Arrhythmias, Eiectrophysiology and Electrocardiography Cardiology 1992:80:205-214
Departments of Internal Medicine and Pharmacology, National Defense Medical Center, and Clinical Research Center at the Veterans General Hospital, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC
Keywords Human atrial tissue Human ventricular tissues Electrical activity Mechanical activity Epinephrine Strophanthidin Diastolic depolarization Delayed afterdepolarizations Triggered activity
Arrhythmogenic Mechanisms in Human Atrial and Ventricular Muscle Fibers
Abstract Mechanisms which may lead to cardiac arrhythmias were studied in atrial and ventricular tissues from human hearts. In human atrial fibers, diastolic depolarization (DD) was consis tently present, but did not induce spontaneous discharge. Epi nephrine enhanced DD. could induce delayed afterdepolariza tions (DADs) and (in combination with strophanthidin) trig ger repetitive activity. The presence of DD modified the recovery of premature action potentials. Human ventricular fibers did not exhibit DD and were more resistent to Ca over load. It is concluded that in atrial tissues the presence of DD may not induce automatic arrhythmias, but it may influence conduction and re-entry rhythms. Cardioactive drugs may induce DADs and repetitive activity in the atria and less easily in the ventricles. The attainment of a threshold may be facili tated when DADs are superimposed.
Introduction In previous reports [1-4], we noted that diastolic depolarization (DD) could be present in human atrial tissue exhibiting dif ferent types of action potentials (AP). This finding elicits several questions which are re lated to the genesis of atrial arrhythmias en countered in cardiac patients. One is whether DD can lead to spontaneous discharge, either
Received: November 28. 1991 Accepted after revision: January 6. 1992
in the absence or presence of epinephrine (EPI). EPI could induce spontaneous activity in atrial fibers either by steepening DD (as it does in pacemaker tissues) or by inducing cal cium overload [5, 6] (and related delayed afterdepolarization, DAD [7]). Therefore, our first aim was to investigate nonautomatic human atrial fibers exhibiting fast responses to determine how frequently DD was present and whether DD would lead
Prof. Chcng-I Lin. BDS. PhD. Chairman Dcpartmenl of Pharmacology National Defense Medical Center PO Box 90C48 Taipei (Taiwan. ROC)
© 1992 S. Karger AG. Basel 0008-6312/92/ 0804-0205$2.75/0
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Tzu-Chiu Yeha c Mario Vassalleb Cheng-1 L inbc
Methods Atrial and ventricular tissues were obtained from patients undergoing cardiac surgery after securing in formed consent prior to surgery. The atrial appendages selected for the present experiments were obtained from 15 patients with congenital and 5 patients with acquired heart disease. The tissues were not automatic and showed fast response APs (maximum upstroke velocity Vmas S* 50 V/s) in normal Tyrode solution. Ventricular tissues were obtained from 3 male (21, 21 and 53 years old) and 1 female patient (41 years old) undergoing cardiac transplant. The atrial appendages (~ I cm2 in size) were ex cised from the right atrium as part of the routine atriotomy procedure and were immediately immersed in cold (4°C) physiological saline solution. Trabeculae with a diameter of ~ 1 mm (range 0.5-1.5 mm) were removed from the specimen and superfused in a tissue bath with Tyrode solution at 37 °C. The composition of the Tyrode solution in mM was: NaCl, 125: KC1, 4: MgCb, 0.5: NaHC03, 24; NaH:P 0 4. 0.5: CaCL, 2.7. and dextrose 5.5. The solution was gassed with a mix ture of 95% CL and 5 % C 0 3(pH ~7.38).
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The preparations were usually driven at a basic cycle length (BCL) of I s. Suprathreshold electrical stimuli of 2 ms duration were provided by a Grass S88 stimulator or a Bloom DPU 101 stimulator. One end of the preparation was immobilized on the floor of the tissue bath by means of one of the stimulating elec trodes. and the other end was connected by means of a silk thread to a Gould UC2 force transducer. Transmembrane potentials were recorded by means of glass microelectrodes filled with 3 M KC1. AP, its first deriv ative (dV/dt) and contractile force were displayed on a digitizing oscilloscope (Tektronix 5223) and recorded on a Gould chart recorder (model 2400S) as well as on a FM Gould recorder (model 6500). The dV/dt was obtained by means of a modified Gould 13-4615-71 differentiator. The taped records were replayed after the experiments by means of an electrostatic recorder (Gould ESI000). The amplitude of the DADs was measured as the difference between the peak and a line drawn between the beginning and the end of DAD. The effective refractory period (ERP) and the restitution of the AP were studied at the BCL of 500 or 1,000 ms: a test stim ulus was applied at progressively longer intervals from the preceding stimulus at the BCL. After each test stimulus, the preparation was driven at the BCL for 8-20 beats to insure complete recovery of the basic AP. The cycle length following the lest stimulus was the same as the BCL [9], Epinephrine bitartrate, theophylline and strophan thidin were obtained from Sigma (USA). Values are expressed as means ± SE. Student’s paired t test was used for statistical analysis, and p < 0.05 was consid ered statistically significant.
Results Human Atrial Muscle Fibers Atrial APs and the Effects o f EPI. Nonauto matic atrial fibers driven at 1 Hz were found to consistently show DD. In figure la, the fast response AP initiated at a resting potential of - 85 mV: phase 3 repolarization undershot the resting potential (to -8 9 mV) and was fol lowed by a subsequent DD. In figure lb, the drive was interrupted to determine whether the fibers would discharge spontaneously: DD eventually attained a stable value (resting poArrhythtnogencsls in Human Tissues
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to spontaneous discharge or DADs and trig gered activity [7] in the presence of EPI and strophanthidin (a cardiotonic steroid). Since DD consistently was found in atrial fibers with fast responses, this elicited the fur ther question as to whether the restitution of the AP during diastole might be influenced by the changing diastolic potential. In turn, pre mature APs with a different maximum up stroke velocity and amplitude may influence arrhythmias due to re-entry. For this reason, test stimuli were applied at progressively lon ger intervals during diastole both in the ab sence and presence of EPI. Since there are several electrophysiological differences between atrial and ventricular tis sues [8], similar experiments were carried out in ventricular preparations obtained from hu man hearts. The results obtained show that atrial and ventricular tissues differ in several respects.
