Electrophysiological Mechanisms of Cocaine-induced Cardiac Arrest A Possible Cause of Sudden Cardiac Death
Hassen T. Sherief, MD, and Robert G. Carpentier, MD
Abstract: The hypothesis that cocaine intoxication results in cardiac arrest by producing a block of the propagation of the action potential, without loss of pacemaker function, was tested in rat cardiac tissues. In spontaneously active sinoatrial preparations, cocaine exerted a dose-dependent negative chronotropic action, which was not modified by atropine or propranolol. Sinus node arrest was never observed. Instead, cocaine produced sinoatrial block. The mechanism of this block involved a fall in the resting potential and a decrease in the amplitude and Vmax of phase 0 of the action potential of atria1 fibers. In sinoatrial preparations and papillary muscles driven at 5 Hz, cocaine depressed the resting potential, the total amplitude, the overshoot of the action potential, and the Vmax of phase 0. Cocaine had a biphasic effect on the atrial action potential duration. The initial shortening was muscarinic. The prolongation was a-adrenergic mediated and probably the result of the inhibition of the transient outof the action poward current I,,. In papillary muscles, only the prolongation tential occurred. In conclusion, the electrophysiological actions of cocaine can explain cardiac sudden death. The fall in the resting potential associated with the decrease in the amplitude of the action potential of contractile fibers will result in a block of the propagation of the action potential. Quiescence of the contractile fibers will occur while the sinus node is still generating action potentials at a rate compatible with life. Key Words: sinus node, membrane potentials, sinoatrial conduction, sinoatriai block, myocardial excitability, arrhythmias.
Cocaine can produce myocardial infarction, pressed contractility, and cardiac arrhythmias,
eluding ventricular fibrillation and cardiac arrest. ’ In spite of the urgent need for information on the mechanisms of the cardiac arrhythmias, there is little in the literature about the acute effects of cocaine on the electrophysiology of the heart. Local anesthetic effects and class I antiarrhythmic actions have been demonstrated in cardiac tissues.2,3 The aim of our investigation was to test the hypothesis that cocaine intoxication can result in sudden cardiac death due to cardiac arrest. The results reported here show that cocaine exerts electrophysiological actions on the rat
dein-
From the Department of Physio1og.v and Biophvsics, College of Medicine. Howard University. Washington, DC This investigation is in partial fulfillment of the requirements for Dr. Sheriefs Ph.D. degree. Supported by grants from the NIHIMBRS and National Heart, Lung and Blood Institute and from the Howard University Faculty Research Prograq. Reprint requests: Dr. Robert G. Carpentier. Department of Physiology and Biophysics, College of Medicine, Howard University, 520 W Street NW, Washington, DC 20059.
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heart that result in cardiac arrest, caused by a block of the propagation of the action potential through the myocardial contractile fibers, without loss of the pacemaker function. When quiescence occurs, the sinus node is still generating action potentials at a rate compatible with life, but these impulses fail to generate conducted action potentials in the contractile fibers.
Materials
and Methods
Adult (300-350 g) male Sprague-Dawley rats from Charles River Breeding Labs Co. were used. The animals were euthanized by decapitation under barbiturate sedation (Sodium Pentobarbital, Sigma Chemical Co., 60 mg/kg intraperitoneally) and the heart was quickly excised. The in vitro preparations and the recording system have been previously described in detai1.4-7 A small strip of the right atrium containing the sinus node and a portion of the crista terminalis was set up, endocardial side up, in a constant-temperature (37’C) tissue bath. The tissue chamber (total volume: 1 ml) was perfused with a physiological solution flowing at 5 ml/min through tubing with a total volume of less than 1 ml. The atria1 strips were superfused with oxygenated and buffered (95% O2 - 5% C02; pH: 7.4) Tyrode’s solution. The physiological solution contained the following (mM): NaCl 137, KC1 5.4, CaCI, 2.7, MgCl, 0.5, NaHC03 11.9, NaH,P04 0.45, and glucose 5.55. All preparations were equilibrated with the Tyrode’s solution for at least 60 minutes before any experimental procedure was carried out.
Studies With Preparations Spontaneously Active The preparations were equilibrated in Tyrode’s solution while beating spontaneously. Therefore, the sinus node rate (SNR) was monitored with standard intracellular microelectrodes filled with 3 M KCl, using multiple cell impalements in different areas of the preparation. Multiple atria1 fibers located between the sinus node and the crista terminalis were impaled. It was also possible to impale sinus node subsidiary pacemaker fibers located in the periphery of the sinus node in the endocardial surface. However, it was not possible to impale true pacemaker cells, which are located intramurally in the rat heart. This procedure allowed us to establish the control
SNR and to determine if there were irregularities or areas of blocked conduction. Only the preparations with a regular and steady control SNR and without areas of block were used in this study. At the end of the equilibration period, the control SNR was recorded. The superfusate was then changed to Tyrode’s solution containing cocaine (cocaine hydrochloride). The following drugs were also employed: atropine sulfate ( lop5 M) and propran0101 hydrochloride ( 10 - ’ M). These concentrations of the muscarinic and P-adrenergic antagonists are known to effectively block muscarinic- and p-adrenergic-mediated actions without affecting directly the SNR.12 All drugs were purchased from Sigma Chemical Co., Missouri. All solutions were kept in the dark and protected from direct light during the superfusion. The SNR was recorded before (control), during, and after (recovery) superfusion with the drug(s). For each experimental series, data are reported as the mean +- the standard error of the mean (SEM). The statistical analysis was done using the Student’s t test for paired data. Values obtained at the end of the exposure to the drug(s) or at the end of the recovery period were compared to those collected during the control period in the same preparation. Significance was set at p values less than 0.0 1. Analysis of variance (ANOVA) was used to compare several means resulting from various interventions. The level of significance is given each time a comparison is made.
