Molecular and Cellular Biochemistry 118: 1-14, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.
The nucleotide metabolism in lactate perfused hearts under ischaemic and reperfused conditions Monique J.M. de Groot, Will A. Coumans and Ger J. van der Vusse Department of Physiology, Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands Received 4 December 1991; accepted 13 August 1992
Abstract It was examined whether lactate influences postischaemic hemodynamic recovery as a function of the duration of ischaemia and whether changes in high-energy phosphate metabolism under ischaemic and reperfused conditions could be held responsible for impairment of cardiac function. To this end, isolated working rat hearts were perfused with either glucose (11 raM), glucose (11 mM) plus lactate (5 raM) or glucose (11 mM) plus pyruvate (5 mM). The extent of ischaemic injury was varied by changing the intervals of ischaemia, i.e. 15, 30 and 45 rain. Perfusion by lactate evoked marked depression of functional recovery after 30 min of ischaemia. Perfusion by pyruvate resulted in marked decline of cardiac function after 45 min of ischaemia, while in glucose perfused hearts hemodynamic performance was still recovered to some extent after 45 rain of ischaemia. Hence, lactate accelerates postischaemic hemodynamic impairment compared to glucose and pyruvate. The marked decline in functional recovery of the lactate perfused hearts cannot be ascribed to the extent of degradation of high-energy phosphates during ischaemia as compared to glucose and pyruvate perfused hearts. Glycolytic ATP formation (evaluated by the rate of lactate production) can neither be responsible for loss of cardiac function in the lactate perfused hearts. Moreover, failure of reenergization during reperfusion, the amount of nucleosides and oxypurines lost or the level of high-energy phosphates at the end of reperfusion cannot explain lactate-induced impairment. Alternatively, the accumulation of endogenous lactate may have contributed to ischaemic damage in the lactate perfused hearts after 30 rain of ischaemia as it was higher in the lactate than in the glucose or pyruvate perfused hearts. It cannot be excluded that possible beneficial effects of the elevated glycolytic ATP formation during 15 to 30 min of ischaemia in the lactate perfused hearts are counterbalanced by the detrimental effects of lactate accumulation. (Mol Cell Biochem 118: 1-14, 1992)
Key words: lactate, pyruvate, ischaemia, reperfusion, rat heart, nucleotides
Introduction The extent of ischaemic and reperfusion damage is in-
fluenced by the nature of exogenous substrates. In this regard, pyruvate has been found beneficial on functional recovery of the postischaemic heart [1,2]. In contrast, exogenous lactate has detrimental effects on the post-
Address for offprints: M.J.M. de Groot, Department of Physiology, Cardiovascular Research Institute Maastricht, University of Limburg, Postbus 616, 6200 MD Maastricht, The Netherlands
ischaemic heart [2-6]. Since lactate concentrations in blood plasma can be increased under several physiological and pathophysiological conditions, e.g. lactateRinger infusion during cardiopulmonary bypass, moderate to severe physical exercise performed by cardiac patients, circulatory shock, hypoxemia and type I glycogenesis [7, 8], it is important to understand the precise nature of the adverse effect of exogenous lactate on postischaemic cardiac function. Earlier studies have revealed that loss of cardiac function after an ischaemic insult is associated with depletion of high-energy phosphates [9-11]. Hence, it is tempting to state that lactate unfavourably influences cardiac high-energy phosphate metabolism under ischaemic and reperfused conditions. Under oxygen restricted circumstances, adenosine triphosphate (ATP) delivery by mitochondria is limited but alternatively, ATP may be obtained by glycolysis. Opie [12] suggested that ATP produced by glycolysis could be important in protecting membrane function and preventing contracture during ischaemia. The generation of ATP by glycolysis, though, is insufficient to meet the total ATP requirement, a consequence of which is the depletion of high-energy phosphate stores during ischaemia [10, 13]. Catabolites of the nucleotides (i.e. adenosine, inosine, hypoxanthine, xanthine and uric acid) are released into the coronary effluents during reperfusion, which contributes to impaired salvage of ATP during the postischaemic state [14-16]. Evaluation of cardiac nucleotide metabolism shows a high degree of complexity. Firstly, measurements of total ATP levels do not identify ATP produced from anaerobic glycolysis. An indirect estimate of glycolytic ATP formation can be made by determining the