Mechanisms of Ageing and Development, 65 (1992) 199-216
199
Elsevier Scientific Publishers Ireland Ltd.
DIGOXIN CARDIOTOXIC1TY IN AGING ANESTHETIZED F344 RATS*
STUART RUCH, RICHARD H. KENNEDY and ERNST SEIFEN Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 (USA) (Received December 13th, 1991) (Revision received March 1lth, 1992)
SUMMARY
It is generally accepted that the sensitivity to toxic effects of digitalis increases with advancing age; however, the relative contribution of pharmacokinetics and pharmacodynamics to this aging-related change is presently unknown. The current study was designed to determine if senescence affects digitalis tolerance in an animal model and if observed changes are mediated by altered cardiac or autonomic nervous system responsiveness. Male, F344 rats of three age groups (4, 14 and 25 months) were anesthetized and infused intravenously with digoxin at a rate of 880 ~tg/kg per rain. Two separate anesthetic regimens were employed: (a) an age-adjusted dose of urethane in spontaneously breathing animals (AR1); and (b) a non-age-adjusted dose of a-chloralose plus urethane in mechanically ventilated rats (AR2). Heart rate, EKG and arterial blood pressure were monitored continuously; baroreceptor reflex function was estimated before and 10 min following the start of digoxin infusion by examining the response to bilateral carotid occlusion. The infusion time required for digoxin-induced AV-dissociation was significantly reduced by senescence in rats anesthetized by AR1. However, doses of digoxin required to elicit ventricular extrasystoles and death were not significantly different among age groups in this anesthetized model and serum digoxin levels did not differ at the time of cardiac arrest. Similarly, AR2 animals showed a significant aging-related decrease in the time to AV-dissociation. However, in contrast to AR1, animals in AR2 displayed an aging-associated increase in the doses of digoxin required to produce ventricular arrhythmias and cardiac arrest. Thus, results suggest that aging in the F344 rat may, Correspondence to: Ernst Seifen, Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Mail Slot 61 l, 4301 West Markham Street, Little Rock, AR 72205, USA. *This work was supported by US Public Health Service Grant AG05237 from the National Institute on Aging and by a grant from the Arkansas Affiliate of the American Heart Association. RHK is the recipient of a Research Career Development Award from the National Institute on Aging. 0047-6374/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
200 by pharmacodynamic mechanisms, promote the sensitivity to digoxin-induced AVdissociation but not to ventricular arrhythmias or cardiac arrest.
Key words: Aging; Rat; Digitalis toxicity; Heart rate; Arterial blood pressure; Baroreceptor reflex
INTRODUCTION Elderly patients are more susceptible to the toxic effects of digitalis glycosides [1-4] and several studies in humans and animal models indicate that this agingrelated alteration is mediated at least in part by changes in pharmacokinetics including a reduction in drug elimination [5-7]. Age-dependent changes in digitalis sensitivity may also result from alterations in the heart or the autonomic nervous system (ANS). In previous studies using anesthetized Fischer-344 (F344) rats [8], isolated rat cardiac tissue [9,10], guinea pig Langendorff-preparations [11] and anesthetized guinea pigs [12] sensitivity to cardiotoxic effects of cardiac glycosides was increased during senescence. In contrast, digitalis tolerance was found not to be affected by advancing age in conscious beagle dogs [13], anesthetized guinea pigs [14] or myocardial tissue isolated from Wistar rats [15]. This disparity in earlier studies may have been mediated by experimental differences including species, anesthetic regimens, presence or absence of respiratory assistance, parameters used to define digitalis-induced cardiotoxicity and potential influence of pharmacokinetic alterations. Thus, the current study was designed to examine aging-associated changes in the cardiotoxicity of digoxin in an animal model. The F344 rat model of aging was chosen, since it has been widely used in this and other laboratories. Two anesthetic regimens, one with and one without respiratory support, were compared. Various markers of cardiotoxicity were monitored including atrioventricular (AV) dissociation, ventricular extrasystoles and cardiac arrest and serum levels of digoxin were determined in order to assure that observed differences were not the result of pharmacokinetic alterations. Effects of anesthetics and digoxin on baroreceptor reflex function were also estimated since alterations in the ANS may influence digitalis toxicity. METHODS
Animals Male, F344 rats, 4, 14 and 25 months of age (weighing approximately 325,440 and 405 g, respectively), were obtained from a colony raised under contract with the National Institute on Aging (Harlan Sprague-Dawley, Inc., Indianapolis, IN). Animals were maintained for at least 7 days in the Division of Laboratory Animal Medicine
201 at the University of Arkansas for Medical Sciences before being used and had free access to food and water. This research project was approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences.