Studies With Preparations Driven at a Constant Rate The preparations were driven electrically at a frequency of 5 Hz by a Grass stimulator (S-88) with an SIU5 isolation unit. Square pulses of voltage 50% above threshold of 1 ms duration were delivered through a pair of silver electrodes in close contact with one end of the preparation. The membrane potentials of atria1 fibers located between the sinus node and the crista terminalis were recorded with intracellular microelectrodes filled with 3 M I 0.2. In other words, the maximum effect was already attained with cocaine 20 mg/l. Figure 3 illustrates the effects of cocaine on atria1 fibers located between the sinus node and the crista terminalis in two experiments in which a single im-
A
palement was maintained throughout the exposure to cocaine. The tracings in the top row show that cocaine 30 mg/l produced a fall in the RP associated with a marked decrease in the APA. Eventually, a sinoatrial block developed, with some action potentials not being conducted to the atria1 tissue. Only a small electrotonic potential was detected each time an action potential was not conducted. Similar sinoatrial blocks were observed within 20 minutes in four of six preparations, while sinus node arrest never occurred. In four additional experiments, the exposure to cocaine was maintained until a total sinoatrial block occurred (between 30 and 45 minutes) and attempts were made to stimulate the preparations with electrical pulses immediately after the atria1 contractile fibers became quiescent. In three cases it was possible to drive the preparations at a rate similar to that of the sinus node with stimuli
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Membrane potentials of atria1 fibers located between the sinus node and the crista terminalis, in preparations with spontaneous activity. Top row: single cell impalement in a preparation exposed to cocaine 30 mg/l. Middle row: single cell impalement in a preparation exposed to cocaine 40 mgil. Tracings in panels c and f (bottom row) are the same as those in panels C and F, but at a higher sensitivity. Voltage calibrations (marks on panels A, D, and c): 0 to - 50 mV for top and middle rows; 25 mV for bottom row. Time calibration (horizontal line below panel D) = 300 ms. Control tracings are in panels A and D. Tracings in panels B and C were recorded at 10 and 20 minutes of exposure to the drug. Tracings in panels E and F were recorded at 4 and 5 minutes of exposure to the drug. Filled circles mark electrotonic potentials determined by sinus node action potentials blocked at the sinoatrial junction. Fig. 3.
Effects of Cocaine
on Cardiac
three to four times the normal threshold intensity. The action potentials recorded from the contractile fibers were similar to those recorded immediately before the block occurred. In one case the preparation was unexcitable. The second row in Figure 3 shows that a similar but more accentuated phenomenon was observed with cocaine 40 mg/l. In all six preparations, a complete sinoatrial block occurred within 5 minutes while the sinus node was still firing regularly. Small regular electrotonic potentials with a constant interval were always present in the transitional area between the sinus node and the crista terminalis. By moving the microelectrode towards the sinus node, it was possible to record action potentials from latent pacemaker fibers. The intervals between the action potentials recorded in this area and the electrotonic potentials recorded in the transitional area were the same. In three additional experiments, attempts were made to stimulate the preparations with electrical pulses immediately after the atria1 contractile
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251
fibers became quiescent. In all cases the preparations were unexcitable.
Effects of Cocaine on Membrane Potentials of Atrial Fibers in Sinoatrial Preparations Driven at a Constant Rate
Cocaine 5 mg/l produced a fall in the RP associated with a decrease in the APA and the Vmax 0 of atria1 fibers (Fig. 4), in the absence of any significant change in the OS or the APD. A higher concentration of cocaine (20 mg/l) produced greater decreases in the magnitudes of the RP, the APA, and the Vmax 0 (Fig. 5). In addition, this concentration of cocaine reduced the OS and produced biphasic changes in the APD. Cocaine 20 mg/l significantly reduced (p < 0.01) the APD measured at - 70 mV from 6 1.5 2 0.8 ms to 55.4 ? 1.4 ms within 1 minute. However, when
-70 1
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4. Effects of cocaine 5 mg/l on membrane potentials of atrial fibers located between the sinus node and terminalis. The preparations were driven electrically at 5 Hz. CON = control values recorded immediately before to the drug; COC = values obtained at the end of a 3-minute exposure to the drug; REC = recovery values, 10 minutes after withdrawal of the drug from the superfusate; each data point represents the mean I SEM *significantly different from control value, p < 0.0 1. obtained from 7 preparations in single impalements; Fig.
the crista exposure obtained of values
Journal
252
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Vol. 24 No. 3 July 1991
-70 -
120-
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ACllON POlENTlM AMPLITUDE
110.