a
b
tential), and the preparation remained quies cent. The presence of a DD raised the question as to whether EPI may increase it and induce spontaneous activity. In figure lc. EPI (0.5 \iM) did not modify the resting potential (-84 mV), but shortened the AP. increased the maximum diastolic potential (MDP: -9 3 mV) and enhanced the slope of DD. How ever. when the drive was interrupted (not shown), the preparation remained quiescent. The changes induced by EPI are more clearly seen in figure Id where the APs recorded in the absence and presence (asterisk) of EPI are superimposed. Figure Id also shows that EPI increased the contractile force. In 12 experiments at l Hz. in Tyrode solu tion Vmax was 86.0 ± 11.3 V/s. take-off poten tial -69.5 ± 2.2 mV and MDP -73.7 ± 6.1 mV. In the presence of EPI (0.5-2.5 pA/). the corresponding values were 53.3 ± 7.2 V/s (p < 0.05). and -64.5 ± 2.7 (p < 0.05) and -72.2 ± 2.9 mV. The difference between MDP and take-off potential was 4.2 ± 0.5 mV in the absence and 7.7 ± 0.9 mV in the
presence of EPI (p < 0.01), reflecting the increase in the DD slope. The results show that atrial fibers with a somewhat diminished resting potential may exhibit DD and that EPI modifies both the AP (shortening) and DD (increased slope). Induction o f DAD by EPI. In several ani mal tissues, catecholamines induce DADs [5, 6], This was investigated in human atrial fibers by temporarily interrupting the drive. In 15 experiments, in Tyrode solution drive at 1 or 2 Hz did not induce DADs. In the pres ence of EPI (2.5 \iM), 1-Hz drive induced DADs in 60.0% and 2-Hz drive in 66.7% of the preparations. The amplitude of DADs was 4.0 ± 1.1 and 4.2 ± 1.2 mV, respectively, at the two rates. The results show that EPI enhances not only DD. but also can induce DADs in human atria as it does in animal pacemaker and nonpacemaker tissues [6, 7, 10]. Potentiation between EPI and Strophan thidin. Since the inhibition of active transport by cardiotonic steroids also induces DADs [5, 11, 12], the effects of EPI were tested in the
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Fig. 1. APs of human atrial fi bers and EPI effects, a AP recorded from an atrial trabecula is shown in control (Tyrode solution). bAPs were recorded at lower gain, and the stimulation was interrupted af ter the third AP. c AP was recorded in the presence of 0.5 \iM EPI. d The superimposed APs and twitch curves were recorded in the absence and presence (asterisk) of EPI.
presence of strophanthidin to determine whether their potentiation leads to triggered activity. In 4 experiments, EPI caused DADs and adding strophanthidin at different con centrations (0.1-1 pM) increased the size of DADs and of aftercontractions. In the pres ence of EPI plus strophanthitin, increasing the driving rate increased the amplitude of both DADs and of aftercontractions. Inter ruption of the drive at BCL of 1.7-2.5 Hz was followed by triggered activity in 6 tests in 3 experiments. These results suggest that release of catecholamines may precipitate triggered rhythms more easily in digitalized patients. AP Restitution during Diastole. The pres ence of DD in human atrial fibers changes the take-off potential during diastole, and there fore may influence the recovery of Vmax and upstroke amplitude. This was tested in the
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absence and presence of EPI with similar results. Procedure and results obtained in the presence of 0.5 \x.M EPI are shown in fig ure 2. The preparation was driven at 1 Hz and test stimuli were applied at the intervals indi cated by the numbers (ms) above the traces. In figure 2a. the test stimulus applied at 260 ms failed to elicit a response whereas the 290ms stimulus evoked a slow response as Vmax and upstroke amplitude were smaller (-58 and -32%, respectively). The 320-ms stimu lus happened to be applied at the same poten tial as the take-off potential of the preceding AP (the last of the regular train), and Vmax as well as upstroke amplitude were the same. In figure 2b. it is apparent that Vmax and upstroke varied as a function of the take-off potential: when the test AP was elicited near MDP, Vmax and upstroke amplitude were
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Fig. 2. Restitution of AP in hu man atrial fibers in the presence of 0.5 p.V/ EPI. In each panel, the top trace shows APs and the bottom trace Vmav a The arrows point to the last AP of the train and the numbers above the APs indicate the interval (in ms) at which the test stimulus was applied, b The test APs recorded at the intervals indicated have been superimposed on DD.
Human Ventricular Muscle Fibers AP and Effects o f EPI and Acetylcholine. The findings in human atrial fibers elicit the question whether similar responses should be present in ventricular muscle fibers. In 26 impalements of ventricular fibers from 4 hu man hearts. AP was not followed by an under shoot, and interruption of drive did not reveal DD. Since EPI modified AP in atrial fibers, its effects were tested also in ventricular fibers. In / preparation, 2.5 \iM EPI substantially increased contractile force, shifted the plateau to more positive values and prolonged the AP. These results arc similar to those induced by norepinephrine and isoproterenol in hu man papillary muscle [14] and ventricular myocytes [15]. EPI also increased Vmax and reduced the twitch duration in this prepara tion. Similar results were observed in ventric ular fibers obtained from another heart. How ever, a reduction in AP duration (APD) and of Vmax were induced by EPI in a 3rd heart. Thus, the effects of EPI were not consistent presumably due to different disease processes in these 3 hearts. In atrial fibers. ACh (5.5 \iM) increases MDP. shifts the plateau to more negative val
ues, shortens APD and decreases contractile force [3]. In contrast, in 5 tests in a ventricular preparation from I heart. ACh (0.55-55 \iM) did not modify Vmax, AP amplitude. APD, MDP or force. These results show that there arc important differences between atrial and ventricular tissues (e.g. in ventricular tissue there is no DD, EPI increases AP amplitude and Vmax and ACh has no effects). AP Restitution during Diastole. In view of the fact that ventricular muscle tissue lacks DD and responds differently to EPI, the resti tution of APs should also be different. In figure 3a. the traces show action poten tials as well as the twitch curves: the dots mark two test APs elicited at different inter vals. The upstroke of the test APs was slightly larger (+2.3 and +2.0%, respectively) in spite of the fact that they initiated at a less negative potential (-5 mV). Also, the twitch after the test AP was larger and this increase subsided with subsequent APs. In figure 3b, the traces recorded at faster speed show that the increase in test AP ampli tude was associated with a decreased Vmax. Also, the test AP was shorter, and the twitch curves were fused. A similar procedure was repeated in the same preparation in the pres ence of 2.5 pM EPI. In figure 3c, AP ampli tude of the test APs was greater than that of the preceding AP (as in the absence of EPI). However, contractile force behaved differ ently in that in the presence of EPI the test contraction was smaller, but the subsequent potentiation was greater. In figure 3d, EPI shifted the plateau to more positive values and prolonged the AP. Also, EPI shortened the time to peak of twitch and this (together with the AP prolongation) allowed force to decay to a larger extent before the next twitch. Induction o f Calcium Overload. The ven tricular preparations were exposed to car dioactive drugs to find out whether DADs
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larger than those of the preceding regular AP. Both parameters decreased gradually with successive test APs as a function of the grad ual decrease in take-off potential brought about by DD. Similar patterns were obtained in 15 experiments. These results indicate that in human atrial fibers the recovery of the fast Na channel does not lag behind the mem brane potential, as found in animal Purkinje fibers [13]. This has obvious implications for excitability and conduction early in diastole in human atria and for re-entry rhythms. In addition. EPI shortened the ERP (ERP was 298.0 ± 17.5 ms in control and 247.3 ± 14.7 ms in the presence of 0.5 \iM EPI. p < 0.01).