Anesthetic regimens Two anesthetic regimens were used. Animals under anesthetic regimen 1 (AR1) were given an age-adjusted i.p. doses of urethane which were established in preliminary experiments using the following, well established procedure [16]: four rats of each age group were given an initial, sub-anesthetic i.p. urethane dose. After 30 min, a standardized footclamp of 40 s duration was applied and purposeful movement of the head and/or legs was monitored [16]. A positive response (purposeful movement) was followed by an additional dose of 50 mg/kg urethane i.p. and the footclamp was examined again after 30 min. Additional 50 mg/kg doses were administered until the footclamp response was negative (no purposeful movement). The minimal anesthetic dose in each animal was calculated as the mean of the total dose and the immediately preceding dose which was associated with a positive footclamp response. This value is similar to MAC values (minimal alveolar concentration) utilized to describe the potency of volatile anesthetics [16]. Minimal anesthetic doses for urethane (855 4- 17, 735 ± 21 and 650 4- 20 mg/kg in 4-, 14and 25-month-old rats, respectively) obtained with this technique showed an ageassociated reduction in anesthetic requirement which was similar to that established for other general anesthetics [17,18]. Under AR 1, animals were given urethane doses 10% greater than the minimal anesthetic doses determined for their ages; these animals were allowed to breath spontaneously and respiratory rate was counted every 5 min. Rats under anesthetic regimen 2 (AR2) were given a standard i.p. dose of o~chloralose plus urethane (45 and 450 mg/kg, respectively); this anesthetic regimen was similar to that used in a previous study with F344 rats [8]. When a surgical depth of anesthesia was obtained, a tracheotomy was performed, a tracheal cannula was inserted and respiration was maintained using a small animal ventilator (Model 683, Harvard Apparatus, South Natick, MA). The FIO2 (fractional inspired oxygen content) during mechanical ventilation was approximately 50%. Optimal ventilator settings were established by monitoring arterial blood gases (pH, Po2, Pfoz and HCO3-); 0.2 ml samples were drawn from a femoral artery before tracheotomy (while the animal was breathing spontaneously) and 20 min after beginning mechanical respiration. Adjustments in ventilator settings were made as necessary to maintain blood gas values within physiological limits. In addition, another sample for arterial gases was obtained 20 min after beginning the digoxin infusion. Measurement of cardiovascular parameters After animals were anesthetized, a femoral vein and artery were cannulated to
202
provide access for drug infusion and arterial blood sampling, respectively. The right carotid artery was cannulated for measurement of systemic arterial pressure and the left carotid artery was tagged to permit ready access for occlusion studies. Subcutaneous needle electrodes were placed across the heart on the chestwall and on the right hindlimb to obtain EKG tracings with prominent P and R waves (Fig. 6). Both EKG and arterial pressure signals were monitored continuously using a strip chart recorder (Model 7700, Hewlett Packard, Lexington, MA). Body temperature, as measured via rectal probe, was maintained at 38 ± I°C by a thermostatically controlled infrared heating lamp. After the recorded cardiovascular parameters stabilized, an i.v. infusion of digoxin was started at a rate of 880 #g/kg per min. Digoxin was dissolved in dimethylsulfoxide (DMSO) and the concentration was adjusted such that each animal received an infusion volume of 0.016 ml/kg per min via a constant rate infusion pump (Harvard Apparatus, Model 901, South Natick, MA). The amount of DMSO infused into each animal during the course of an