-75
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Fig. 5. Effects of cocaine 20 mgil on membrane potentials of atria1 fibers located between the sinus node and the crista terminalis. The preparations were driven electrically at 5 Hz. CON = control values recorded immediately before exposure to the drug; COC = values obtained at the end of a 3-minute exposure to the drug; REC = recovery values, obtained 10 minutes after withdrawal of the drug from the superfusate; each data point represents the mean k SEM of values * = significantly different from control value, p < 0.01. obtained from 6 preparations in single impalements;
atropine was added to the superfusate, there was no difference (p > 0.05) between the control value (64.4 k 1.3 ms) and the value collected 1 minute after cocaine was added to the Tyrode’s solution (6 1.7 k 1.9 ms). In other words, the muscarinic blockade abolished the initial shortening action of cocaine on the APD. The addition of atropine to the superfusate also accentuated the subsequent prolonging action of cocaine on APD. In the absence of atropine, the APD measured at - 70 mV was prolonged by 6.5 2 2.1 ms (p < 0.01) at the end of the 3-minute exposure to cocaine, while in the presence of the muscarinic antagonist, the APD was prolonged by 16.5 I 3.6 ms (Fig. 6). The mechanism of the prolonging action of cocaine on APD was studied in preparations superfused with Tyrode’s solution containing either phentolamine or atropine and 4-aminopyridine. Figure 6 shows that cocaine did not prolong the APD when tested in the presence of the cw-adrenergic antagonist phentolamine. Figure 6 also shows that 4aminopyridine,
25
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Fig. 6. Effects of cocaine 20 mg/l on the action potential duration of atria1 fibers, measured at - 70 mV of repolarization (APD-70) in preparations driven at 5 Hz. TYR = cocaine alone in Tyrode’s solution; PHEN = cocaine in the presence of phentolamine; ATROP = cocaine in the presence of atropine; ATROP and 4-AP = cocaine in the presence of atropine and 4-aminopyridine; each value is the mean k SEM of values obtained from 6 preparations in *significantly different from control single impalements; value, p < 0.0 1.
Effects of Cocaine -70
-75
1
;i
Electrophysiology
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253
ACTION POTENTLAL AMPLITUDE
RESTING POTENTIAL
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7. Effects of cocaine 20 mg/l on membrane potentials of ventricular fibers in papillary muscles driven at 5 Hz. CON = control values recorded immediately before exposure to the drug; COC = values obtained at the end of a 3-minute exposure to the drug; REC = recovery values, obtained 10 minutes after withdrawal of the drug from the superfusate; each data point represents the mean 2 SEM of values obtained from 6 preparations in single impalements; *significantly different from control value, p < 0.0 1.
which inhibits the transient outward current I,,,” abolished the prolonging action of cocaine on APD.
Effects of Cocaine on Membrane Potentials of Papillary Muscles
The effects of cocaine 20 mg/l on the magnitude of membrane potentials of papillary muscles were very similar to those described for atria1 muscle (Fig. 7). On the other hand, the initial shortening of APD was not observed, while the subsequent prolongation was present. The control value for the APD measured at - 70 mV was 50.2 2 1.6 ms. At 1 minute of exposure to cocaine, it was still 49.2 + 2.1 ms (not different from control value, p > 0.05). At the end of the 3-minute exposure to the drug, the APD was 57.3 + 2.2 ms (significantly different from control value, p < 0.01).
Discussion
The “recreational” use of cocaine has reached epidemic proportions in the United States.12 It is now well known that cocaine intoxication often results in cardiac sudden death.’ Our results show that the concentrations used in our experiments produced cardiac arrest in the rat. It might be argued that the concentrations used were very high. Interestingly, the levels of blood cocaine attained in acute intoxication are much higher than previously believed. A recent autopsy study indicates that the concentration of cocaine in blood in fatal acute intoxications was on average 5.3 ? 8.1 mg/l, in one case reaching 24 mg/l. ’ 3 It is also true that it is impossible to precisely compare in vitro superfusate levels of cocaine with the levels actually present in the cardiac extracellular space in vivo. In terms of the mechanisms responsible for the cardiotoxic effects of cocaine, the emphasis has been
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on the adrenergic and anesthetic effects of the drug, which would act synergistically to elicit and maintain ventricular fibrillation or similar “malignant arrhythmias.“’ Our results show that cocaine can also produce sudden cardiac death through a different mechanism-cardiac arrest. In the rat, a rapid increase in extracellular cocaine concentration in vitro to levels in the range of those found in fatal acute intoxication produces a decrease in the amplitude of the action potentials and a fall in the resting potential of atria1 and ventricular fibers. Eventually, a block of the propagation of the action potentials to the contractile cells occurs, while the sinus node is still generating action potentials at a rate compatible with life. With the highest concentration tested, all preparations become unexcitable, as shown by the fact that it is not possible to trigger action potentials with electrical stimulation. The absence of a positive chronotropic effect of cocaine on the rat sinus node indicates a species difference. In dogs, intravenous cocaine produces adrenergically mediated rapid increases in blood pressure, cardiac output, and heart rate. 14,15On the other hand, in anesthetized and conscious rats, intravenous cocaine also elicits a biphasic presser/depressor effect, but this is associated with no change or a slight decrease in heart rate. l6 Our investigation of the direct effects of cocaine on the SNR in vitro supports the hypothesis that cocaine does not exert positive chronotropic actions on the rat sinus node. The reason for this absence of adrenergic-mediated, cocaineinduced stimulation of sinus node automaticity is still unknown. Our results indicate that cocaine does not produce sinus node arrest in the rat. The maximum negative chronotropic action was attained with 20 mg/l. Cocaine 30 and 40 mg/l had a similar effect on the SNR but produced a sinoatrial block. The mechanism of the atria1 arrest is similar to that of hypothermia.’ In both cases, the cause of the atria1 arrest is a block of conduction between the sinus node and the atrium and not a cessation of pacemaker activity of the sinus node. The mechanism of this block involves a fall in the resting potential and a decrease in the amplitude and Vmax of phase 0 of the action potential of atria1 fibers. The changes in the action potential are the consequence of both the anesthetic action of cocaine on the fast sodium system2,3 and the fall in the resting potential. ” Our results also show that cocaine exerts similar effects on membrane potentials of atria1 and ventricular fibers. It is therefore reasonable to postulate that acute cocaine intoxication will result in cardiac arrest in the absence of sinus node arrest, even if latent ectopic pacemaker fibers are firing, simply because the propagation of the action potentials
through the myocardium is blocked. Of course this does not exclude other possible mechanisms of cardiac arrest that may result in “malignant arrhythmias.“’ In preparations driven at a low rate (43/min), Przywara and Dambach found that cocaine 60 p.M (20 mg/l) did not affect the resting potential or the overshoot of the action potential of atria1 and ventricular fibers from rabbit hearts. Species differences and/or frequency-dependence of the actions of cocaine on these two parameters would explain the difference between their results and those reported here. Voltage clamp studies will be required to characterize the possible actions of cocaine on the currents responsible for the resting potential and the overshoot of the action potenial. The effects of cocaine on the duration of the action potential are of interest. The drug had a biphasic effect on the APD of atria1 fibers, while it had only an APD-prolonging action in the papillary muscles. The initial brief shortening effect on the APD of atria1 fibers was blocked by atropine, indicating that it is a muscarinic action. The subsequent APD-prolonging action, which has been described recently by others,’ was abolished by the c-w-adrenergic antagonist phentolamine. This is not surprising: ol-adrenergic stimulation is known to prolong the APD of rat cardiac fibers, both in multicellular preparations’0,‘8 and in isolated myocytes.” The fact that this APDprolonging effect of cocaine was also blocked by 4aminopyridine provides further support to the concept that this a is an a-adrenergic-mediated action resulting in the inhibition of the current I,,,. Tohse et al.” recently showed that the inhibition of I,,, is the primary cause of the prolongation of the APD produced by a-adrenergic stimulation. The results of this investigation may be of clinical significance. We have shown that cocaine may produce sudden cardiac death by a mechanism other than the production of “malignant arrhytmias.“’ It is true that the electrophysiology of the rat myocardium is different from that of other mammals and this has to be considered before any speculation is made about possible effects of cocaine on human myocardium. However, with these results in vitro, it becomes necessary to fully investigate this possible mechanism in the entire animal. Interestingly, in a recent study in dogs, in which the plasma concentrations of cocaine attained were similar to the ones used in our experiments, a cocaine-mediated impairment of cardiac conduction was proposed as a potential mechanism for sudden death.20 Thus, there seems to already be enough experimental evidence to at least consider cardiac arrest as one of the pos-
Effects
sible causes of sudden ication in humans.
death
of Cocaine
in acute cocaine
on Cardiac
intox-
References 1. Billman GE: Mechanisms responsible for the cardiotoxic effects of cocaine. FASEB J. 4:2469, 1990 2. Weidmann S: Effects of calcium ions and local anesthetics on electrical properties of Purkinje fibres. J Physiol (Lond) 129:568, 1955 3. Przywara DA, Dambach GE: Direct actions of cocaine on cardiac cellular electrical activity. Circ Res 65: 185, 1989 4. Carpentier RG, Posner P, Bloom S: Sinoatrial automaticity and transmembrane potentials in hamsters on a magnesium-deficient diet. J Cardiovasc Pharmacol 7:919, 1985 A: Acute and 5. Carpentier RG, Gallardo-Carpentier chronic effects of ethanol on sinoatrial electrophysiology in the rat heart. J Cardiovasc Pharmacol 10:616, 1987 6. Salvatici RP, Carry1 OR, Isaacson RL et al: Actions and interactions of dihydropyridines and ethanol on the rat sinus node. Cardiovasc Drugs Ther 3:177, 1989 Salvatici RP, Isaacson RL, Carpentier RG: Actions and interactions of calcium modulators and ethanol on atria1 membrane potentials. J Electrocardiol 23: 157, 1990 Brown RA, Carpentier RG: Effects of acetaldehyde on membrane potentials of sinus node pacemaker fibers. Alcohol 7:33, 1990 Carpentier RG, Vassalle M: Restoration of electrical activity of guinea pig atria during hypothermia: effects of norepinephrine and electrical stimulation on membrane potentials. Circ Res 3 1: 507, 1972