Fig. 3. Restitution of the AP in human ventricular muscle libers in the absence (a. b) and presence (c. d) of 2.5 [i\f EPI. a. c APs and twitch curves are shown. The dots indicate test APs. b. d The first three APs are shown at faster time base.
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increase in diastolic force (dashed line). In the first panel of figure 4c. the drive was inter rupted. and DAD became obvious as the de polarization peaked and then decayed (up ward arrow) while at the same time a shallow aftercontraction appeared (downward arrow). In the second panel, the driving rate was increased (arrow above the APs): on stoppage of the faster drive, the last AP was followed by a larger DAD (upward arrow) and aftercon traction (downward arrow). Apparently, DAD was superimposed on DD since the membrane potential remained less negative than the MDP during the period of quies cence. The drive was repeatedly interrupted and DD and DADs (but no triggered activity) were present.
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and related arrhythmias could be induced (as in human Purkinje fibers [8]). While ventricu lar tissue was found to be more resistant to calcium overload. DADs could be induced but only by a combination of cardioactive drugs. In figure 4. after the control traces were recorded (4a), the preparation was exposed to strophanthidin (up to 1 pM). but neither DD nor DADs were induced (not shown). There after, theophylline (1 m.V/) and EPI (I \iM) were added to the strophanthidin solution. In figure 4b, the combination of these drugs de creased the resting potential, diminished AP amplitude and APD (at the plateau), induced an undershoot at the end of phase 3 repolari zation and caused DD. At the same time, the twitch was markedly increased. That DD was in part due to a DAD is suggested by the
Discussion DD in Human AtriaI and Ventricular Tissues The presence of DD in atrial fibers elicit the question as to whether this is a normal finding in human tissue or is the result of dis ease. The present results show that even when AP is a fast response, the resting potential is lower than that found in animal normal atrial tissue, suggesting that presence of DD is the result of abnormally low resting potential. The resting potential could be decreased because the intracellular K is decreased (less negative Ek) and/or gK is decreased. Both fac tors may be involved. For MDP to be more
negative than the diminished resting poten tial. it is only necessary that gK increases dur ing the AP (i.e. a time-dependent increase in the delayed rectifier current I k ). An increase in gK during AP would lead to an undershoot at MDP since increase in gK would allow the membrane to approach (the diminished) Ek. Still. MDP would be less negative than a nor mal resting potential if Ek is less negative. Apparently, ventricular muscle cells do not exhibit DD because the resting potential was better maintained (-90 mV) than in atrial cell (-74 mV). The role of a diminished resting potential in the induction of DD is suggested by the fact that also in ventricular tissue the decrease in resting potential caused by several
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Fig. 4. Induction of DAD in hu man ventricular muscle fibers. The traces were recorded in Tyrode so lution (a), in the presence of 0.5 u.\/strophanthidin plus I m.Y/ the ophylline plus 1 p.V/ EPI (b. c). c In the first panel, drive was inter rupted after the second AP. In the second panel (slow speed record ing). the rate of drive was increased at the arrow, and the drive was interrupted after the fourth AP re corded at faster speed. Dashed lines emphasize the changes in rest ing force during diastole.
Action o f EPI on AP o f Atria! and Ventricular Tissues EPI acts on both atrial and ventricular tis sues, but its effects are bound to depend on the characteristics of these tissues. Thus, in the present experiments, EPI decreased APD5o and increased DD slope in atrial fi bers, whereas it increased the positivity of the plateau and AP amplitude in most of the ven tricular tissues (without inducing DD). The explanation for these differences is not apparent. However, it is known that in atrial pacemarkers, catecholamines increase I k (as well as Isj) [17]. The increase in ISj in human atrial fibers is indicated by the increase in force. A simultaneous increase in I k would indeed bring about a fast repolarization of the AP and a steeper DD (as a larger Ik decays during diastole). For ventricular fibers. EPI may have ex actly the same effects on the two currents. Thus, contractile force increases also in this tissue. However, in animal ventricular tissues. I k is smaller than in other cardiac tissues [18], Therefore, the increase in ISj may prevail over that of the smaller IKand account for the posi tive shift of the plateau, AP prolongation and absence of DD. Effect o f EPI on ERP EPI affects ERP differently in atrial and ventricular muscle fibers in relation to the presence of DD in the former and the differ ent response of APD to EPI. Thus, EPI de creased Vmax of basic atrial APs, probably in relation to the decreased take-off potential due to the steepening of DD slope. And EPI increased Vmax in the ventricular preparation,
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possibly because the resting potential was the same and the intracellular sodium activity might have been decreased [ 19]. In atrial fibers, the role of the take-off potential in determining Vmax is shown by the fact that both Vmax and AP amplitude were markedly increased when APs were elicited at MDP. Both were decreased when the test AP originated during the repolarization phase at potentials positive to the resting potential. The height of the plateau of the test AP is much less affected as the slow channels are not dependent on the changes of the take-off potential at negative values. In ventricular muscle fibers, instead. Vmax was cither decreased or the same depending on whether take-off potential was less than the resting potential or similar to it. Still, AP amplitude of the test APs was somewhat greater than that of the regular beats and more so the earlier the extrabeat. The reason for the increase in amplitude of the extrabeat is un clear but it is unlikely to be related to a larger fast Na current as Vmax was decreased. In this connection, it is important to note that a larger AP amplitude had been observed in canine ventricular epicardium. but not endo cardium [20]. The greater AP amplitude in early diastole had been related to an incom plete time-dependent recovery of the tran sient outward K current It0 [20]. The fact that EPI shortened ERP in atrial, but prolonged it in ventricular muscle fibers is clearly related to the fact that EPI decreased APD in atrial but not in ventricular fibers [ 14; present results]. The reason why APD should be affected differently in these two tissues is not apparent. One possibility is that EPI in creases Isj and I k in both tissues, but more Ik in atrial and Isj in ventricular fibers. This would result in a shortening and in a prolon gation of AP. repsectively. in the two tissues.