experiment never exceeded 1.0 ml. Immediately before and at 10-min intervals following the start of infusion, the left common carotid artery was clamped for 45 s effectively creating a bilateral carotid occlusion (the right carotid was cannulated), and maximal changes in heart rate and blood pressure were recorded. In addition to examining effects of digoxin on heart rate, arterial pressure and baroreceptor reflex, the EKG was monitored for the onset of AV-dissociation, ventricular arrhythmias and cardiac arrest. AV-dissociation was defined by either: (a) a variable duration PR-interval; or (b) repetitive P waves in the absence of a QRS complex. Ventricular arrhythmia was defined by QRS complexes exceeding 30-ms duration which showed an irregular rhythm for more than 30 s. Cardiac arrest was defined as either ventricular fibrillation lasting longer than 30 s, asystole or a mean arterial pressure less than 10 mmHg. Serum digoxin measurements In AR1 experiments a tracer amount of [3H]digoxin ( ~ 1 #Ci/ml) was added to the infusate. Immediately upon the onset of cardiac arrest, a thoracotomy was performed and a venous blood sample was obtained from the inferior vena cava. The blood sample was centrifuged for I0 rain at 20 × g and a 0.5-ml aliquot of serum was solubilized with 2 ml of NCS Tissue Solubilizer (Amersham Corp., Arlington Heights, IL). The solubilized serum was added to 10 ml of scintillation fluid (Liquiscint, National Diagnostics, Manville, N.J.) and radioactivity was determined via liquid scintillation spectrometry (Tri-Carb 300, Packard Instrument Company, Meriden, CT). The serum digoxin concentration (Cs) at the time of death was calculated using the following equation: Cs = DPMs/DPMI
x VOll/VOls × CI
where DPM represents disintegrations per min, vol represents original sample volumes and C represents digoxin concentration; the subscripts indicate serum (s) or infusate (1).
203
Chemicals [3H)Digoxin (specific activity: 42 mCi/#mol) was purchased from D u p o n t New England Nuclear Products (Boston, MA). Digoxin was obtained f r o m Sigma Chemical C o m p a n y (St. Louis, MO). All other chemicals were reagent grade.
Statistical evaluation D a t a are expressed as means ± S.E.M. Statistical evaluation was done by analysis o f variance with individual values being c o m p a r e d by D u n c a n ' s multiple range test. Criterion for significance was a P value smaller than 0.05.
600 I
b d
4
mo
14
mo
25 mo
500 b c
400
300
a
200
T
100
0 AR1AR2 Confrol
AR1AR2 Maximum
AR1AR2
End
Fig. 1. Heart rate in 4- (open bars), 14- (hatched bars) and 25-month-old (solid bars) F344 rats. Values were obtained before digoxin infusion (control), during the maximal positive chronotropic response (maximum) and approximately 5 min before cardiac arrest (end). Animals were anesthetized with urethane (AR1; n = 8-11) or chloralose-urethane (AR2; n = 6). Vertical bars represent S.E.M. Superscripts indicate statistically significant differences: a4 vs. 14 months; b4 vs. 25 months; c14 vs. 25 months; dvariable vs. its control value; eAR1 vs. AR2.
204 RESULTS
Effects of digoxin on heart rate As shown in Fig. 1, control heart rate (HR) in ARI was greater in 4-month-old than in 14- and 25-month-old animals (392 ~- 13,356 4- 11 and 347 4- 10 beats/min for 4-, 14- and 25-month-olds, respectively). A similar trend in control HR was observed in AR2 (418 4- 24, 381 4- 11 and 380 4- 16 beats/min for 4-, 14- and 25month-old rats, respectively); however, this trend was not statistically significant. Control HR in the individual age groups was not significantly different when compared in the two anesthetic regimens. In ARI, digoxin infusion elicited a positive chronotropic effect which reached maximum values within 10-20 min in all age groups (Fig. 1). Maximal observed HR
200 e
~
25 rno
d
150 b
100 13..