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10. Brown RA, Carpentier RG: Alpha-adrenoceptor-mediated effects of norepinephrine on the guinea pig sinus node. J Electrocardiol 2 I:2 13, i 988 11. Tohse N, Nakaya H, Hattori Y et al: Inhibitory effect mediated by alpha, -adrenoceptors on transient outward current in isolated rat ventricular cells. Pflugers Arch 415:575, 1990 12. Abelson HI, Miller JD: A decade of trends in cocaine use in the household population. NIDA Res Monogr 61:25, 1985 13. Virmani R, Robinowitz M, Smialek JE et al: Cardiovascular effects of cocaine: an autopsy study of 40 patients. Am Heart J 115: 1068, 1988 14. Catravas JD, Waters EW, Walz MA et al: Acute cocaine intoxication in the conscious dog: pathophysiologic profile of acute lethality. Arch Int Pharmacodyn Ther 235:328, 1978 15. Bedotto JB, Lee RW, Lancaster LD et al: Cocaine and cardiovascular function in dogs: effects on heart and peripheral circulation. J Am Coil Cardiol 11: 1337, 1988 16. Pitts DK, Udom CE, Marvah J: Cardiovascular effects of cocaine in anesthetized and conscious rats. Life Sci 40: 1099, 1987 17. Weidmann S: Effect of the cardiac membrane potential on the rapid availability of the sodium-carrying system. J Physiol (Lond) 127:213, 1955 18. Tohse N, Hattori Y, Nakaya H et al: Effects of alphaadrenoceptor stimulation on electrophysiological properties and mechanics in rat papillary muscle. Gen Pharmacol 18:539, 1987 regulation of 19. Vogel SM, Terzic A: Alpha-adrenergic action potentials in isolated rat cardiomyocytes. Eur J Pharmacol 164:231, 1989 20. Kabas JS, Blanchard SM, Matsuyama Y et al. Cocainemediated impairment of cardiac conduction in the dog: a potential mechanism for sudden death after cocaine. J Pharmacol Exp Ther 252: 185, 1990
Hassen T. Sherief, MD, and Robert G. Carpentier, MD
Abstract: The hypothesis that cocaine intoxication results in cardiac arrest by producing a block of the propagation of the action potential, without loss of pacemaker function, was tested in rat cardiac tissues. In spontaneously active sinoatrial preparations, cocaine exerted a dose-dependent negative chronotropic action, which was not modified by atropine or propranolol. Sinus node arrest was never observed. Instead, cocaine produced sinoatrial block. The mechanism of this block involved a fall in the resting potential and a decrease in the amplitude and Vmax of phase 0 of the action potential of atria1 fibers. In sinoatrial preparations and papillary muscles driven at 5 Hz, cocaine depressed the resting potential, the total amplitude, the overshoot of the action potential, and the Vmax of phase 0. Cocaine had a biphasic effect on the atrial action potential duration. The initial shortening was muscarinic. The prolongation was a-adrenergic mediated and probably the result of the inhibition of the transient outof the action poward current I,,. In papillary muscles, only the prolongation tential occurred. In conclusion, the electrophysiological actions of cocaine can explain cardiac sudden death. The fall in the resting potential associated with the decrease in the amplitude of the action potential of contractile fibers will result in a block of the propagation of the action potential. Quiescence of the contractile fibers will occur while the sinus node is still generating action potentials at a rate compatible with life. Key Words: sinus node, membrane potentials, sinoatrial conduction, sinoatriai block, myocardial excitability, arrhythmias.
Cocaine can produce myocardial infarction, pressed contractility, and cardiac arrhythmias,
eluding ventricular fibrillation and cardiac arrest. ’ In spite of the urgent need for information on the mechanisms of the cardiac arrhythmias, there is little in the literature about the acute effects of cocaine on the electrophysiology of the heart. Local anesthetic effects and class I antiarrhythmic actions have been demonstrated in cardiac tissues.2,3 The aim of our investigation was to test the hypothesis that cocaine intoxication can result in sudden cardiac death due to cardiac arrest. The results reported here show that cocaine exerts electrophysiological actions on the rat
dein-
From the Department of Physio1og.v and Biophvsics, College of Medicine. Howard University. Washington, DC This investigation is in partial fulfillment of the requirements for Dr. Sheriefs Ph.D. degree. Supported by grants from the NIHIMBRS and National Heart, Lung and Blood Institute and from the Howard University Faculty Research Prograq. Reprint requests: Dr. Robert G. Carpentier. Department of Physiology and Biophysics, College of Medicine, Howard University, 520 W Street NW, Washington, DC 20059.
247
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of Electrocardiology
Vol. 24 No. 3 July 1991
heart that result in cardiac arrest, caused by a block of the propagation of the action potential through the myocardial contractile fibers, without loss of the pacemaker function. When quiescence occurs, the sinus node is still generating action potentials at a rate compatible with life, but these impulses fail to generate conducted action potentials in the contractile fibers.
Materials
and Methods
Adult (300-350 g) male Sprague-Dawley rats from Charles River Breeding Labs Co. were used. The animals were euthanized by decapitation under barbiturate sedation (Sodium Pentobarbital, Sigma Chemical Co., 60 mg/kg intraperitoneally) and the heart was quickly excised. The in vitro preparations and the recording system have been previously described in detai1.4-7 A small strip of the right atrium containing the sinus node and a portion of the crista terminalis was set up, endocardial side up, in a constant-temperature (37’C) tissue bath. The tissue chamber (total volume: 1 ml) was perfused with a physiological solution flowing at 5 ml/min through tubing with a total volume of less than 1 ml. The atria1 strips were superfused with oxygenated and buffered (95% O2 - 5% C02; pH: 7.4) Tyrode’s solution. The physiological solution contained the following (mM): NaCl 137, KC1 5.4, CaCI, 2.7, MgCl, 0.5, NaHC03 11.9, NaH,P04 0.45, and glucose 5.55. All preparations were equilibrated with the Tyrode’s solution for at least 60 minutes before any experimental procedure was carried out.
