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Development of a cardiotoxicity screening model using the isolated perfused rat heart
Fd Chem. Toxic. Vol. 24, No. 6/7, pp. 597-598, 1986
0278-6915/86 $3.00+ 0.00 Copyright © 1986 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
DEVELOPMENT OF A CARDIOTOXICITY SCREENING MODEL USING THE ISOLATED P E R F U S E D RAT HEART A. COCKBURN Toxicology Department, Beecham Pharmaceuticals, Research Division, Honeypot Lane, Stock, Nr Ingatestone, Essex CM4 9PE, England
Introduction In preclinical animal studies, the first sign of cardiotoxicity is often limited to sporadic deaths or the findings of terminal histopathology (Marshall & Lewis, 1973; Molello, Gerbig & Robinson, 1973). The poor predictability of myocardial involvement, despite the use of such established diagnostic techniques as physical examination, electrocardiography, measurement of the so-called 'heart-specific' enzymes and heart-weight analysis, is clearly cause for concern and has resulted in the development of a number of elaborate in vitro and in vivo experimental models. Even so, compounds with side effects on the heart have still proved refractory to detection and it is salutary that the cardiotoxic effects of isoprenaline, orciprenaline, hydralazine and diazoxide were first observed in man (Balazs, 1973). We aimed, therefore, to develop a reliable method for the detection of agents with a direct effect on the heart and to validate the procedure with established positive controls. The Langendorff (1895) isolated perfused rat heart system was selected as the basis for this model, as it permits the direct monitoring of parameters of structural and functional integrity free from extracardiac haemodynamic, neural and humoral influences.
Experimental Male Sprague-Dawley rats of the CD strain, weighing 200-300 g, were obtained from Charles River (Manston). Reagents used in the analysis of the perfusate for creatine kinase (CK) activity were obtained from Boehringer Mannheim GmbH (Mannheim, FRG), chemicals used in the preparation of the Krebs-Henseleit bicarbonate perfusion medium (Krebs & Henseleit, 1932) from Fisons Ltd (Loughborough), isoprenaline hydrochloride from Sigma Chemical Co. Ltd (Kingston upon Thames) and heparin BP (1000U/ml) from Evans Medical (Liverpool). The perfusion system was of the non-recirculating type and consisted essentially of an oxygenator, a constant-flow peristaltic pump, an in-line Swinnex 0.45-/1m-pore cellulose acetate filter (Millipore (UK) Ltd, London), a warming coil and bubble trap (Aimer Scientific Supplies, London) and a heart chamber. Both the warming coil and the heart chamber were jacketed and connected via a Capex III circulating pump (Charles Austen Pumps Ltd, Weybridge, Surrey) to a thermostatically controlled water-bath, which kept the temperature of the perfusate leaving
the aortic cannula at 36.5 + 0.5°C. The oxygenator contained perfusate into which O2-CO2 (95:5) was continuously bubbled at 700 ml/min. This provided an oxygen tension of approximately 450 mm Hg. To prepare the heart, rats were lightly anaesthetized with diethyl ether and killed by cervical dislocation. The chest was opened, the heart was lifted upwards and forwards and the great vessels were cut 5 mm distal to the base. The heart was blotted dry, trimmed of any filamentous tissue, transferred to a pre-weighed petri dish containing 0.9% heparinized saline chilled to +4°C, and weighed. During this procedure, spontaneous contractions ceased. The perfusion apparatus was allowed to run for 20 minutes before excision of the heart. The aorta was then slipped onto the cannula and ligatured into place. The heart resumed contraction within seconds. After its mounting on the perfusion cannula, the heart was allowed 30 minutes to stabilize. Test substances were infused at a rate of 0,08 ml/minute by means of a syringe pump over the following 100 minutes and the heart was then allowed a further period of 30 minutes in which to recover. A variety of parameters were measured on the freshly isolated heart to monitor the physiological viability and stability of the preparation. Any hearts not responding within the initial 30-minute stabilization period were rejected. CK activity was measured in 1-2-ml samples of coronary effluent collected during the periods of stabilization (at 1, 5, 15 and 30 minutes), infusion (35, 45, 60, 105 and 130 minutes) and recovery (145 and 160 minutes). Surface electrocardiograms (ECG) were differentially recorded at a sensitivity of 0.4 mV/mm between the aortic stump and ventricular apex, using lead III. Heart rate was calculated directly from ECG paper traces recorded at 25ram/second on a Hewlett Packard model B Electrocardiograph. Coronary flow rate remained between a maximum and minimum of 5.2 and 4.4 ml/minute. Perfusion pressure was recorded as an index of coronary resistance using a mercury manometer connected to a sidearm on the aortic cannula. Oedema formation was quantified by heart-weight measurement before and after the experiment. In order to evaluate the diagnostic potential of the model, isoprenaline (a well-documented cardiotoxin), ischaemia and trauma by lancing were chosen as three distinct means of inducing cardiac damage. Isoprenaline was infused at levels of 1 to 100mg over the 100-minute infusion period approximately equivalent to concentrations of 2 to 200/~g/ml in the