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cardiotonic drugs was associated with DD (upon which a DAD was superimposed). These findings arc in agreement with the pos sibility of inducing DD in single myocytes by applied depolarization [ 16].
Conclusions The present results suggest that the pres ence of DD in atrial fibers may be the result of decreased resting potential and of a larger delayed rectifier current I k than in the ventri cle. The activation of a larger I k during the AP would allow the potential to undershoot the diminished resting potential. This also would account for the fact that EPI shortened the
tissues are more prone to the development of Ca overload than ventricular tissues, as it was easier to induce DAD (and triggered activity) in atrial than in ventricular muscle fibers. The results raise the possibility that in digitalized patients, release of catecholamines might more easily result in atrial triggered tachyar rhythmias. This will be facilitated by the over drive imposed by the faster sinus node rate under the influence of catecholamines.
A P .
While the DD did not attain the threshold and initiate spontaneous activity even in the presence of EPI. it did affect markedly prema ture APs. This elicits the possibility that in the atria, extrasystoles may be conducted at a fas ter speed if originating near the MDP. In turn, this may affect the rate at which the atria are activated in the presence of atrial flutter. The present results indicate that, in the presence of cardiotonic agents, human atrial
Acknowledgements We wish to express our appreciations to Dr. B.N. Chiang for general support and to Drs. K.K. Cheng and J. Wei for the supply of specimens. The present study was supported in part by grants from IBMS, Academia Sinicia. Taipei, and National Science Coun cil (grants 76-0412-BO 16-35 and 79-0412-B016-74). Taipei, Taiwan. ROC.
References 6 Valenzuela F. Vassalle M: Over drive excitation and cellular calcium load in canine cardiac Purkinje fi bers. J Elcctrocardiol 1985:18:2134. 7 Wit AL. Cranefield PF: Triggered activity in cardiac muscle fibers of the simian mitral valve. Circ Res 1976:38:85-98. 8 Dangman KH. Danilo P, Hordof AJ. Mary-Rabine L, Reder RF. Rosen MR: Elcctrophvsiologicchar acteristics of human ventricular and Purkinje fibers. Circulation 1982: 65:362-368. 9 Yeh TC. Lin CL Chiang BN: Me chanical restitution of human atrial myocardium in health and disease: in Yang WJ. Lee CJ (eds): Biomedi cal Engineering. Washington. Hemi sphere Publishing. 1989, pp 133— 144.
10 Vassallc M. Carpentier R: Over drive excitation: The initiation of spontaneous activity in Purkinje fi bers following a fast drive in the presence of norepinephrine. Pflü gers Arch 1972:332:198-205. 11 Lin CL Kotake H. Vassalle M: On the mechanism underlying the oscil latory current in cardiac Purkinje fibers. J Cardiovasc Pharmacol 1986:8:906-914. 12 Kass RS. Lederer WJ. Tsien RW, Weingart R: Role of calcium ions in transient inward currents and after contractions induced by strophan thidin in cardiac Purkinje fibres. J Physiol (Lond) 1978:281:187-208. 13 Weidmann S: The effect of the car diac membrane potential on the rapid availability of the sodium carrying system. J Physiol (Lond) 1955;127:213-224. Downloaded by: Stockholm University Library 130.237.165.40 - 1/10/2019 5:57:13 AM
1 Lin Cl. Chiu TH. Chiang BN. Cheng KK: Electromechanical effects of caffeine in isolated human atrial fibres. Cardiovasc Res 1985; 19: 727-733. 2 Lin CL Chuang IN, Cheng KK. Chiang BN: Arrhvthmogenic effects of theophylline in human atrial tis sue. Int J Cardiol 1987:17:289-297. 3 Hou ZY. Lin CL Vassallc M. Chiang BN. Cheng KK: Role of acetylcho line in induction of repetitive activ ity in human atrial fibers. Am J Physiol 1989;256:H74-H84. 4 Lin CL Tao PL. Chang YF, Chiang BN: Pacemaker activity is modu lated by tissue levels of cyclic adeno sine 3',5'-monophosphate in human atrial fibers, tnt J Cardiol 1989:25: 39-46. 5 Vassalle M. Mugelli A: An oscilla tory current in sheep cardiac Pur kinje fibers. Circ Res 1981:48:618631.
16 Valenzuela F. Vassallc M: Role of membrane potential in B r"-in duced automaticity in guinea pig cardiac myocytes. Cardiovasc Res 1991:25:421-430. 17 Noble D: The surprising heart: A review of recent progress in cardiac electrophysiology. J Physiol (Lond) 1984:353:1-50.
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18 Trautwein VV: Membrane currents in cardiac muscle fibers. Physiol Rev 1973:53:793-835. 19 Lee CO. Vassalle M: Modulation of intracellular N a‘ activity and car diac force by norepinephrine and C a -\ Am J Physiol 1983;244:C110C 1 1 4.
20 Litovsky SH. Antzelevitch C: Tran sient outward current prominent in canine ventricular epicardium but not endocardium. Circ Res 1988:62: 116-126.
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14 Eckel L, Gristwood RW. Nawrath H, Owen DAA: Inotropic and electrophysiological cfTects of histamine on human ventricular heart muscle. J Physiol (Lond) 1982:330:111123. 15 Mitchell MR. Powell T. Sturridgc ME. Terrar DA. Twist VW: Elec trical properties and response to noradrenaline of individual heart cells isolated from human ventricu lar tissue. Cardiovasc Res 1986:20: 869-876.