glycolytic flux rate including glucose (glycogen) utilization and lactate production. Secondly, the total tissue content of ATP is an indication for the amount of ATP in cytoplasm as about 90% of total ATP is present in cytoplasm [17]. Cytoplasmic hydrolysis of ATP into adenosine diphosphate (ADP) and adenosine monophosphate (AMP) is difficult to detect as the majority of ADP and AMP is bound to actomyosin or compartmentalized in mitochondria [17, 18]. Zucchi and colleagues [19] suggested that detection of the degradation products of ATP (adenosine, inosine, hypoxanthine, xanthine and uric acid) in the coronary effluents may be a more simple and sensitive method to evaluate cardiac energy metabolism. The amount of degradation products released should be related to the cytoplasmic AMP concentration, which in turn is derived from cytoplas-
mic ATP [17, 18]. Moreover, purine release reflects breakdown of myocardial guanine nucleotides. These compounds may also be relevant for energy delivery [201. It was the aim of the present study to evaluate whether the effect of lactate on postischaemic function was dependent on the time of ischaemia; to elucidate whether changes in glycolytic ATP production and cardiac high-energy phosphate levels during ischaemia or failure of reenergization during reperfusion could be held responsible for lactate-induced impairment; to compare the above mentioned changes in the lactate perfused hearts with glucose and pyruvate perfused hearts; to delineate whether there exists a general relationship between high-energy phosphate metabolism and hemodynamic recovery in the hearts subjected to ischaemia and reperfusion. Isolated working rat hearts were perfused with either glucose (11 mM) plus lactate (5 raM), glucose (11 mM) plus pyruvate (5mM) or glucose as sole substrate (11 mM). The hearts were subjected to 15, 30 or 45 min of global no-flow ischaemia in order to vary the severity of ischaemic insult. In a subset of experiments ischaemic hearts were reperfused for 35 min. Cardiac function was monitored by the product of heart rate and left ventricular developed pressure. Glycolytic ATP production was indirectly evaluated by glycogen breakdown and lactate formation during global ischaemia. Changes of the tissue content of high-energy phosphates were assessed by creatine phosphate (CP), ATP and guanosine triphosphate (GTP) measured before ischaemia, after ischaemia and after reperfusion. Breakdown of high-energy phosphates was evaluated by tissue accumulation and/or coronary release of nucleosides and oxypurines. In addition, attention was paid to the sum of nucleotides and their degradation products at the end of reperfusion compared to the sum of these compounds at the end of ischaemia.
Methods Isolated working heart preparation Hearts were obtained from male Lewis rats (10 weeks old, body weight range 250-350 g) under light ether anaesthesia. Isolation and cannulation were performed as previously described [21]. Working hearts were perfused with a modified Krebs-Henseleit bicarbonate buffer (37°C, pH range 7.35-7.45) equilibrated with
95% 02/5% CO 2 (pO2 > 75 kPa). The perfusion buffer contained (in mM): NaC1 (130.0), KCI (5.6), CaCI2 (2.2), MgCI2 (1.2), NaH2PO4 (1.2), NaHCO3 (25.0). D(+) glucose (11mM) was added as a basal substrate. L (+)-Lactate (5 mM) or pyruvate (5 mM, sodium salt) was added as cosubstrate where indicated. The lactate buffer was neutralized by sodiumhydroxide.
Experimental protocol During a stabilization period of 10 min, the hearts were retrogradely perfused according to the Langendorff technique. Then, the hearts were subjected to antegrade perfusion for 30 rain. Left atrial filling pressure was set at 1 kPa and diastolic aortic pressure at 8 kPa. The aortic impedance used resulted in an aortic pressure pulse of about 4.5 kPa [22]. After 30 min of normoxic perfusion, hearts were subjected to either 15, 30 or 45 min of ischaemia [temperature kept constant (37 ° C) by a water jacket and environment moistured and gassed by nitrogen (95% N2 and 5% CO2) bubbling in a water bath] and reperfused for additional 35 min (retrogradely during the first 5 min and antegradely thereafter). Since the hearts were perfused in the assisted mode [22], coronary perfusion pressure (8 kPa) was maintained even when reperfused hearts were unable to provide their own coronary flow. Hemodynamic characteristics [aortic pressure, left ventricular pressure (systolic and end-diastolic) and electrogram] were continuously monitored on a six-channel recorder (Schwarzer) as has been described earlier [21,22]. The pressurerate product was defined as the product of left ventricular developed pressure (difference between systolic and end-diastolic pressure) and heart rate.