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Durotion of perfusion (rain) Fig. 1. Mean creatine kinase (CK) release (a) before (stabilization period), (b) during and (c) after infusion of isolated perfused rat hearts with 0 (0), 1 (A), 5(E), 25 (O), 50 (Z~) or 100 (D) mg isoprenaline. Each point is the mean for at least four hearts and the SEMs (omitted from the graph to avoid confusion) are, for the 12 time points in sequence: for the control curve--13.7, 7.0, 8.4, 5.8, 5.2, 0, 0, 0, 0, 0, 5.0, 2.3; for l-rag curve--10.5, 4.4, 4.0, 3.7, 3.6, 3.5, 2.3, 3.5, 5.3, 3.7, 8.0, 15.3; for 5-mg curve---8.2, 8.6, 9.6, 4.4, 5.9, 11.5, 16.9, 13.3, 14.0, 36, 31.4, 31.6; for 25-rag curve--2.3, 4.8, 4.9, 6.0, 5.0, 9.8, 6.7, 3.1, 10.0, 14.3, 19.1, 16.7; for the 50-rag curve--10.5, 8.0, 5.3, 2.8, 3.8, 11.1, 9.2, 15.8, 8.5, 14.1, 17.5, 8.2; and for the 100-rag curve---5.1, 2.7, 4.0, 3.9, 4.3, 8.0, 7.0, 3.7, 9.0, 11.0, 8.2, 4.8.
infusate. Coronary emboli were induced by the injection of small volumes of air (c. 0.1 ml) into the aortic reservoir and thence into the coronary circulation in an attempt to produce an experimental model of coronary thrombosis. Physical injury was caused by passing a No. 1 gauge needle through the ventricular apex immediately after the recovery period. Results and Discussion
Under control conditions each of the parameters of viability and stability remained stable, and enzyme release was barely detectable for periods of up to 3 hours. Infusion of isoprenaline caused alterations in each of these indices, with progressive increases in CK activity commencing as early as 15 minutes after the start of the infusion (Fig. 1). Except at the highest dose level isoprenaline increased heart rate, T-wave amplitude and ST segment elevation, the effects being most pronounced during the first 30 minutes of the infusion. An increase of 2 mm Hg in perfusion pressure occurred over the 100-minute test period at all isoprenaline infusions of more than 2/~g/ml. Similar or more marked changes were also induced by microemboli, or by physically damaging the heart by lancing, the latter serving to validate grossly the analytical aspect of each experiment. It is therefore concluded that the modified Langendorff isolated perfused rat heart system can provide a rapid and sensitive method for the detection and ranking of structural and/or functional effects following diverse cardiac insults. The system is robust and can distinguish chemicals that have a
direct action on the heart from those that act indirectly on the regulating influences that normally control heart action. It is proposed that, used alone or in conjunction with conventional screening procedures, this technique can significantly increase the detection of compounds having pharmacological and/or toxicological activity on the heart. Moreover the system has the potential to be developed further to provide useful insight into the pathophysiological mechanisms of compounds such as the anthracyclines, the clinical utility of which is limited by their cardiotoxic effects (Lahtinen, Uusitupa, Kuikka & Lansimies, 1982). REFERENCES
Balazs T. (1973). In Experimental Model Systems in Toxicology and Their Significance in Man. Edited by W. A. M. Duncan. Vol. 15. p. 71. Excerpta Medica, New York. Krebs H. A. & Henseleit K. (1932). t0ntersuchungen iiber die Harnstoffbildung in Tierkorper. Z. Physiol. Chem. 210, 33. Lahtinen R., Uusitupa M., Kuikka J. & Lansimies E. (1982). Non-invasive evaluation of anthracycline-induced cardiotoxicity in man. Acta reed. scand. 212, 201. Langendorff O. (1985). Untersuchungen am /iberlebenden Sfiugetierherzen. Pflfigers. Arch. ges. Physiol. 61, 291. Marshall F. N. & Lewis J. E. (1973). Sensitization to epinephrine-induced ventricular fibrillation produced by probucol in dogs. Toxic. appl. Pharmac. 24, 594. MoleUo J. A., Gerbig C. G. & Robinson V. B. (1973). Toxicity of [4,4'-(isopropylidenodithio)bis-(2,6-di-t-butylphenol)] Probucol, in mice, rats, dogs and monkeys: Demonstration of a species-specific phenomenon. Toxic. appl. Pharmac. 24, 590.