Departments of Internal Medicine and Pharmacology, National Defense Medical Center, and Clinical Research Center at the Veterans General Hospital, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, ROC
Keywords Human atrial tissue Human ventricular tissues Electrical activity Mechanical activity Epinephrine Strophanthidin Diastolic depolarization Delayed afterdepolarizations Triggered activity
Arrhythmogenic Mechanisms in Human Atrial and Ventricular Muscle Fibers
Abstract Mechanisms which may lead to cardiac arrhythmias were studied in atrial and ventricular tissues from human hearts. In human atrial fibers, diastolic depolarization (DD) was consis tently present, but did not induce spontaneous discharge. Epi nephrine enhanced DD. could induce delayed afterdepolariza tions (DADs) and (in combination with strophanthidin) trig ger repetitive activity. The presence of DD modified the recovery of premature action potentials. Human ventricular fibers did not exhibit DD and were more resistent to Ca over load. It is concluded that in atrial tissues the presence of DD may not induce automatic arrhythmias, but it may influence conduction and re-entry rhythms. Cardioactive drugs may induce DADs and repetitive activity in the atria and less easily in the ventricles. The attainment of a threshold may be facili tated when DADs are superimposed.
Introduction In previous reports [1-4], we noted that diastolic depolarization (DD) could be present in human atrial tissue exhibiting dif ferent types of action potentials (AP). This finding elicits several questions which are re lated to the genesis of atrial arrhythmias en countered in cardiac patients. One is whether DD can lead to spontaneous discharge, either
Received: November 28. 1991 Accepted after revision: January 6. 1992
in the absence or presence of epinephrine (EPI). EPI could induce spontaneous activity in atrial fibers either by steepening DD (as it does in pacemaker tissues) or by inducing cal cium overload [5, 6] (and related delayed afterdepolarization, DAD [7]). Therefore, our first aim was to investigate nonautomatic human atrial fibers exhibiting fast responses to determine how frequently DD was present and whether DD would lead
Prof. Chcng-I Lin. BDS. PhD. Chairman Dcpartmenl of Pharmacology National Defense Medical Center PO Box 90C48 Taipei (Taiwan. ROC)
© 1992 S. Karger AG. Basel 0008-6312/92/ 0804-0205$2.75/0
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Tzu-Chiu Yeha c Mario Vassalleb Cheng-1 L inbc
Methods Atrial and ventricular tissues were obtained from patients undergoing cardiac surgery after securing in formed consent prior to surgery. The atrial appendages selected for the present experiments were obtained from 15 patients with congenital and 5 patients with acquired heart disease. The tissues were not automatic and showed fast response APs (maximum upstroke velocity Vmas S* 50 V/s) in normal Tyrode solution. Ventricular tissues were obtained from 3 male (21, 21 and 53 years old) and 1 female patient (41 years old) undergoing cardiac transplant. The atrial appendages (~ I cm2 in size) were ex cised from the right atrium as part of the routine atriotomy procedure and were immediately immersed in cold (4°C) physiological saline solution. Trabeculae with a diameter of ~ 1 mm (range 0.5-1.5 mm) were removed from the specimen and superfused in a tissue bath with Tyrode solution at 37 °C. The composition of the Tyrode solution in mM was: NaCl, 125: KC1, 4: MgCb, 0.5: NaHC03, 24; NaH:P 0 4. 0.5: CaCL, 2.7. and dextrose 5.5. The solution was gassed with a mix ture of 95% CL and 5 % C 0 3(pH ~7.38).
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The preparations were usually driven at a basic cycle length (BCL) of I s. Suprathreshold electrical stimuli of 2 ms duration were provided by a Grass S88 stimulator or a Bloom DPU 101 stimulator. One end of the preparation was immobilized on the floor of the tissue bath by means of one of the stimulating elec trodes. and the other end was connected by means of a silk thread to a Gould UC2 force transducer. Transmembrane potentials were recorded by means of glass microelectrodes filled with 3 M KC1. AP, its first deriv ative (dV/dt) and contractile force were displayed on a digitizing oscilloscope (Tektronix 5223) and recorded on a Gould chart recorder (model 2400S) as well as on a FM Gould recorder (model 6500). The dV/dt was obtained by means of a modified Gould 13-4615-71 differentiator. The taped records were replayed after the experiments by means of an electrostatic recorder (Gould ESI000). The amplitude of the DADs was measured as the difference between the peak and a line drawn between the beginning and the end of DAD. The effective refractory period (ERP) and the restitution of the AP were studied at the BCL of 500 or 1,000 ms: a test stim ulus was applied at progressively longer intervals from the preceding stimulus at the BCL. After each test stimulus, the preparation was driven at the BCL for 8-20 beats to insure complete recovery of the basic AP. The cycle length following the lest stimulus was the same as the BCL [9], Epinephrine bitartrate, theophylline and strophan thidin were obtained from Sigma (USA). Values are expressed as means ± SE. Student’s paired t test was used for statistical analysis, and p < 0.05 was consid ered statistically significant.
Results Human Atrial Muscle Fibers Atrial APs and the Effects o f EPI. Nonauto matic atrial fibers driven at 1 Hz were found to consistently show DD. In figure la, the fast response AP initiated at a resting potential of - 85 mV: phase 3 repolarization undershot the resting potential (to -8 9 mV) and was fol lowed by a subsequent DD. In figure lb, the drive was interrupted to determine whether the fibers would discharge spontaneously: DD eventually attained a stable value (resting poArrhythtnogencsls in Human Tissues
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to spontaneous discharge or DADs and trig gered activity [7] in the presence of EPI and strophanthidin (a cardiotonic steroid). Since DD consistently was found in atrial fibers with fast responses, this elicited the fur ther question as to whether the restitution of the AP during diastole might be influenced by the changing diastolic potential. In turn, pre mature APs with a different maximum up stroke velocity and amplitude may influence arrhythmias due to re-entry. For this reason, test stimuli were applied at progressively lon ger intervals during diastole both in the ab sence and presence of EPI. Since there are several electrophysiological differences between atrial and ventricular tis sues [8], similar experiments were carried out in ventricular preparations obtained from hu man hearts. The results obtained show that atrial and ventricular tissues differ in several respects.