Biochemical analysis At the end of either the preischaemic, ischaemic or postischaemic phase, ventricular tissue was rapidly dissected from atrial tissue with a surgical blade and immediately freeze clamped with the use of aluminum tongues, precooled in liquid nitrogen. Throughout the preischaemic and postischaemic phase of the experimental protocol, coronary effluents were collected and sampled. Tissue and effluent samples were stored at - 8 0 ° C until analysis.
Tissue Creatine phosphate, adenine nucleotides, guanine nucleotides, nucleosides, oxypurines, glucose and lactate: small aliquots of deeply frozen ventricle tissue were freeze dried. Metabolites were extracted by an ice-cold mixture of perchloric acid (3 mM) and dithiothreitol (5 mM) and subsequently, neutralized by potassiumbicarbonate [23]. Creatine phosphate was determined fluorometrically [24]. Tissue contents of adenosine tri-, di- and monophosphate (ATP, ADP, AMP), guanosine tri-, di- and monophosphate (GTP, GDP, GMP), inosine monophosphate (IMP), purine nucleosides (adenosine, inosine) and oxypurines (hypoxanthine, xanthine, uric acid) were assayed by high performance liquid chromatography (HPLC; Varian 5500) [16, 25]. Glucose and lactate were determined fluorometrically [24, 261 . Glycogen: Small parts of the frozen ventricles were freeze dried. After adding 1 N hydrochloric acid to the freeze dried material, glycogen was hydrolyzed at 100°C for 3hrs. The samples were neutralized and, subsequently, glucose residues were measured fluorometrically [21, 24]. The values obtained were corrected for the content of free glucose in the tissue.
Coronary effluent The release of nucleosides and oxypurines in coronary effluents was monitored by an isocratic HPLC procedure [16, 27].
Normalization of data Measurements in tissue samples are expressed in moles per gram dry weight. The dry weight of cardiac tissue was determined by freeze drying overnight. Data concerning release of nucleosides and oxypurines in the coronary effluent are expressed in moles per gram wet weight (release per heart corresponds to release per gram wet tissue). For conversion of the release of degradation products per gram wet weight to release of degradation products per gram dry weight an average, empirically determined, factor of 6.25 was used.
Statistical analysis Results are expressed as median values and 95% confidence limits throughout. The number of experiments varied from 6 to 8. Differences within groups were evaluated for significance using Wilcoxon's matchedpairs signed rank test. Differences between groups were evaluated for significance by Mann Whitney U test. P values less than 0.05 were considered statistically significant.
Results Hemodynamics Under preischaemic, normoxic conditions the pressurerate product was not influenced by lactate perfusion compared to glucose or pyruvate perfusion (Table 1). However, the effect of lactate turned out detrimental under ischaemic/reperfused conditions. Whilst after 15 min of ischaemia the recovery of the pressure-rate product was still 96% of the preischaemic value, a marked decrease was observed after 30 min of ischaemia (recovery 5%) in the lactate perfused hearts. In the glucose and pyruvate perfused hearts, the recovery of the pressure rate product was still 78 and 98%, respectively after 30 min of ischaemia and reperfusion. Following 45 min of ischaemia and reperfusion a marked decline of the pressure-rate product was also obvious in the pyruvate perfused hearts (recovery 0%), whereas in the glucose perfused hearts the pressure rate product was less severely reduced (recovery 59% of the preischaemic value).