0278-6915/86 $3.00+ 0.00 Copyright © 1986 Pergamon Journals Ltd
Printed in Great Britain. All rights reserved
DEVELOPMENT OF A CARDIOTOXICITY SCREENING MODEL USING THE ISOLATED P E R F U S E D RAT HEART A. COCKBURN Toxicology Department, Beecham Pharmaceuticals, Research Division, Honeypot Lane, Stock, Nr Ingatestone, Essex CM4 9PE, England
Introduction In preclinical animal studies, the first sign of cardiotoxicity is often limited to sporadic deaths or the findings of terminal histopathology (Marshall & Lewis, 1973; Molello, Gerbig & Robinson, 1973). The poor predictability of myocardial involvement, despite the use of such established diagnostic techniques as physical examination, electrocardiography, measurement of the so-called 'heart-specific' enzymes and heart-weight analysis, is clearly cause for concern and has resulted in the development of a number of elaborate in vitro and in vivo experimental models. Even so, compounds with side effects on the heart have still proved refractory to detection and it is salutary that the cardiotoxic effects of isoprenaline, orciprenaline, hydralazine and diazoxide were first observed in man (Balazs, 1973). We aimed, therefore, to develop a reliable method for the detection of agents with a direct effect on the heart and to validate the procedure with established positive controls. The Langendorff (1895) isolated perfused rat heart system was selected as the basis for this model, as it permits the direct monitoring of parameters of structural and functional integrity free from extracardiac haemodynamic, neural and humoral influences.
Experimental Male Sprague-Dawley rats of the CD strain, weighing 200-300 g, were obtained from Charles River (Manston). Reagents used in the analysis of the perfusate for creatine kinase (CK) activity were obtained from Boehringer Mannheim GmbH (Mannheim, FRG), chemicals used in the preparation of the Krebs-Henseleit bicarbonate perfusion medium (Krebs & Henseleit, 1932) from Fisons Ltd (Loughborough), isoprenaline hydrochloride from Sigma Chemical Co. Ltd (Kingston upon Thames) and heparin BP (1000U/ml) from Evans Medical (Liverpool). The perfusion system was of the non-recirculating type and consisted essentially of an oxygenator, a constant-flow peristaltic pump, an in-line Swinnex 0.45-/1m-pore cellulose acetate filter (Millipore (UK) Ltd, London), a warming coil and bubble trap (Aimer Scientific Supplies, London) and a heart chamber. Both the warming coil and the heart chamber were jacketed and connected via a Capex III circulating pump (Charles Austen Pumps Ltd, Weybridge, Surrey) to a thermostatically controlled water-bath, which kept the temperature of the perfusate leaving
the aortic cannula at 36.5 + 0.5°C. The oxygenator contained perfusate into which O2-CO2 (95:5) was continuously bubbled at 700 ml/min. This provided an oxygen tension of approximately 450 mm Hg. To prepare the heart, rats were lightly anaesthetized with diethyl ether and killed by cervical dislocation. The chest was opened, the heart was lifted upwards and forwards and the great vessels were cut 5 mm distal to the base. The heart was blotted dry, trimmed of any filamentous tissue, transferred to a pre-weighed petri dish containing 0.9% heparinized saline chilled to +4°C, and weighed. During this procedure, spontaneous contractions ceased. The perfusion apparatus was allowed to run for 20 minutes before excision of the heart. The aorta was then slipped onto the cannula and ligatured into place. The heart resumed contraction within seconds. After its mounting on the perfusion cannula, the heart was allowed 30 minutes to stabilize. Test substances were infused at a rate of 0,08 ml/minute by means of a syringe pump over the following 100 minutes and the heart was then allowed a further period of 30 minutes in which to recover. A variety of parameters were measured on the freshly isolated heart to monitor the physiological viability and stability of the preparation. Any hearts not responding within the initial 30-minute stabilization period were rejected. CK activity was measured in 1-2-ml samples of coronary effluent collected during the periods of stabilization (at 1, 5, 15 and 30 minutes), infusion (35, 45, 60, 105 and 130 minutes) and recovery (145 and 160 minutes). Surface electrocardiograms (ECG) were differentially recorded at a sensitivity of 0.4 mV/mm between the aortic stump and ventricular apex, using lead III. Heart rate was calculated directly from ECG paper traces recorded at 25ram/second on a Hewlett Packard model B Electrocardiograph. Coronary flow rate remained between a maximum and minimum of 5.2 and 4.4 ml/minute. Perfusion pressure was recorded as an index of coronary resistance using a mercury manometer connected to a sidearm on the aortic cannula. Oedema formation was quantified by heart-weight measurement before and after the experiment. In order to evaluate the diagnostic potential of the model, isoprenaline (a well-documented cardiotoxin), ischaemia and trauma by lancing were chosen as three distinct means of inducing cardiac damage. Isoprenaline was infused at levels of 1 to 100mg over the 100-minute infusion period approximately equivalent to concentrations of 2 to 200/~g/ml in the
597 F C T 2416-7--K
598
A. COCKBURN
11o
(o)
(b)
(c)
I
80
"tO I .-_
60
--'5Oi
°°
I!