a
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tential), and the preparation remained quies cent. The presence of a DD raised the question as to whether EPI may increase it and induce spontaneous activity. In figure lc. EPI (0.5 \iM) did not modify the resting potential (-84 mV), but shortened the AP. increased the maximum diastolic potential (MDP: -9 3 mV) and enhanced the slope of DD. How ever. when the drive was interrupted (not shown), the preparation remained quiescent. The changes induced by EPI are more clearly seen in figure Id where the APs recorded in the absence and presence (asterisk) of EPI are superimposed. Figure Id also shows that EPI increased the contractile force. In 12 experiments at l Hz. in Tyrode solu tion Vmax was 86.0 ± 11.3 V/s. take-off poten tial -69.5 ± 2.2 mV and MDP -73.7 ± 6.1 mV. In the presence of EPI (0.5-2.5 pA/). the corresponding values were 53.3 ± 7.2 V/s (p < 0.05). and -64.5 ± 2.7 (p < 0.05) and -72.2 ± 2.9 mV. The difference between MDP and take-off potential was 4.2 ± 0.5 mV in the absence and 7.7 ± 0.9 mV in the
presence of EPI (p < 0.01), reflecting the increase in the DD slope. The results show that atrial fibers with a somewhat diminished resting potential may exhibit DD and that EPI modifies both the AP (shortening) and DD (increased slope). Induction o f DAD by EPI. In several ani mal tissues, catecholamines induce DADs [5, 6], This was investigated in human atrial fibers by temporarily interrupting the drive. In 15 experiments, in Tyrode solution drive at 1 or 2 Hz did not induce DADs. In the pres ence of EPI (2.5 \iM), 1-Hz drive induced DADs in 60.0% and 2-Hz drive in 66.7% of the preparations. The amplitude of DADs was 4.0 ± 1.1 and 4.2 ± 1.2 mV, respectively, at the two rates. The results show that EPI enhances not only DD. but also can induce DADs in human atria as it does in animal pacemaker and nonpacemaker tissues [6, 7, 10]. Potentiation between EPI and Strophan thidin. Since the inhibition of active transport by cardiotonic steroids also induces DADs [5, 11, 12], the effects of EPI were tested in the
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Fig. 1. APs of human atrial fi bers and EPI effects, a AP recorded from an atrial trabecula is shown in control (Tyrode solution). bAPs were recorded at lower gain, and the stimulation was interrupted af ter the third AP. c AP was recorded in the presence of 0.5 \iM EPI. d The superimposed APs and twitch curves were recorded in the absence and presence (asterisk) of EPI.
presence of strophanthidin to determine whether their potentiation leads to triggered activity. In 4 experiments, EPI caused DADs and adding strophanthidin at different con centrations (0.1-1 pM) increased the size of DADs and of aftercontractions. In the pres ence of EPI plus strophanthitin, increasing the driving rate increased the amplitude of both DADs and of aftercontractions. Inter ruption of the drive at BCL of 1.7-2.5 Hz was followed by triggered activity in 6 tests in 3 experiments. These results suggest that release of catecholamines may precipitate triggered rhythms more easily in digitalized patients. AP Restitution during Diastole. The pres ence of DD in human atrial fibers changes the take-off potential during diastole, and there fore may influence the recovery of Vmax and upstroke amplitude. This was tested in the
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absence and presence of EPI with similar results. Procedure and results obtained in the presence of 0.5 \x.M EPI are shown in fig ure 2. The preparation was driven at 1 Hz and test stimuli were applied at the intervals indi cated by the numbers (ms) above the traces. In figure 2a. the test stimulus applied at 260 ms failed to elicit a response whereas the 290ms stimulus evoked a slow response as Vmax and upstroke amplitude were smaller (-58 and -32%, respectively). The 320-ms stimu lus happened to be applied at the same poten tial as the take-off potential of the preceding AP (the last of the regular train), and Vmax as well as upstroke amplitude were the same. In figure 2b. it is apparent that Vmax and upstroke varied as a function of the take-off potential: when the test AP was elicited near MDP, Vmax and upstroke amplitude were
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Fig. 2. Restitution of AP in hu man atrial fibers in the presence of 0.5 p.V/ EPI. In each panel, the top trace shows APs and the bottom trace Vmav a The arrows point to the last AP of the train and the numbers above the APs indicate the interval (in ms) at which the test stimulus was applied, b The test APs recorded at the intervals indicated have been superimposed on DD.
Human Ventricular Muscle Fibers AP and Effects o f EPI and Acetylcholine. The findings in human atrial fibers elicit the question whether similar responses should be present in ventricular muscle fibers. In 26 impalements of ventricular fibers from 4 hu man hearts. AP was not followed by an under shoot, and interruption of drive did not reveal DD. Since EPI modified AP in atrial fibers, its effects were tested also in ventricular fibers. In / preparation, 2.5 \iM EPI substantially increased contractile force, shifted the plateau to more positive values and prolonged the AP. These results arc similar to those induced by norepinephrine and isoproterenol in hu man papillary muscle [14] and ventricular myocytes [15]. EPI also increased Vmax and reduced the twitch duration in this prepara tion. Similar results were observed in ventric ular fibers obtained from another heart. How ever, a reduction in AP duration (APD) and of Vmax were induced by EPI in a 3rd heart. Thus, the effects of EPI were not consistent presumably due to different disease processes in these 3 hearts. In atrial fibers. ACh (5.5 \iM) increases MDP. shifts the plateau to more negative val
ues, shortens APD and decreases contractile force [3]. In contrast, in 5 tests in a ventricular preparation from I heart. ACh (0.55-55 \iM) did not modify Vmax, AP amplitude. APD, MDP or force. These results show that there arc important differences between atrial and ventricular tissues (e.g. in ventricular tissue there is no DD, EPI increases AP amplitude and Vmax and ACh has no effects). AP Restitution during Diastole. In view of the fact that ventricular muscle tissue lacks DD and responds differently to EPI, the resti tution of APs should also be different. In figure 3a. the traces show action poten tials as well as the twitch curves: the dots mark two test APs elicited at different inter vals. The upstroke of the test APs was slightly larger (+2.3 and +2.0%, respectively) in spite of the fact that they initiated at a less negative potential (-5 mV). Also, the twitch after the test AP was larger and this increase subsided with subsequent APs. In figure 3b, the traces recorded at faster speed show that the increase in test AP ampli tude was associated with a decreased Vmax. Also, the test AP was shorter, and the twitch curves were fused. A similar procedure was repeated in the same preparation in the pres ence of 2.5 pM EPI. In figure 3c, AP ampli tude of the test APs was greater than that of the preceding AP (as in the absence of EPI). However, contractile force behaved differ ently in that in the presence of EPI the test contraction was smaller, but the subsequent potentiation was greater. In figure 3d, EPI shifted the plateau to more positive values and prolonged the AP. Also, EPI shortened the time to peak of twitch and this (together with the AP prolongation) allowed force to decay to a larger extent before the next twitch. Induction o f Calcium Overload. The ven tricular preparations were exposed to car dioactive drugs to find out whether DADs
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larger than those of the preceding regular AP. Both parameters decreased gradually with successive test APs as a function of the grad ual decrease in take-off potential brought about by DD. Similar patterns were obtained in 15 experiments. These results indicate that in human atrial fibers the recovery of the fast Na channel does not lag behind the mem brane potential, as found in animal Purkinje fibers [13]. This has obvious implications for excitability and conduction early in diastole in human atria and for re-entry rhythms. In addition. EPI shortened the ERP (ERP was 298.0 ± 17.5 ms in control and 247.3 ± 14.7 ms in the presence of 0.5 \iM EPI. p < 0.01).