Consumption of glycogen and production of lactate At the onset of ischaemia, glycogen stores were higher in the lactate and pyruvate perfused hearts (about 180 p~mol glucose equivalents per g dry weight) than in the glucose perfused hearts (109/~mol glucose equivalents per g dry weight, Fig. 1). After 15 min of ischaerfiia, the tissue glycogen content was decreased by 83.9, 91.9 and 98.2/~mol glucose equivalents/g dry weight in the glucose, lactate and pyruvate perfused hearts, respectively. From 15 to 30 min of ischaemia, glycogen consumption was nihil in the glucose and pyruvate perfused hearts, whereas in the lactate perfused hearts glycogen utilization was still considerable (Fig. 1). Af-
ter 45 rain of ischaemia, 96.1, 145.2 and 119.3/zmol glucose equivalents/g dry weight were set free from the cardiac glycogen pool in glucose, lactate and pyruvate perfused hearts, respectively (Fig. 1, calculated by median values). The preischaemic lactate content in cardiac tissue was higher in the lactate than in the glucose or pyruvate peffused hearts (Fig. 2). When a correction was made for the lactate amount in extracellular fluid (corrected value is 13.0 ~mol per g dry weight), the tissue lactate content was still slightly higher in the lactate perfused hearts compared to the glucose or pyruvate peffused hearts ( p < 0.05 and 0.05
The nucleotide metabolism in lactate perfused hearts under ischaemic and reperfused conditions Monique J.M. de Groot, Will A. Coumans and Ger J. van der Vusse Department of Physiology, Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands Received 4 December 1991; accepted 13 August 1992
Abstract It was examined whether lactate influences postischaemic hemodynamic recovery as a function of the duration of ischaemia and whether changes in high-energy phosphate metabolism under ischaemic and reperfused conditions could be held responsible for impairment of cardiac function. To this end, isolated working rat hearts were perfused with either glucose (11 raM), glucose (11 mM) plus lactate (5 raM) or glucose (11 mM) plus pyruvate (5 mM). The extent of ischaemic injury was varied by changing the intervals of ischaemia, i.e. 15, 30 and 45 rain. Perfusion by lactate evoked marked depression of functional recovery after 30 min of ischaemia. Perfusion by pyruvate resulted in marked decline of cardiac function after 45 min of ischaemia, while in glucose perfused hearts hemodynamic performance was still recovered to some extent after 45 rain of ischaemia. Hence, lactate accelerates postischaemic hemodynamic impairment compared to glucose and pyruvate. The marked decline in functional recovery of the lactate perfused hearts cannot be ascribed to the extent of degradation of high-energy phosphates during ischaemia as compared to glucose and pyruvate perfused hearts. Glycolytic ATP formation (evaluated by the rate of lactate production) can neither be responsible for loss of cardiac function in the lactate perfused hearts. Moreover, failure of reenergization during reperfusion, the amount of nucleosides and oxypurines lost or the level of high-energy phosphates at the end of reperfusion cannot explain lactate-induced impairment. Alternatively, the accumulation of endogenous lactate may have contributed to ischaemic damage in the lactate perfused hearts after 30 rain of ischaemia as it was higher in the lactate than in the glucose or pyruvate perfused hearts. It cannot be excluded that possible beneficial effects of the elevated glycolytic ATP formation during 15 to 30 min of ischaemia in the lactate perfused hearts are counterbalanced by the detrimental effects of lactate accumulation. (Mol Cell Biochem 118: 1-14, 1992)
Key words: lactate, pyruvate, ischaemia, reperfusion, rat heart, nucleotides
Introduction The extent of ischaemic and reperfusion damage is in-
fluenced by the nature of exogenous substrates. In this regard, pyruvate has been found beneficial on functional recovery of the postischaemic heart [1,2]. In contrast, exogenous lactate has detrimental effects on the post-
Address for offprints: M.J.M. de Groot, Department of Physiology, Cardiovascular Research Institute Maastricht, University of Limburg, Postbus 616, 6200 MD Maastricht, The Netherlands
ischaemic heart [2-6]. Since lactate concentrations in blood plasma can be increased under several physiological and pathophysiological conditions, e.g. lactateRinger infusion during cardiopulmonary bypass, moderate to severe physical exercise performed by cardiac patients, circulatory shock, hypoxemia and type I glycogenesis [7, 8], it is important to understand the precise nature of the adverse effect of exogenous lactate on postischaemic cardiac function. Earlier studies have revealed that loss of cardiac function after an ischaemic insult is associated with depletion of high-energy phosphates [9-11]. Hence, it is tempting to state that lactate unfavourably influences cardiac high-energy phosphate metabolism under ischaemic and reperfused conditions. Under oxygen restricted circumstances, adenosine triphosphate (ATP) delivery by mitochondria is limited but alternatively, ATP may be obtained by glycolysis. Opie [12] suggested that ATP produced by glycolysis could be important in protecting membrane function and preventing contracture during ischaemia. The generation of ATP by glycolysis, though, is insufficient to meet the total ATP requirement, a consequence of which is the depletion of high-energy phosphate stores during ischaemia [10, 13]. Catabolites of the nucleotides (i.e. adenosine, inosine, hypoxanthine, xanthine and uric acid) are released into