J Mol Cell Cardiol22,815-826


( 1990)

Release from Isolated Rat Heart: A New Approach Study of Energy Metabolism



Ugo Limbruno ls3, Rosa Poddight’, Giovanni Ronca3

1Istituto di Cardiologia,

‘Scuola Superiore S. Anna, and ‘Istituto University of Piss, Italy

(Received 19 Ju& 1989, acceped in revisedform


to The



di Chimica Biologica,

16 February 1990)

R. ZUCCHI, U. LIYBRUNO, R. PODDIGHE, M. MARIANI AND G. RONCA. Purine Release from Isolated Rat Heart: A New Approach to the Study of Energy Metabolism. 3oumalof Molecular and Cellular Cardiology (1990) 22, 815-826. The rate of release of purines (adenosine, inosine, hypoxanthine, xanthine and uric acid) from isolated working rat hearts was measured and compared to tissue concentrations of high energy phosphate compounds. Hearts were subjected to different workloads, and perfusions were performed: with normal oxygen supply (group 1); with the addition ofinsulin to the standard perfusion buffer, which contained glucose as energy source (group 2); in hypoxic conditions (group 3). In each group purine release increased (P < 0.01) at higher workload and was closely related to indices of mechanical performance such as cardiac output or minute work (r = 0.902 and 0.858 in group 1, T = 0.902 and 0.851 in group 2, r = 0.851 and 0.881 in group 3, P < 0.001 in each case). Work had no effect on adenine nucleotides but produced a significant (P < 0.01) reduction in phosphocreatine/creatine ratio. The comparison of different groups showed that at any level of heart performance purine release was higher (P < 0.001) in group 3~s. group 1, and lower (P < 0.001) in group 2 vs. group 1. High energy phosphates were reduced in group 3 vs. group 1 but were unchanged in group 2 vs. group 1. We conclude that in the isolated heart purine release is directly related to the rate of energy consumption, and inversely related to the rate of energy production. Purine release provides a sensitive method to evaluate myocardial energy metabolism, which is more sensitive than measurement of high energy phosphates.

Introduction Myocardial energy metabolism is usually evaluated through the measurement of high energy phosphate compounds and of the adenyla te charge [q. Although universally followed, this approach has low sensitivity, since thesevariables are unchanged in a wide range of physiological and pathological conditions. High energy phosphates and adenylate charge were found to be constant [35,5O] or only slightly modified [41] at different levels of heart work and minimally reduced (less than 10%) after partial [,‘#I or short-term [54] ischemia. The correct expression of myocardial energy metabolism is the phosphorylation state of ATP in the cytosol [ZZ, 401, and whole tissuelevels of adenine nucleotides are very rough estimates of their cytosolic concentrations






AMP and ADP is bound or compartimenKEY WORDS: Energy metabolism; * Please address all correspondence 0022-2828/90/070815

+ 12 $03.00/O

talized (e.g. intramitochondrial) [13], while 90% of the ATP pool is free in the cytosol 113, 401. Even with sophisticated techniques an accurate measurement of free cytosolic AMP and ADP is extremely difficult and requires theoretical assumptions about cytosolic solvent spaceand intracellular pH [13]. Isolated perfused hearts release purine catabolites [2, 7, 8, 11, 17, 42, 44, 45, 47, 501. Owing to the mechanism of substrate regulation purine release (PR) should be proportional to cytosolic AMP concentration, which is in turn


to the



of cytosolic ATP because of the myokinase reaction (Fig. 1). So PR should increasein any condition associatedwith increase in the rate of energy consumption or decreasein the rate of energy production. A closerelationship between adenosineproduction and myocardial energy metabolism

Purine release; Adenosine; ATP, Phosphocreatine; Hypoxia; Insulin. to: R. Zucchi, Istituto di Chimica Biologica, via Roma 55, 56100 Pisa, Italy. 0 1990 Academic

Press Limited


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FIGURE 1. Principal pathways ofpurine metabolism in the heart. Enzymes involved: 1, myokinase (EC; 2, 5’nucleotidase (EC; 3, AMPdeaminase (EC; 4, adenosine deaminase (EC; 5, purine nucleoside phosphorylase (EC; 6, xanthine oxidoreductase (EC; 7, S-adenosylhomocysteine (SAH)hydrolase (EC; 8, adenosine kinase (EC Dashed lines, quantitatively minor pathways or pathways whose relevance is not well known; l , catabcrlites which can be released from the heart.