Fig. 3. Restitution of the AP in human ventricular muscle libers in the absence (a. b) and presence (c. d) of 2.5 [i\f EPI. a. c APs and twitch curves are shown. The dots indicate test APs. b. d The first three APs are shown at faster time base.
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increase in diastolic force (dashed line). In the first panel of figure 4c. the drive was inter rupted. and DAD became obvious as the de polarization peaked and then decayed (up ward arrow) while at the same time a shallow aftercontraction appeared (downward arrow). In the second panel, the driving rate was increased (arrow above the APs): on stoppage of the faster drive, the last AP was followed by a larger DAD (upward arrow) and aftercon traction (downward arrow). Apparently, DAD was superimposed on DD since the membrane potential remained less negative than the MDP during the period of quies cence. The drive was repeatedly interrupted and DD and DADs (but no triggered activity) were present.
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and related arrhythmias could be induced (as in human Purkinje fibers [8]). While ventricu lar tissue was found to be more resistant to calcium overload. DADs could be induced but only by a combination of cardioactive drugs. In figure 4. after the control traces were recorded (4a), the preparation was exposed to strophanthidin (up to 1 pM). but neither DD nor DADs were induced (not shown). There after, theophylline (1 m.V/) and EPI (I \iM) were added to the strophanthidin solution. In figure 4b, the combination of these drugs de creased the resting potential, diminished AP amplitude and APD (at the plateau), induced an undershoot at the end of phase 3 repolari zation and caused DD. At the same time, the twitch was markedly increased. That DD was in part due to a DAD is suggested by the
Discussion DD in Human AtriaI and Ventricular Tissues The presence of DD in atrial fibers elicit the question as to whether this is a normal finding in human tissue or is the result of dis ease. The present results show that even when AP is a fast response, the resting potential is lower than that found in animal normal atrial tissue, suggesting that presence of DD is the result of abnormally low resting potential. The resting potential could be decreased because the intracellular K is decreased (less negative Ek) and/or gK is decreased. Both fac tors may be involved. For MDP to be more
negative than the diminished resting poten tial. it is only necessary that gK increases dur ing the AP (i.e. a time-dependent increase in the delayed rectifier current I k ). An increase in gK during AP would lead to an undershoot at MDP since increase in gK would allow the membrane to approach (the diminished) Ek. Still. MDP would be less negative than a nor mal resting potential if Ek is less negative. Apparently, ventricular muscle cells do not exhibit DD because the resting potential was better maintained (-90 mV) than in atrial cell (-74 mV). The role of a diminished resting potential in the induction of DD is suggested by the fact that also in ventricular tissue the decrease in resting potential caused by several
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Fig. 4. Induction of DAD in hu man ventricular muscle fibers. The traces were recorded in Tyrode so lution (a), in the presence of 0.5 u.\/strophanthidin plus I m.Y/ the ophylline plus 1 p.V/ EPI (b. c). c In the first panel, drive was inter rupted after the second AP. In the second panel (slow speed record ing). the rate of drive was increased at the arrow, and the drive was interrupted after the fourth AP re corded at faster speed. Dashed lines emphasize the changes in rest ing force during diastole.
Action o f EPI on AP o f Atria! and Ventricular Tissues EPI acts on both atrial and ventricular tis sues, but its effects are bound to depend on the characteristics of these tissues. Thus, in the present experiments, EPI decreased APD5o and increased DD slope in atrial fi bers, whereas it increased the positivity of the plateau and AP amplitude in most of the ven tricular tissues (without inducing DD). The explanation for these differences is not apparent. However, it is known that in atrial pacemarkers, catecholamines increase I k (as well as Isj) [17]. The increase in ISj in human atrial fibers is indicated by the increase in force. A simultaneous increase in I k would indeed bring about a fast repolarization of the AP and a steeper DD (as a larger Ik decays during diastole). For ventricular fibers. EPI may have ex actly the same effects on the two currents. Thus, contractile force increases also in this tissue. However, in animal ventricular tissues. I k is smaller than in other cardiac tissues [18], Therefore, the increase in ISj may prevail over that of the smaller IKand account for the posi tive shift of the plateau, AP prolongation and absence of DD. Effect o f EPI on ERP EPI affects ERP differently in atrial and ventricular muscle fibers in relation to the presence of DD in the former and the differ ent response of APD to EPI. Thus, EPI de creased Vmax of basic atrial APs, probably in relation to the decreased take-off potential due to the steepening of DD slope. And EPI increased Vmax in the ventricular preparation,
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possibly because the resting potential was the same and the intracellular sodium activity might have been decreased [ 19]. In atrial fibers, the role of the take-off potential in determining Vmax is shown by the fact that both Vmax and AP amplitude were markedly increased when APs were elicited at MDP. Both were decreased when the test AP originated during the repolarization phase at potentials positive to the resting potential. The height of the plateau of the test AP is much less affected as the slow channels are not dependent on the changes of the take-off potential at negative values. In ventricular muscle fibers, instead. Vmax was cither decreased or the same depending on whether take-off potential was less than the resting potential or similar to it. Still, AP amplitude of the test APs was somewhat greater than that of the regular beats and more so the earlier the extrabeat. The reason for the increase in amplitude of the extrabeat is un clear but it is unlikely to be related to a larger fast Na current as Vmax was decreased. In this connection, it is important to note that a larger AP amplitude had been observed in canine ventricular epicardium. but not endo cardium [20]. The greater AP amplitude in early diastole had been related to an incom plete time-dependent recovery of the tran sient outward K current It0 [20]. The fact that EPI shortened ERP in atrial, but prolonged it in ventricular muscle fibers is clearly related to the fact that EPI decreased APD in atrial but not in ventricular fibers [ 14; present results]. The reason why APD should be affected differently in these two tissues is not apparent. One possibility is that EPI in creases Isj and I k in both tissues, but more Ik in atrial and Isj in ventricular fibers. This would result in a shortening and in a prolon gation of AP. repsectively. in the two tissues.