the coronary effluents during reperfusion, which contributes to impaired salvage of ATP during the postischaemic state [14-16]. Evaluation of cardiac nucleotide metabolism shows a high degree of complexity. Firstly, measurements of total ATP levels do not identify ATP produced from anaerobic glycolysis. An indirect estimate of glycolytic ATP formation can be made by determining the glycolytic flux rate including glucose (glycogen) utilization and lactate production. Secondly, the total tissue content of ATP is an indication for the amount of ATP in cytoplasm as about 90% of total ATP is present in cytoplasm [17]. Cytoplasmic hydrolysis of ATP into adenosine diphosphate (ADP) and adenosine monophosphate (AMP) is difficult to detect as the majority of ADP and AMP is bound to actomyosin or compartmentalized in mitochondria [17, 18]. Zucchi and colleagues [19] suggested that detection of the degradation products of ATP (adenosine, inosine, hypoxanthine, xanthine and uric acid) in the coronary effluents may be a more simple and sensitive method to evaluate cardiac energy metabolism. The amount of degradation products released should be related to the cytoplasmic AMP concentration, which in turn is derived from cytoplas-
mic ATP [17, 18]. Moreover, purine release reflects breakdown of myocardial guanine nucleotides. These compounds may also be relevant for energy delivery [201. It was the aim of the present study to evaluate whether the effect of lactate on postischaemic function was dependent on the time of ischaemia; to elucidate whether changes in glycolytic ATP production and cardiac high-energy phosphate levels during ischaemia or failure of reenergization during reperfusion could be held responsible for lactate-induced impairment; to compare the above mentioned changes in the lactate perfused hearts with glucose and pyruvate perfused hearts; to delineate whether there exists a general relationship between high-energy phosphate metabolism and hemodynamic recovery in the hearts subjected to ischaemia and reperfusion. Isolated working rat hearts were perfused with either glucose (11 mM) plus lactate (5 raM), glucose (11 mM) plus pyruvate (5mM) or glucose as sole substrate (11 mM). The hearts were subjected to 15, 30 or 45 min of global no-flow ischaemia in order to vary the severity of ischaemic insult. In a subset of experiments ischaemic hearts were reperfused for 35 min. Cardiac function was monitored by the product of heart rate and left ventricular developed pressure. Glycolytic ATP production was indirectly evaluated by glycogen breakdown and lactate formation during global ischaemia. Changes of the tissue content of high-energy phosphates were assessed by creatine phosphate (CP), ATP and guanosine triphosphate (GTP) measured before ischaemia, after ischaemia and after reperfusion. Breakdown of high-energy phosphates was evaluated by tissue accumulation and/or coronary release of nucleosides and oxypurines. In addition, attention was paid to the sum of nucleotides and their degradation products at the end of reperfusion compared to the sum of these compounds at the end of ischaemia.
Methods Isolated working heart preparation Hearts were obtained from male Lewis rats (10 weeks old, body weight range 250-350 g) under light ether anaesthesia. Isolation and cannulation were performed as previously described [21]. Working hearts were perfused with a modified Krebs-Henseleit bicarbonate buffer (37°C, pH range 7.35-7.45) equilibrated with
95% 02/5% CO 2 (pO2 > 75 kPa). The perfusion buffer contained (in mM): NaC1 (130.0), KCI (5.6), CaCI2 (2.2), MgCI2 (1.2), NaH2PO4 (1.2), NaHCO3 (25.0). D(+) glucose (11mM) was added as a basal substrate. L (+)-Lactate (5 mM) or pyruvate (5 mM, sodium salt) was added as cosubstrate where indicated. The lactate buffer was neutralized by sodiumhydroxide.
Experimental protocol During a stabilization period of 10 min, the hearts were retrogradely perfused according to the Langendorff technique. Then, the hearts were subjected to antegrade perfusion for 30 rain. Left atrial filling pressure was set at 1 kPa and diastolic aortic pressure at 8 kPa. The aortic impedance used resulted in an aortic pressure pulse of about 4.5 kPa [22]. After 30 min of normoxic perfusion, hearts were subjected to either 15, 30 or 45 min of ischaemia [temperature kept constant (37 ° C) by a water jacket and environment moistured and gassed by nitrogen (95% N2 and 5% CO2) bubbling in a water bath] and reperfused for additional 35 min (retrogradely during the first 5 min and antegradely thereafter). Since the hearts were perfused in the assisted mode [22], coronary perfusion pressure (8 kPa) was maintained even when reperfused hearts were unable to provide their own coronary flow. Hemodynamic characteristics [aortic pressure, left ventricular pressure (systolic and end-diastolic) and electrogram] were continuously monitored on a six-channel recorder (Schwarzer) as has been described earlier [21,22]. The pressurerate product was defined as the product of left ventricular developed pressure (difference between systolic and end-diastolic pressure) and heart rate.
Biochemical analysis At the end of either the preischaemic, ischaemic or postischaemic phase, ventricular tissue was rapidly dissected from atrial tissue with a surgical blade and immediately freeze clamped with the use of aluminum tongues, precooled in liquid nitrogen. Throughout the preischaemic and postischaemic phase of the experimental protocol, coronary effluents were collected and sampled. Tissue and effluent samples were stored at - 8 0 ° C until analysis.