was first proposed by Berne [lZ]. Adenosine releaseincreasesremarkably during ischemia or hypoxia [II, 44,47J and was reported to be proportional to heart work [8, 16, 20, ,251,but the latter finding was disputed [32]. Overall PR should reflect energy metabolism more accurately than adenosinerelease, becauseit overcomes the problems deriving from adenosine compartmentation in the heart [40, 46] and from the breakdown of AMP through the IMP-inosine pathway [2]. Furthermore, PR takes into account the catabolism of guanine nucleotides, which might be relevant to cellular energy metabolism [2]. In this work we have tested the hypothesis that PR is a sensitive index of myocardial energy metabolism, measuring PR and high energy phosphate compounds under conditions in which both energy consumption and energy production were varied. Methods Animals and perfusion technique

Sprague-Dawley rats, fed with standard diet (275 to 300 g body weight), were anesthetized with a mixture of ether and air. After injection of 1000 IU sodium heparin in the femoral vein, the heart was quickly excised and perfused according to the working heart technique [3q. The preload (height of the atria1 chamber) and the afterload (height of the aortic chamber) were set at different values in

different groups of perfusions, as indicated below. Aortic flow (AF) and coronary flow (CF) were measured collecting the overflow from the aortic chamber and the ellluent from the heart chamber into graduated cylinders. Aortic pressure was monitored by a membrane transducer (P 23 ID, Gould, Oxnard, CA) connected to a side arm of the aortic cannula. Heart rate was calculated from aortic pressuretracings, recorded on paper every 5 min. Cardiac output (CO) was determined asthe sum of AF and CF, minute work (MW) as the product of cardiac output and peak systolic aortic pressure. At the end of each experiment the ventricular mass was separated from the atria and great vesselsand its dry weight was determined. Hearts of a limited range of weight (160 to 200 mg dry weight) were always used. Krebs-Henseleit bicarbonate buffer was the standard perfusion medium. Its composition was the following (mM): NaCl 118, NaHCOs 25, KC1 4.5, KHzP04 1.2, MgS04 1.2, CaC122.5, glucose 11.The buffer was equilibrated with a mixture of oxygen (95%) and carbon dioxide (5%). Temperature was kept between 37 and 37.4”C, and the pH was 7.4. Assay of purines and determination of purine release Purines were assayed in the perfusion fluid every 5 to 10 min by high performance liquid chromatography (HPLC; BIP-I Liquid



and Energy

Chromatographer equipped with a Uvidec100-111 variable length spectrophotometer, Jasco, Tokyo, Japan) as previously described [.!%I. Briefly, a p-Bondapak (Waters Associates, Milford, MA) C-18 column (4 x 300 mm, particle size 10 pm) was eluted isocratitally with 50 mM NHbHzPOd, pH 6.0 at a flow rate of 1.5 ml/min, and the concentrations of adenosine, inosine, hypoxanthine, xanthine and uric acid were measured on the basis of the absorption at 254 or 292 nm by standards. comparison with appropriate Adenine, guanine and guanosine were undetectable. Assays were usually performed just after sample collection, and in any case within 24 h, storing the samples at 4°C. Serial determinations showed that such storage did not modify the results of the assay. In preliminary experiments PR was measured both during recirculating and during open (not recirculating) perfusion. In the first 25 min of working heart perfusion the coronary ellluent was discarded, then it was recirculated for 65 min. Samples of perfusion buffer were taken simultaneously from the aortic efluent and coronary effluent. Release of each was calculated as (coronary substance concentration - aortic concentration) x CF (method A). During recirculating perfusion release was also calculated as: (rate of increase in concentration) X (volume of recirculating fluid) (method B). Assay

of nucleotides,

creatine and phosphocreatine

Hearts were freeze-clamped by Wollenberger clamps cooled in liquid nitrogen. Tissue was then freeze-dried, and ATP, ADP, AMP, creatine and phosphocreatine were determined by HPLC technique according to Sellevold et al. [48], with minor modifications. Briefly, samples of dried ventricle were homogenized by a mechanical homogenizer (Ultraturrax, Janke and Kunkel, Staufen, FRG), and 6 to 10 mg of the resulting power were dissolved into 1 ml 0.42 M perchloric acid and stirred intermittently on ice for 10 min. After precipitation (with 200 to 220 ~1 2 N KOH) and centrifugation (5 min in a chilled Eppendorf centrifuge) the supernatant was used for HPLC assay. The column was the same as used for the purine catabolite assay, the mobile phase contained 215 mM KHz PO4, 2.3