199
Elsevier Scientific Publishers Ireland Ltd.
DIGOXIN CARDIOTOXIC1TY IN AGING ANESTHETIZED F344 RATS*
STUART RUCH, RICHARD H. KENNEDY and ERNST SEIFEN Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 (USA) (Received December 13th, 1991) (Revision received March 1lth, 1992)
SUMMARY
It is generally accepted that the sensitivity to toxic effects of digitalis increases with advancing age; however, the relative contribution of pharmacokinetics and pharmacodynamics to this aging-related change is presently unknown. The current study was designed to determine if senescence affects digitalis tolerance in an animal model and if observed changes are mediated by altered cardiac or autonomic nervous system responsiveness. Male, F344 rats of three age groups (4, 14 and 25 months) were anesthetized and infused intravenously with digoxin at a rate of 880 ~tg/kg per rain. Two separate anesthetic regimens were employed: (a) an age-adjusted dose of urethane in spontaneously breathing animals (AR1); and (b) a non-age-adjusted dose of a-chloralose plus urethane in mechanically ventilated rats (AR2). Heart rate, EKG and arterial blood pressure were monitored continuously; baroreceptor reflex function was estimated before and 10 min following the start of digoxin infusion by examining the response to bilateral carotid occlusion. The infusion time required for digoxin-induced AV-dissociation was significantly reduced by senescence in rats anesthetized by AR1. However, doses of digoxin required to elicit ventricular extrasystoles and death were not significantly different among age groups in this anesthetized model and serum digoxin levels did not differ at the time of cardiac arrest. Similarly, AR2 animals showed a significant aging-related decrease in the time to AV-dissociation. However, in contrast to AR1, animals in AR2 displayed an aging-associated increase in the doses of digoxin required to produce ventricular arrhythmias and cardiac arrest. Thus, results suggest that aging in the F344 rat may, Correspondence to: Ernst Seifen, Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Mail Slot 61 l, 4301 West Markham Street, Little Rock, AR 72205, USA. *This work was supported by US Public Health Service Grant AG05237 from the National Institute on Aging and by a grant from the Arkansas Affiliate of the American Heart Association. RHK is the recipient of a Research Career Development Award from the National Institute on Aging. 0047-6374/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
200 by pharmacodynamic mechanisms, promote the sensitivity to digoxin-induced AVdissociation but not to ventricular arrhythmias or cardiac arrest.
Key words: Aging; Rat; Digitalis toxicity; Heart rate; Arterial blood pressure; Baroreceptor reflex
INTRODUCTION Elderly patients are more susceptible to the toxic effects of digitalis glycosides [1-4] and several studies in humans and animal models indicate that this agingrelated alteration is mediated at least in part by changes in pharmacokinetics including a reduction in drug elimination [5-7]. Age-dependent changes in digitalis sensitivity may also result from alterations in the heart or the autonomic nervous system (ANS). In previous studies using anesthetized Fischer-344 (F344) rats [8], isolated rat cardiac tissue [9,10], guinea pig Langendorff-preparations [11] and anesthetized guinea pigs [12] sensitivity to cardiotoxic effects of cardiac glycosides was increased during senescence. In contrast, digitalis tolerance was found not to be affected by advancing age in conscious beagle dogs [13], anesthetized guinea pigs [14] or myocardial tissue isolated from Wistar rats [15]. This disparity in earlier studies may have been mediated by experimental differences including species, anesthetic regimens, presence or absence of respiratory assistance, parameters used to define digitalis-induced cardiotoxicity and potential influence of pharmacokinetic alterations. Thus, the current study was designed to examine aging-associated changes in the cardiotoxicity of digoxin in an animal model. The F344 rat model of aging was chosen, since it has been widely used in this and other laboratories. Two anesthetic regimens, one with and one without respiratory support, were compared. Various markers of cardiotoxicity were monitored including atrioventricular (AV) dissociation, ventricular extrasystoles and cardiac arrest and serum levels of digoxin were determined in order to assure that observed differences were not the result of pharmacokinetic alterations. Effects of anesthetics and digoxin on baroreceptor reflex function were also estimated since alterations in the ANS may influence digitalis toxicity. METHODS
Animals Male, F344 rats, 4, 14 and 25 months of age (weighing approximately 325,440 and 405 g, respectively), were obtained from a colony raised under contract with the National Institute on Aging (Harlan Sprague-Dawley, Inc., Indianapolis, IN). Animals were maintained for at least 7 days in the Division of Laboratory Animal Medicine
201 at the University of Arkansas for Medical Sciences before being used and had free access to food and water. This research project was approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences.