Io °
~J
l
0 5
15
IJ 30 35 45
J
i
T
T
T
60
80
105
130
i
J
145
160
Durotion of perfusion (rain) Fig. 1. Mean creatine kinase (CK) release (a) before (stabilization period), (b) during and (c) after infusion of isolated perfused rat hearts with 0 (0), 1 (A), 5(E), 25 (O), 50 (Z~) or 100 (D) mg isoprenaline. Each point is the mean for at least four hearts and the SEMs (omitted from the graph to avoid confusion) are, for the 12 time points in sequence: for the control curve--13.7, 7.0, 8.4, 5.8, 5.2, 0, 0, 0, 0, 0, 5.0, 2.3; for l-rag curve--10.5, 4.4, 4.0, 3.7, 3.6, 3.5, 2.3, 3.5, 5.3, 3.7, 8.0, 15.3; for 5-mg curve---8.2, 8.6, 9.6, 4.4, 5.9, 11.5, 16.9, 13.3, 14.0, 36, 31.4, 31.6; for 25-rag curve--2.3, 4.8, 4.9, 6.0, 5.0, 9.8, 6.7, 3.1, 10.0, 14.3, 19.1, 16.7; for the 50-rag curve--10.5, 8.0, 5.3, 2.8, 3.8, 11.1, 9.2, 15.8, 8.5, 14.1, 17.5, 8.2; and for the 100-rag curve---5.1, 2.7, 4.0, 3.9, 4.3, 8.0, 7.0, 3.7, 9.0, 11.0, 8.2, 4.8.
infusate. Coronary emboli were induced by the injection of small volumes of air (c. 0.1 ml) into the aortic reservoir and thence into the coronary circulation in an attempt to produce an experimental model of coronary thrombosis. Physical injury was caused by passing a No. 1 gauge needle through the ventricular apex immediately after the recovery period. Results and Discussion
Under control conditions each of the parameters of viability and stability remained stable, and enzyme release was barely detectable for periods of up to 3 hours. Infusion of isoprenaline caused alterations in each of these indices, with progressive increases in CK activity commencing as early as 15 minutes after the start of the infusion (Fig. 1). Except at the highest dose level isoprenaline increased heart rate, T-wave amplitude and ST segment elevation, the effects being most pronounced during the first 30 minutes of the infusion. An increase of 2 mm Hg in perfusion pressure occurred over the 100-minute test period at all isoprenaline infusions of more than 2/~g/ml. Similar or more marked changes were also induced by microemboli, or by physically damaging the heart by lancing, the latter serving to validate grossly the analytical aspect of each experiment. It is therefore concluded that the modified Langendorff isolated perfused rat heart system can provide a rapid and sensitive method for the detection and ranking of structural and/or functional effects following diverse cardiac insults. The system is robust and can distinguish chemicals that have a
direct action on the heart from those that act indirectly on the regulating influences that normally control heart action. It is proposed that, used alone or in conjunction with conventional screening procedures, this technique can significantly increase the detection of compounds having pharmacological and/or toxicological activity on the heart. Moreover the system has the potential to be developed further to provide useful insight into the pathophysiological mechanisms of compounds such as the anthracyclines, the clinical utility of which is limited by their cardiotoxic effects (Lahtinen, Uusitupa, Kuikka & Lansimies, 1982). REFERENCES
Balazs T. (1973). In Experimental Model Systems in Toxicology and Their Significance in Man. Edited by W. A. M. Duncan. Vol. 15. p. 71. Excerpta Medica, New York. Krebs H. A. & Henseleit K. (1932). t0ntersuchungen iiber die Harnstoffbildung in Tierkorper. Z. Physiol. Chem. 210, 33. Lahtinen R., Uusitupa M., Kuikka J. & Lansimies E. (1982). Non-invasive evaluation of anthracycline-induced cardiotoxicity in man. Acta reed. scand. 212, 201. Langendorff O. (1985). Untersuchungen am /iberlebenden Sfiugetierherzen. Pflfigers. Arch. ges. Physiol. 61, 291. Marshall F. N. & Lewis J. E. (1973). Sensitization to epinephrine-induced ventricular fibrillation produced by probucol in dogs. Toxic. appl. Pharmac. 24, 594. MoleUo J. A., Gerbig C. G. & Robinson V. B. (1973). Toxicity of [4,4'-(isopropylidenodithio)bis-(2,6-di-t-butylphenol)] Probucol, in mice, rats, dogs and monkeys: Demonstration of a species-specific phenomenon. Toxic. appl. Pharmac. 24, 590.