mM tetrabutylammonium hydrogen sulphate and 3% acetonitrile, pH 6.25. The flow rate was 1 ml/min and the spectrophotometer was set at 206 nm. The purity of the creatine peak was checked by incubation with creatinase (EC Oxygen consumption To measure oxygen consumption, the pulmonary artery was cannulated and samples of perfusate were collected from the aortic and the pulmonary cannulas without exposure to air, as described by Neely et al. [.%I. Oxygen tension was measured with an oxygen electrode (YSI 5300, YSI Scientific, Yellow Springs, OH) calibrated with air. Oxygen consumption was calculated as described elsewhere [367 and expressed as pmol/min/g ventricular dry weight. As an index of efficiency we measured the ratio of mechanical work (pressure-volume work + kinetic work), calculated according to Neely et al. [Xl and the energy’ equivalent of the oxygen consumed, calculated according to Biinger et al. [14]. Other analytical procedures Glucose and lactate were assayed according to Trinder [.52] and No11 [39] (commercial kits by Menarini, Florence, Italy, and Boehringer, Mannheim, FRG). Glucose uptake was determined during recirculating perfusions as the product of buffer volume and decrease in glucose concentration. Creatine kinase (CK) and lactate dehydrogenase (LDH) were assayed according to Meiattini et al. [32] and Bergemeyer and Burnt [IO] (commercial kits by Sclavo, Siena, Italy). Experimental


To obtain a wide range of work loads, hearts were perfused at 6, 10 or 20 cm of preload and 43,66 or 90 cm of afterload. In addition some hearts were perfused in the Langendorlf mode [271, setting the perfusion reservoir 80 cm above the heart (mean aortic pressure, 62 mmHg). To assess the effect of changes in the rate of energy production such perfusions were performed: in standard conditions (normal oxygen supply, no insulin: group l), in the


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presence of 4 IU/l bovine insulin (group 2), and during hypoxia (group 3). Hypoxia was produced by a reduction of the rate of oxygen supply adjusted in order to get an 80% decrease in aortic flow. With this procedure no necrosis was produced, since no significant enzyme release was detected (see below). Each perfusion was performed in the recirculating mode and lasted 60 min. The volume of perfusion fluid was measured each time and averaged 180 ml. Samples of perfusion buffer (c. 0.5 ml) were collected every 5 min and PR was determined by method B. In parallel experiments hearts were freezeclamped after 10 min of perfusion and adenine nucleotides, creatine and phosphocreatine were measured as described above. In these experiments PR was determined by method A. In a few working hearts (preload 10 cm, afterload 66 cm, n = 3) we studied the effect on’ PR of an isolated increase in coronary flow. Coronary flow was increased by the addition of 10 mg/l isosorbide dinitrate to the perfusion buffer.


et PI.

Statistical analysis

All data are expressedasmean & S.E.M. Different groups were compared by Student’s t-test for paired or unpaired data (two groups) or analysis of variance and Fisher’s F-test (more than two groups) [5J. Regressionanalysis was performed by standard techniques [5]. Results

In open perfusions the major purine catabolites released were uric acid, inosine and hypoxanthine. With normal oxygen supply uric acid releaseaccounted for about 70% of overall PR (Fig. 2), while this percentage was substantially lower during hypoxia (data not shown). When the coronary effluent was recirculated, purine compounds were further catabolized to uric acid at each passage through the heart [4.2]. So after 10 to 20 min (the lag was two to three times longer during hypoxia) PR was virtually equal to uric acid release (Fig. 2), while overall PR was unchanged. Calculations basedeither on arteriovenous differences (method A) or on the in-










FIGURE 2. Determination of PR in a working heart experiment. After 25 min of open (not recirculating) perfusion, the coronary effluent was recirculated. Hemodynamic performance remained stable all through the experiment. Bars represent the release of uric acid, xanthine, hypoxanthine, inosine and adenosine (subdivision of bars from bottom upwards) calculated according to method A (the time course is shown) and method B (mean values over the recirculating period).



and Energy

crease in purine cata,bolites concentrations (method B) gave the same value for PR. Method B required fewer HPLC runs and was preferentially usedin subsequentexperiments. Prolonged perfusions showed that PR remained stable for at least 90 min. In standard conditions (group 1) PR was significantly higher in working than in Langendorff perfused hearts (Table 1). In the different working subgroups PR increased either when the preload was raised at the same afterload (P

Purine release from isolated rat heart: a new approach to the study of energy metabolism.

The rate of release of purines (adenosine, inosine, hypoxanthine, xanthine and uric acid) from isolated working rat hearts was measured and compared t...
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