Anesthetic regimens Two anesthetic regimens were used. Animals under anesthetic regimen 1 (AR1) were given an age-adjusted i.p. doses of urethane which were established in preliminary experiments using the following, well established procedure [16]: four rats of each age group were given an initial, sub-anesthetic i.p. urethane dose. After 30 min, a standardized footclamp of 40 s duration was applied and purposeful movement of the head and/or legs was monitored [16]. A positive response (purposeful movement) was followed by an additional dose of 50 mg/kg urethane i.p. and the footclamp was examined again after 30 min. Additional 50 mg/kg doses were administered until the footclamp response was negative (no purposeful movement). The minimal anesthetic dose in each animal was calculated as the mean of the total dose and the immediately preceding dose which was associated with a positive footclamp response. This value is similar to MAC values (minimal alveolar concentration) utilized to describe the potency of volatile anesthetics [16]. Minimal anesthetic doses for urethane (855 4- 17, 735 ± 21 and 650 4- 20 mg/kg in 4-, 14and 25-month-old rats, respectively) obtained with this technique showed an ageassociated reduction in anesthetic requirement which was similar to that established for other general anesthetics [17,18]. Under AR 1, animals were given urethane doses 10% greater than the minimal anesthetic doses determined for their ages; these animals were allowed to breath spontaneously and respiratory rate was counted every 5 min. Rats under anesthetic regimen 2 (AR2) were given a standard i.p. dose of o~chloralose plus urethane (45 and 450 mg/kg, respectively); this anesthetic regimen was similar to that used in a previous study with F344 rats [8]. When a surgical depth of anesthesia was obtained, a tracheotomy was performed, a tracheal cannula was inserted and respiration was maintained using a small animal ventilator (Model 683, Harvard Apparatus, South Natick, MA). The FIO2 (fractional inspired oxygen content) during mechanical ventilation was approximately 50%. Optimal ventilator settings were established by monitoring arterial blood gases (pH, Po2, Pfoz and HCO3-); 0.2 ml samples were drawn from a femoral artery before tracheotomy (while the animal was breathing spontaneously) and 20 min after beginning mechanical respiration. Adjustments in ventilator settings were made as necessary to maintain blood gas values within physiological limits. In addition, another sample for arterial gases was obtained 20 min after beginning the digoxin infusion. Measurement of cardiovascular parameters After animals were anesthetized, a femoral vein and artery were cannulated to
202
provide access for drug infusion and arterial blood sampling, respectively. The right carotid artery was cannulated for measurement of systemic arterial pressure and the left carotid artery was tagged to permit ready access for occlusion studies. Subcutaneous needle electrodes were placed across the heart on the chestwall and on the right hindlimb to obtain EKG tracings with prominent P and R waves (Fig. 6). Both EKG and arterial pressure signals were monitored continuously using a strip chart recorder (Model 7700, Hewlett Packard, Lexington, MA). Body temperature, as measured via rectal probe, was maintained at 38 ± I°C by a thermostatically controlled infrared heating lamp. After the recorded cardiovascular parameters stabilized, an i.v. infusion of digoxin was started at a rate of 880 #g/kg per min. Digoxin was dissolved in dimethylsulfoxide (DMSO) and the concentration was adjusted such that each animal received an infusion volume of 0.016 ml/kg per min via a constant rate infusion pump (Harvard Apparatus, Model 901, South Natick, MA). The amount of DMSO infused into each animal during the course of an