Studies With Preparations Spontaneously Active The preparations were equilibrated in Tyrode’s solution while beating spontaneously. Therefore, the sinus node rate (SNR) was monitored with standard intracellular microelectrodes filled with 3 M KCl, using multiple cell impalements in different areas of the preparation. Multiple atria1 fibers located between the sinus node and the crista terminalis were impaled. It was also possible to impale sinus node subsidiary pacemaker fibers located in the periphery of the sinus node in the endocardial surface. However, it was not possible to impale true pacemaker cells, which are located intramurally in the rat heart. This procedure allowed us to establish the control
SNR and to determine if there were irregularities or areas of blocked conduction. Only the preparations with a regular and steady control SNR and without areas of block were used in this study. At the end of the equilibration period, the control SNR was recorded. The superfusate was then changed to Tyrode’s solution containing cocaine (cocaine hydrochloride). The following drugs were also employed: atropine sulfate ( lop5 M) and propran0101 hydrochloride ( 10 - ’ M). These concentrations of the muscarinic and P-adrenergic antagonists are known to effectively block muscarinic- and p-adrenergic-mediated actions without affecting directly the SNR.12 All drugs were purchased from Sigma Chemical Co., Missouri. All solutions were kept in the dark and protected from direct light during the superfusion. The SNR was recorded before (control), during, and after (recovery) superfusion with the drug(s). For each experimental series, data are reported as the mean +- the standard error of the mean (SEM). The statistical analysis was done using the Student’s t test for paired data. Values obtained at the end of the exposure to the drug(s) or at the end of the recovery period were compared to those collected during the control period in the same preparation. Significance was set at p values less than 0.0 1. Analysis of variance (ANOVA) was used to compare several means resulting from various interventions. The level of significance is given each time a comparison is made.
Studies With Preparations Driven at a Constant Rate The preparations were driven electrically at a frequency of 5 Hz by a Grass stimulator (S-88) with an SIU5 isolation unit. Square pulses of voltage 50% above threshold of 1 ms duration were delivered through a pair of silver electrodes in close contact with one end of the preparation. The membrane potentials of atria1 fibers located between the sinus node and the crista terminalis were recorded with intracellular microelectrodes filled with 3 M I 0.2. In other words, the maximum effect was already attained with cocaine 20 mg/l. Figure 3 illustrates the effects of cocaine on atria1 fibers located between the sinus node and the crista terminalis in two experiments in which a single im-
A
palement was maintained throughout the exposure to cocaine. The tracings in the top row show that cocaine 30 mg/l produced a fall in the RP associated with a marked decrease in the APA. Eventually, a sinoatrial block developed, with some action potentials not being conducted to the atria1 tissue. Only a small electrotonic potential was detected each time an action potential was not conducted. Similar sinoatrial blocks were observed within 20 minutes in four of six preparations, while sinus node arrest never occurred. In four additional experiments, the exposure to cocaine was maintained until a total sinoatrial block occurred (between 30 and 45 minutes) and attempts were made to stimulate the preparations with electrical pulses immediately after the atria1 contractile fibers became quiescent. In three cases it was possible to drive the preparations at a rate similar to that of the sinus node with stimuli
C
B
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Membrane potentials of atria1 fibers located between the sinus node and the crista terminalis, in preparations with spontaneous activity. Top row: single cell impalement in a preparation exposed to cocaine 30 mg/l. Middle row: single cell impalement in a preparation exposed to cocaine 40 mgil. Tracings in panels c and f (bottom row) are the same as those in panels C and F, but at a higher sensitivity. Voltage calibrations (marks on panels A, D, and c): 0 to - 50 mV for top and middle rows; 25 mV for bottom row. Time calibration (horizontal line below panel D) = 300 ms. Control tracings are in panels A and D. Tracings in panels B and C were recorded at 10 and 20 minutes of exposure to the drug. Tracings in panels E and F were recorded at 4 and 5 minutes of exposure to the drug. Filled circles mark electrotonic potentials determined by sinus node action potentials blocked at the sinoatrial junction. Fig. 3.
Effects of Cocaine
on Cardiac
three to four times the normal threshold intensity. The action potentials recorded from the contractile fibers were similar to those recorded immediately before the block occurred. In one case the preparation was unexcitable. The second row in Figure 3 shows that a similar but more accentuated phenomenon was observed with cocaine 40 mg/l. In all six preparations, a complete sinoatrial block occurred within 5 minutes while the sinus node was still firing regularly. Small regular electrotonic potentials with a constant interval were always present in the transitional area between the sinus node and the crista terminalis. By moving the microelectrode towards the sinus node, it was possible to record action potentials from latent pacemaker fibers. The intervals between the action potentials recorded in this area and the electrotonic potentials recorded in the transitional area were the same. In three additional experiments, attempts were made to stimulate the preparations with electrical pulses immediately after the atria1 contractile
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fibers became quiescent. In all cases the preparations were unexcitable.
