine triphosphate (ATP) depletion from 17.08 +/- 2.26 to 0.63 +/- 0.11 nmol/mg within 8 minutes, and the cells went into contracture. In this time, the cells lost 1.65 +/- 0.1 nmol Ca/mg of total rapidly exchangeable cellular calcium, and the level of free cytosolic calcium as measured by indo1 rose from 47.4 +/- 16.3 nM to 79.8 +/- 27.6 nM. The subsequent rate of rise of intracellular free calcium concentration was just 4 nM/min for at least 40 minutes. Therefore, we investigated the effect of ATP depletion on the rate of calcium entry. In cells loaded with sodium by ouabain treatment without calcium, the initial rate of calcium influx on calcium addition was inhibited by 82-84% when cellular ATP was depleted, as measured by 45Ca or indo1. Quin2 also showed a strong inhibition of calcium influx by ATP depletion, but itself also caused a strong inhibition of calcium influx. The rate of calcium influx declined even further in ATP-depleted cells after the initial influx: Between 1 and 12 minutes after calcium addition, the residual 45Ca uptake rate of the first minute was inhibited by an additional 90%. We conclude that ATP depletion per se does not quickly elevate cytoplasmic free calcium and that such an elevation is prevented by a very strong inhibition of the rate of calcium entry.

Control of thallium and sodium fluxes in isolated adult rat heart cells by anthopleurin-A, verapamil and magnesium.

Anthopleurin-A stimulated the initial rate of 201thallium uptake by isolated adult rat heart cells by a factor of 3.41 +/- 0.56, and induced a unique pattern of spontaneous beating activity. Ouabain inhibited the basal uptake rate by 58 +/- 11% and all the anthopleurin-A stimulated rate. The Km for thallium uptake was 0.95 +/- 0.26 mM, and was not changed by anthopleurin-A. Accumulated thallium was quickly released from cells by EDTA addition. Such release was inhibited 87 +/- 10% by verapamil. Thallium reuptake was initiated by restoration of magnesium to the medium. Reuptake was mostly inhibited by ouabain, but the residual ouabain-insensitive uptake remained. The ouabain-insensitive uptake was inhibited by ATP depletion. Anthopleurin-A stimulated the rate of 22Na entry into cells by a factor of 3.17 +/- 1.65, and EDTA stimulated the rate of entry by a factor of 29.5 +/- 13.0. The EDTA-induced 22Na entry was inhibited 86 +/- 11% by verapamil. From this we draw three conclusions: The major pathway for thallium uptake is the Na-K pump. The rate of uptake by this route, like the rate of K+ uptake, is governed by the rate of cellular sodium influx; A residual ouabain-insensitive uptake route also exists which appears to require ATP but not a monovalent ion gradient; Removal of Mg and Ca induces a verapamil-sensitive monovalent channel activity which is both massive and reversible.

Arterial blood-flow waveform measurement in intact animals: new digital radiographic technique.

With a new radiographic method to determine the pulsatile flow pattern in an imaged artery, flow velocity is determined by tracking the movement of contrast material along the artery over time. Flow velocity is multiplied by an automatically determined cross-sectional area of the artery to determine blood flow. Pulsatile blood-flow waveforms were determined by radiographic and electromagnetic techniques in each femoral artery of four dogs while flow conditions were varied. Peak and average blood flows measured by the two techniques were highly correlated (r = .97 and .95, respectively). The closest agreement between the flow waveforms measured by the electromagnetic and radiographic techniques was found under normal flow conditions. The radiographically derived flow waveforms tended to be noisy at low blood-flow rates, and instantaneous blood-flow rates exceeding 700 ml/min were underestimated.