experiment never exceeded 1.0 ml. Immediately before and at 10-min intervals following the start of infusion, the left common carotid artery was clamped for 45 s effectively creating a bilateral carotid occlusion (the right carotid was cannulated), and maximal changes in heart rate and blood pressure were recorded. In addition to examining effects of digoxin on heart rate, arterial pressure and baroreceptor reflex, the EKG was monitored for the onset of AV-dissociation, ventricular arrhythmias and cardiac arrest. AV-dissociation was defined by either: (a) a variable duration PR-interval; or (b) repetitive P waves in the absence of a QRS complex. Ventricular arrhythmia was defined by QRS complexes exceeding 30-ms duration which showed an irregular rhythm for more than 30 s. Cardiac arrest was defined as either ventricular fibrillation lasting longer than 30 s, asystole or a mean arterial pressure less than 10 mmHg. Serum digoxin measurements In AR1 experiments a tracer amount of [3H]digoxin ( ~ 1 #Ci/ml) was added to the infusate. Immediately upon the onset of cardiac arrest, a thoracotomy was performed and a venous blood sample was obtained from the inferior vena cava. The blood sample was centrifuged for I0 rain at 20 × g and a 0.5-ml aliquot of serum was solubilized with 2 ml of NCS Tissue Solubilizer (Amersham Corp., Arlington Heights, IL). The solubilized serum was added to 10 ml of scintillation fluid (Liquiscint, National Diagnostics, Manville, N.J.) and radioactivity was determined via liquid scintillation spectrometry (Tri-Carb 300, Packard Instrument Company, Meriden, CT). The serum digoxin concentration (Cs) at the time of death was calculated using the following equation: Cs = DPMs/DPMI
x VOll/VOls × CI
where DPM represents disintegrations per min, vol represents original sample volumes and C represents digoxin concentration; the subscripts indicate serum (s) or infusate (1).
203
Chemicals [3H)Digoxin (specific activity: 42 mCi/#mol) was purchased from D u p o n t New England Nuclear Products (Boston, MA). Digoxin was obtained f r o m Sigma Chemical C o m p a n y (St. Louis, MO). All other chemicals were reagent grade.
Statistical evaluation D a t a are expressed as means ± S.E.M. Statistical evaluation was done by analysis o f variance with individual values being c o m p a r e d by D u n c a n ' s multiple range test. Criterion for significance was a P value smaller than 0.05.
600 I
b d
4
mo
14
mo
25 mo
500 b c
400
300
a
200
T
100
0 AR1AR2 Confrol
AR1AR2 Maximum
AR1AR2
End
Fig. 1. Heart rate in 4- (open bars), 14- (hatched bars) and 25-month-old (solid bars) F344 rats. Values were obtained before digoxin infusion (control), during the maximal positive chronotropic response (maximum) and approximately 5 min before cardiac arrest (end). Animals were anesthetized with urethane (AR1; n = 8-11) or chloralose-urethane (AR2; n = 6). Vertical bars represent S.E.M. Superscripts indicate statistically significant differences: a4 vs. 14 months; b4 vs. 25 months; c14 vs. 25 months; dvariable vs. its control value; eAR1 vs. AR2.
204 RESULTS
Effects of digoxin on heart rate As shown in Fig. 1, control heart rate (HR) in ARI was greater in 4-month-old than in 14- and 25-month-old animals (392 ~- 13,356 4- 11 and 347 4- 10 beats/min for 4-, 14- and 25-month-olds, respectively). A similar trend in control HR was observed in AR2 (418 4- 24, 381 4- 11 and 380 4- 16 beats/min for 4-, 14- and 25month-old rats, respectively); however, this trend was not statistically significant. Control HR in the individual age groups was not significantly different when compared in the two anesthetic regimens. In ARI, digoxin infusion elicited a positive chronotropic effect which reached maximum values within 10-20 min in all age groups (Fig. 1). Maximal observed HR
200 e
~
25 rno
d
150 b
100 13..