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cardiotonic drugs was associated with DD (upon which a DAD was superimposed). These findings arc in agreement with the pos sibility of inducing DD in single myocytes by applied depolarization [ 16].
Conclusions The present results suggest that the pres ence of DD in atrial fibers may be the result of decreased resting potential and of a larger delayed rectifier current I k than in the ventri cle. The activation of a larger I k during the AP would allow the potential to undershoot the diminished resting potential. This also would account for the fact that EPI shortened the
tissues are more prone to the development of Ca overload than ventricular tissues, as it was easier to induce DAD (and triggered activity) in atrial than in ventricular muscle fibers. The results raise the possibility that in digitalized patients, release of catecholamines might more easily result in atrial triggered tachyar rhythmias. This will be facilitated by the over drive imposed by the faster sinus node rate under the influence of catecholamines.
A P .
While the DD did not attain the threshold and initiate spontaneous activity even in the presence of EPI. it did affect markedly prema ture APs. This elicits the possibility that in the atria, extrasystoles may be conducted at a fas ter speed if originating near the MDP. In turn, this may affect the rate at which the atria are activated in the presence of atrial flutter. The present results indicate that, in the presence of cardiotonic agents, human atrial
Acknowledgements We wish to express our appreciations to Dr. B.N. Chiang for general support and to Drs. K.K. Cheng and J. Wei for the supply of specimens. The present study was supported in part by grants from IBMS, Academia Sinicia. Taipei, and National Science Coun cil (grants 76-0412-BO 16-35 and 79-0412-B016-74). Taipei, Taiwan. ROC.
References 6 Valenzuela F. Vassalle M: Over drive excitation and cellular calcium load in canine cardiac Purkinje fi bers. J Elcctrocardiol 1985:18:2134. 7 Wit AL. Cranefield PF: Triggered activity in cardiac muscle fibers of the simian mitral valve. Circ Res 1976:38:85-98. 8 Dangman KH. Danilo P, Hordof AJ. Mary-Rabine L, Reder RF. Rosen MR: Elcctrophvsiologicchar acteristics of human ventricular and Purkinje fibers. Circulation 1982: 65:362-368. 9 Yeh TC. Lin CL Chiang BN: Me chanical restitution of human atrial myocardium in health and disease: in Yang WJ. Lee CJ (eds): Biomedi cal Engineering. Washington. Hemi sphere Publishing. 1989, pp 133— 144.
10 Vassallc M. Carpentier R: Over drive excitation: The initiation of spontaneous activity in Purkinje fi bers following a fast drive in the presence of norepinephrine. Pflü gers Arch 1972:332:198-205. 11 Lin CL Kotake H. Vassalle M: On the mechanism underlying the oscil latory current in cardiac Purkinje fibers. J Cardiovasc Pharmacol 1986:8:906-914. 12 Kass RS. Lederer WJ. Tsien RW, Weingart R: Role of calcium ions in transient inward currents and after contractions induced by strophan thidin in cardiac Purkinje fibres. J Physiol (Lond) 1978:281:187-208. 13 Weidmann S: The effect of the car diac membrane potential on the rapid availability of the sodium carrying system. J Physiol (Lond) 1955;127:213-224. Downloaded by: Stockholm University Library 130.237.165.40 - 1/10/2019 5:57:13 AM
1 Lin Cl. Chiu TH. Chiang BN. Cheng KK: Electromechanical effects of caffeine in isolated human atrial fibres. Cardiovasc Res 1985; 19: 727-733. 2 Lin CL Chuang IN, Cheng KK. Chiang BN: Arrhvthmogenic effects of theophylline in human atrial tis sue. Int J Cardiol 1987:17:289-297. 3 Hou ZY. Lin CL Vassallc M. Chiang BN. Cheng KK: Role of acetylcho line in induction of repetitive activ ity in human atrial fibers. Am J Physiol 1989;256:H74-H84. 4 Lin CL Tao PL. Chang YF, Chiang BN: Pacemaker activity is modu lated by tissue levels of cyclic adeno sine 3',5'-monophosphate in human atrial fibers, tnt J Cardiol 1989:25: 39-46. 5 Vassalle M. Mugelli A: An oscilla tory current in sheep cardiac Pur kinje fibers. Circ Res 1981:48:618631.
16 Valenzuela F. Vassallc M: Role of membrane potential in B r"-in duced automaticity in guinea pig cardiac myocytes. Cardiovasc Res 1991:25:421-430. 17 Noble D: The surprising heart: A review of recent progress in cardiac electrophysiology. J Physiol (Lond) 1984:353:1-50.
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18 Trautwein VV: Membrane currents in cardiac muscle fibers. Physiol Rev 1973:53:793-835. 19 Lee CO. Vassalle M: Modulation of intracellular N a‘ activity and car diac force by norepinephrine and C a -\ Am J Physiol 1983;244:C110C 1 1 4.
20 Litovsky SH. Antzelevitch C: Tran sient outward current prominent in canine ventricular epicardium but not endocardium. Circ Res 1988:62: 116-126.
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14 Eckel L, Gristwood RW. Nawrath H, Owen DAA: Inotropic and electrophysiological cfTects of histamine on human ventricular heart muscle. J Physiol (Lond) 1982:330:111123. 15 Mitchell MR. Powell T. Sturridgc ME. Terrar DA. Twist VW: Elec trical properties and response to noradrenaline of individual heart cells isolated from human ventricu lar tissue. Cardiovasc Res 1986:20: 869-876.