Effects of Cocaine on Membrane Potentials of Atrial Fibers in Sinoatrial Preparations Driven at a Constant Rate
Cocaine 5 mg/l produced a fall in the RP associated with a decrease in the APA and the Vmax 0 of atria1 fibers (Fig. 4), in the absence of any significant change in the OS or the APD. A higher concentration of cocaine (20 mg/l) produced greater decreases in the magnitudes of the RP, the APA, and the Vmax 0 (Fig. 5). In addition, this concentration of cocaine reduced the OS and produced biphasic changes in the APD. Cocaine 20 mg/l significantly reduced (p < 0.01) the APD measured at - 70 mV from 6 1.5 2 0.8 ms to 55.4 ? 1.4 ms within 1 minute. However, when
-70 1
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4. Effects of cocaine 5 mg/l on membrane potentials of atrial fibers located between the sinus node and terminalis. The preparations were driven electrically at 5 Hz. CON = control values recorded immediately before to the drug; COC = values obtained at the end of a 3-minute exposure to the drug; REC = recovery values, 10 minutes after withdrawal of the drug from the superfusate; each data point represents the mean I SEM *significantly different from control value, p < 0.0 1. obtained from 7 preparations in single impalements; Fig.
the crista exposure obtained of values
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-70 -
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Fig. 5. Effects of cocaine 20 mgil on membrane potentials of atria1 fibers located between the sinus node and the crista terminalis. The preparations were driven electrically at 5 Hz. CON = control values recorded immediately before exposure to the drug; COC = values obtained at the end of a 3-minute exposure to the drug; REC = recovery values, obtained 10 minutes after withdrawal of the drug from the superfusate; each data point represents the mean k SEM of values * = significantly different from control value, p < 0.01. obtained from 6 preparations in single impalements;
atropine was added to the superfusate, there was no difference (p > 0.05) between the control value (64.4 k 1.3 ms) and the value collected 1 minute after cocaine was added to the Tyrode’s solution (6 1.7 k 1.9 ms). In other words, the muscarinic blockade abolished the initial shortening action of cocaine on the APD. The addition of atropine to the superfusate also accentuated the subsequent prolonging action of cocaine on APD. In the absence of atropine, the APD measured at - 70 mV was prolonged by 6.5 2 2.1 ms (p < 0.01) at the end of the 3-minute exposure to cocaine, while in the presence of the muscarinic antagonist, the APD was prolonged by 16.5 I 3.6 ms (Fig. 6). The mechanism of the prolonging action of cocaine on APD was studied in preparations superfused with Tyrode’s solution containing either phentolamine or atropine and 4-aminopyridine. Figure 6 shows that cocaine did not prolong the APD when tested in the presence of the cw-adrenergic antagonist phentolamine. Figure 6 also shows that 4aminopyridine,
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Fig. 6. Effects of cocaine 20 mg/l on the action potential duration of atria1 fibers, measured at - 70 mV of repolarization (APD-70) in preparations driven at 5 Hz. TYR = cocaine alone in Tyrode’s solution; PHEN = cocaine in the presence of phentolamine; ATROP = cocaine in the presence of atropine; ATROP and 4-AP = cocaine in the presence of atropine and 4-aminopyridine; each value is the mean k SEM of values obtained from 6 preparations in *significantly different from control single impalements; value, p < 0.0 1.
Effects of Cocaine -70
-75
1
;i
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ACTION POTENTLAL AMPLITUDE
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7. Effects of cocaine 20 mg/l on membrane potentials of ventricular fibers in papillary muscles driven at 5 Hz. CON = control values recorded immediately before exposure to the drug; COC = values obtained at the end of a 3-minute exposure to the drug; REC = recovery values, obtained 10 minutes after withdrawal of the drug from the superfusate; each data point represents the mean 2 SEM of values obtained from 6 preparations in single impalements; *significantly different from control value, p < 0.0 1.
which inhibits the transient outward current I,,,” abolished the prolonging action of cocaine on APD.
Effects of Cocaine on Membrane Potentials of Papillary Muscles
The effects of cocaine 20 mg/l on the magnitude of membrane potentials of papillary muscles were very similar to those described for atria1 muscle (Fig. 7). On the other hand, the initial shortening of APD was not observed, while the subsequent prolongation was present. The control value for the APD measured at - 70 mV was 50.2 2 1.6 ms. At 1 minute of exposure to cocaine, it was still 49.2 + 2.1 ms (not different from control value, p > 0.05). At the end of the 3-minute exposure to the drug, the APD was 57.3 + 2.2 ms (significantly different from control value, p < 0.01).