Tissue Creatine phosphate, adenine nucleotides, guanine nucleotides, nucleosides, oxypurines, glucose and lactate: small aliquots of deeply frozen ventricle tissue were freeze dried. Metabolites were extracted by an ice-cold mixture of perchloric acid (3 mM) and dithiothreitol (5 mM) and subsequently, neutralized by potassiumbicarbonate [23]. Creatine phosphate was determined fluorometrically [24]. Tissue contents of adenosine tri-, di- and monophosphate (ATP, ADP, AMP), guanosine tri-, di- and monophosphate (GTP, GDP, GMP), inosine monophosphate (IMP), purine nucleosides (adenosine, inosine) and oxypurines (hypoxanthine, xanthine, uric acid) were assayed by high performance liquid chromatography (HPLC; Varian 5500) [16, 25]. Glucose and lactate were determined fluorometrically [24, 261 . Glycogen: Small parts of the frozen ventricles were freeze dried. After adding 1 N hydrochloric acid to the freeze dried material, glycogen was hydrolyzed at 100°C for 3hrs. The samples were neutralized and, subsequently, glucose residues were measured fluorometrically [21, 24]. The values obtained were corrected for the content of free glucose in the tissue.
Coronary effluent The release of nucleosides and oxypurines in coronary effluents was monitored by an isocratic HPLC procedure [16, 27].
Normalization of data Measurements in tissue samples are expressed in moles per gram dry weight. The dry weight of cardiac tissue was determined by freeze drying overnight. Data concerning release of nucleosides and oxypurines in the coronary effluent are expressed in moles per gram wet weight (release per heart corresponds to release per gram wet tissue). For conversion of the release of degradation products per gram wet weight to release of degradation products per gram dry weight an average, empirically determined, factor of 6.25 was used.
Statistical analysis Results are expressed as median values and 95% confidence limits throughout. The number of experiments varied from 6 to 8. Differences within groups were evaluated for significance using Wilcoxon's matchedpairs signed rank test. Differences between groups were evaluated for significance by Mann Whitney U test. P values less than 0.05 were considered statistically significant.
Results Hemodynamics Under preischaemic, normoxic conditions the pressurerate product was not influenced by lactate perfusion compared to glucose or pyruvate perfusion (Table 1). However, the effect of lactate turned out detrimental under ischaemic/reperfused conditions. Whilst after 15 min of ischaemia the recovery of the pressure-rate product was still 96% of the preischaemic value, a marked decrease was observed after 30 min of ischaemia (recovery 5%) in the lactate perfused hearts. In the glucose and pyruvate perfused hearts, the recovery of the pressure rate product was still 78 and 98%, respectively after 30 min of ischaemia and reperfusion. Following 45 min of ischaemia and reperfusion a marked decline of the pressure-rate product was also obvious in the pyruvate perfused hearts (recovery 0%), whereas in the glucose perfused hearts the pressure rate product was less severely reduced (recovery 59% of the preischaemic value).
Consumption of glycogen and production of lactate At the onset of ischaemia, glycogen stores were higher in the lactate and pyruvate perfused hearts (about 180 p~mol glucose equivalents per g dry weight) than in the glucose perfused hearts (109/~mol glucose equivalents per g dry weight, Fig. 1). After 15 min of ischaerfiia, the tissue glycogen content was decreased by 83.9, 91.9 and 98.2/~mol glucose equivalents/g dry weight in the glucose, lactate and pyruvate perfused hearts, respectively. From 15 to 30 min of ischaemia, glycogen consumption was nihil in the glucose and pyruvate perfused hearts, whereas in the lactate perfused hearts glycogen utilization was still considerable (Fig. 1). Af-
ter 45 rain of ischaemia, 96.1, 145.2 and 119.3/zmol glucose equivalents/g dry weight were set free from the cardiac glycogen pool in glucose, lactate and pyruvate perfused hearts, respectively (Fig. 1, calculated by median values). The preischaemic lactate content in cardiac tissue was higher in the lactate than in the glucose or pyruvate peffused hearts (Fig. 2). When a correction was made for the lactate amount in extracellular fluid (corrected value is 13.0 ~mol per g dry weight), the tissue lactate content was still slightly higher in the lactate perfused hearts compared to the glucose or pyruvate peffused hearts ( p < 0.05 and 0.05