Discussion
The “recreational” use of cocaine has reached epidemic proportions in the United States.12 It is now well known that cocaine intoxication often results in cardiac sudden death.’ Our results show that the concentrations used in our experiments produced cardiac arrest in the rat. It might be argued that the concentrations used were very high. Interestingly, the levels of blood cocaine attained in acute intoxication are much higher than previously believed. A recent autopsy study indicates that the concentration of cocaine in blood in fatal acute intoxications was on average 5.3 ? 8.1 mg/l, in one case reaching 24 mg/l. ’ 3 It is also true that it is impossible to precisely compare in vitro superfusate levels of cocaine with the levels actually present in the cardiac extracellular space in vivo. In terms of the mechanisms responsible for the cardiotoxic effects of cocaine, the emphasis has been
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on the adrenergic and anesthetic effects of the drug, which would act synergistically to elicit and maintain ventricular fibrillation or similar “malignant arrhythmias.“’ Our results show that cocaine can also produce sudden cardiac death through a different mechanism-cardiac arrest. In the rat, a rapid increase in extracellular cocaine concentration in vitro to levels in the range of those found in fatal acute intoxication produces a decrease in the amplitude of the action potentials and a fall in the resting potential of atria1 and ventricular fibers. Eventually, a block of the propagation of the action potentials to the contractile cells occurs, while the sinus node is still generating action potentials at a rate compatible with life. With the highest concentration tested, all preparations become unexcitable, as shown by the fact that it is not possible to trigger action potentials with electrical stimulation. The absence of a positive chronotropic effect of cocaine on the rat sinus node indicates a species difference. In dogs, intravenous cocaine produces adrenergically mediated rapid increases in blood pressure, cardiac output, and heart rate. 14,15On the other hand, in anesthetized and conscious rats, intravenous cocaine also elicits a biphasic presser/depressor effect, but this is associated with no change or a slight decrease in heart rate. l6 Our investigation of the direct effects of cocaine on the SNR in vitro supports the hypothesis that cocaine does not exert positive chronotropic actions on the rat sinus node. The reason for this absence of adrenergic-mediated, cocaineinduced stimulation of sinus node automaticity is still unknown. Our results indicate that cocaine does not produce sinus node arrest in the rat. The maximum negative chronotropic action was attained with 20 mg/l. Cocaine 30 and 40 mg/l had a similar effect on the SNR but produced a sinoatrial block. The mechanism of the atria1 arrest is similar to that of hypothermia.’ In both cases, the cause of the atria1 arrest is a block of conduction between the sinus node and the atrium and not a cessation of pacemaker activity of the sinus node. The mechanism of this block involves a fall in the resting potential and a decrease in the amplitude and Vmax of phase 0 of the action potential of atria1 fibers. The changes in the action potential are the consequence of both the anesthetic action of cocaine on the fast sodium system2,3 and the fall in the resting potential. ” Our results also show that cocaine exerts similar effects on membrane potentials of atria1 and ventricular fibers. It is therefore reasonable to postulate that acute cocaine intoxication will result in cardiac arrest in the absence of sinus node arrest, even if latent ectopic pacemaker fibers are firing, simply because the propagation of the action potentials
through the myocardium is blocked. Of course this does not exclude other possible mechanisms of cardiac arrest that may result in “malignant arrhythmias.“’ In preparations driven at a low rate (43/min), Przywara and Dambach found that cocaine 60 p.M (20 mg/l) did not affect the resting potential or the overshoot of the action potential of atria1 and ventricular fibers from rabbit hearts. Species differences and/or frequency-dependence of the actions of cocaine on these two parameters would explain the difference between their results and those reported here. Voltage clamp studies will be required to characterize the possible actions of cocaine on the currents responsible for the resting potential and the overshoot of the action potenial. The effects of cocaine on the duration of the action potential are of interest. The drug had a biphasic effect on the APD of atria1 fibers, while it had only an APD-prolonging action in the papillary muscles. The initial brief shortening effect on the APD of atria1 fibers was blocked by atropine, indicating that it is a muscarinic action. The subsequent APD-prolonging action, which has been described recently by others,’ was abolished by the c-w-adrenergic antagonist phentolamine. This is not surprising: ol-adrenergic stimulation is known to prolong the APD of rat cardiac fibers, both in multicellular preparations’0,‘8 and in isolated myocytes.” The fact that this APDprolonging effect of cocaine was also blocked by 4aminopyridine provides further support to the concept that this a is an a-adrenergic-mediated action resulting in the inhibition of the current I,,,. Tohse et al.” recently showed that the inhibition of I,,, is the primary cause of the prolongation of the APD produced by a-adrenergic stimulation. The results of this investigation may be of clinical significance. We have shown that cocaine may produce sudden cardiac death by a mechanism other than the production of “malignant arrhytmias.“’ It is true that the electrophysiology of the rat myocardium is different from that of other mammals and this has to be considered before any speculation is made about possible effects of cocaine on human myocardium. However, with these results in vitro, it becomes necessary to fully investigate this possible mechanism in the entire animal. Interestingly, in a recent study in dogs, in which the plasma concentrations of cocaine attained were similar to the ones used in our experiments, a cocaine-mediated impairment of cardiac conduction was proposed as a potential mechanism for sudden death.20 Thus, there seems to already be enough experimental evidence to at least consider cardiac arrest as one of the pos-
Effects
sible causes of sudden ication in humans.
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References 1. Billman GE: Mechanisms responsible for the cardiotoxic effects of cocaine. FASEB J. 4:2469, 1990 2. Weidmann S: Effects of calcium ions and local anesthetics on electrical properties of Purkinje fibres. J Physiol (Lond) 129:568, 1955 3. Przywara DA, Dambach GE: Direct actions of cocaine on cardiac cellular electrical activity. Circ Res 65: 185, 1989 4. Carpentier RG, Posner P, Bloom S: Sinoatrial automaticity and transmembrane potentials in hamsters on a magnesium-deficient diet. J Cardiovasc Pharmacol 7:919, 1985 A: Acute and 5. Carpentier RG, Gallardo-Carpentier chronic effects of ethanol on sinoatrial electrophysiology in the rat heart. J Cardiovasc Pharmacol 10:616, 1987 6. Salvatici RP, Carry1 OR, Isaacson RL et al: Actions and interactions of dihydropyridines and ethanol on the rat sinus node. Cardiovasc Drugs Ther 3:177, 1989 Salvatici RP, Isaacson RL, Carpentier RG: Actions and interactions of calcium modulators and ethanol on atria1 membrane potentials. J Electrocardiol 23: 157, 1990 Brown RA, Carpentier RG: Effects of acetaldehyde on membrane potentials of sinus node pacemaker fibers. Alcohol 7:33, 1990 Carpentier RG, Vassalle M: Restoration of electrical activity of guinea pig atria during hypothermia: effects of norepinephrine and electrical stimulation on membrane potentials. Circ Res 3 1: 507, 1972
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