The effect of acidosis on the ECG of the rat heart

The effect of acidosis on the ECG of the rat heart A. Aberra*, K. Komukai, F. C. Howarth and C. H. Orchard School of Biomedical Sciences, University of Leeds, Leeds LS2 9NL, UK, * Faculty of Medicine, Addis Ababa University, PO Box 9886, Addis Ababa, Ethiopia and Faculty of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, United Arab Emirates (Manuscript received 14 April 2000; accepted 17 October 2000) We have investigated the effect of acidosis on the ECG in isolated rat heart to determine whether acidosis has marked effects on the ECG, and have used pharmacological agents to investigate possible mechanisms whereby acidosis alters the ECG. Acidosis produced a marked decrease in heart rate and an increase in P R interval with little apparent effect on the duration of the QRS complex. The effects of acidosis did not appear to be due to acidosis-induced changes in transmitter release from severed autonomic nerve terminals within the heart. Experimental Physiology (2001) 86.1, 27 31. Acidosis alters the electrical activity of cardiac muscle, having marked effects on most of the membrane currents that have been studied (Orchard & Kentish, 1990; Orchard & Cingolani, 1994) and hence on the configuration of the action potential (e.g. Fry & Poole-Wilson, 1981; Kurachi, 1982). However, the effect of acidosis on the action potential will not be the same in all regions of the heart, because of regional differences in the expression of the ion channels underlying the membrane currents (Casis et al. 1998; Cheng et al. 1999). Acidosis-induced changes in any current that is not uniformly distributed throughout the heart will result in non-uniform changes in action potential configuration. In addition the effect of acidosis can depend on local conditions. For example, the transient outward current (Ito) is unaffected by acidosis at normal resting potentials, but becomes larger during acidosis when the diastolic membrane potential is depolarised (Hulme & Orchard, 2000). Since some regions of the heart have a relatively positive diastolic membrane potential (e.g. the sinoatrial (SA) and atrioventricular (AV) nodes), Ito will increase in response to acidosis only in those regions which express Ito and have a depolarised diastolic membrane potential. It appears likely, therefore, that regional variations in protein expression and, for example, diastolic potential will result in regional differences in the response of the action potential configuration to acidosis. The resulting dispersion of action potential configuration would be expected to alter the ECG and may underlie some of the arrhythmias which have been reported during and after acidosis (Antzelevitch et al. 1994; Orchard & Cingolani, 1994). However, the effect of acidosis on the ECG is unpredictable for a number of reasons. First, the effect of acidosis on Ito, and possibly on other currents, depends on the diastolic membrane potential (e.g. Hulme & Orchard, 2000), which varies in different regions of the heart, and is itself altered by acidosis (Orchard et al. 1987), and it is unknown whether there are regional effects of acidosis on diastolic potential. Second, although regional differences in action potential configuration have been reported using cells isolated from different regions of the heart (Antzelevitch et al. 1991) electrotonic current spread in coupled cells will tend to reduce these regional differences (Li et al. 1999). Third, the effects of acidosis reported previously may depend on the recording conditions used: the L-type Ca current, for example, has been reported to decrease during acidosis when recorded using whole cell patch clamp techniques (Irisawa & Sato, 1986), but not when using less invasive methods, such as the perforated patch clamp technique (Hulme & Orchard, 1998). Finally, the ECG monitored in vivo will be affected by other factors, such as the reflex effects initiated by the chemoreceptors, and the rise in plasma [K ] that occurs during acidosis. In the present study we have, therefore, investigated the effect of acidosis on the ECG in isolated rat heart to determine whether acidosis has marked effects on the ECG, and have used pharmacological agents to investigate possible mechanisms whereby acidosis alters the ECG. METHODS Preparation Wistar rats ( 250 g) of either sex were killed by stunning followed by cervical dislocation. The heart was rapidly removed and washed in Tyrode solution (see below for composition). The aorta was cannulated and retrogradely perfused with Tyrode solution at 37 C andatarateof7ml min. Recording electrodes were attached to the apex and base of the heart and the recorded ECG was analysed using a MacLab system. In clinical terms the apexïbase ECG is equivalent to a lead II ECG. Publication of The Physiological Society Corresponding author: chris.howarth@uaeu.ac.ae 2051

28 A. Aberra, K. Komukai, F. C. Howarth and C. H. Orchard Exp. Physiol. 86.1 Solutions The solution used for perfusion of the hearts during these experiments contained (mò): NaCl, 108; KCl, 5; NaµHPOÚ.12HµO, 1; MgSOÚ.7HµO, 1; sodium acetate, 20; glucose 10; CaClµ, 1; Hepes, 10; also 5 U l insulin. ph was first adjusted to 7.40 using NaOH. HCl was then used to decrease the ph of an aliquot of this solution to 6.50. Propranolol was kept as a stock solution and added to the perfusate to give a final concentration of 1 ² 10 Ç Ò. Atropine was kept as a stock solution and added to the perfusate to give a final concentration of 1 ² 10 Ç Ò. 4-Aminopyridine (4AP) was kept as a stock solution and added to the perfusate to give a final concentration of 10 mò. The ph of the perfusate was checked, and adjusted if necessary, after such additions. Protocols and measurements Two series of experiments were carried out. Series 1. Hearts were bathed with control solution until they appeared to be in a steady state. The perfusate was then switched to the acid solution for 5 min before returning to control ph for 10 min. Propranolol was then added to the perfusate; after 10 min exposure to propranolol at control ph the heart was exposed to acid solution containing propranolol for 5 min, before returning to control ph, still in the presence of propranolol for 10 min. The propranolol-containing solution was then washed out for 10 min and the protocol was then repeated using atropine instead of propranolol. Series 2. Hearts were bathed with control solution until they appeared to be in a steady state. The perfusate was then switched to the acid solution for 5 min before returning to control ph for 10 min. The perfusate was then switched to one containing 4AP; after 10 min exposure to 4AP at control ph the heart was exposed to acid solution containing 4AP for 5 min, before returning to control ph. The ECG was recorded continuously during these protocols. The ECG was then measured off-line, using the MacLab cursor system. Averaged measurements of 10 ECG records were made every 2 min while the hearts were being perfused with solution at control ph and every 1 min during perfusion with the acid solution. The following measurements were made: P R interval, R R interval (to monitor heart rate) and the duration of the QRS complex as shown in Fig. 1. It proved impossible to obtain reliable measurements of the T wave, making assessment of the Q T interval impossible. Statistics The data are presented as the mean ± s.e.m. Data at the end of the acid exposure were compared with data either just before exposure to the acid solution or with data obtained at the end of a control exposure to the acid solution. In either case, the comparison was made with data obtained in the same hearts, so that a paired t test was used for statistical comparisons. P values < 0.05 were considered significant. RESULTS The effect of acidosis on the ECG The data presented in Fig. 2 show that acidosis markedly slowed heart rate and prolonged the P R interval but had little apparent effect on the duration of the QRS complex. Figure 2A shows the time course of changes in heart rate before, during and after 5 min perfusion with the acid solution. The data show that acidosis caused a rapid and reversible decrease in resting heart rate, from 171.6 ± 12.0 beats min just before exposure to acidosis, to 75.4 ± 13.9 beats min after 5 min exposure to the acid solution (P <0.01, n = 10). This decrease in heart rate was accompanied by an increase in Figure 1 Typical example of ECG trace during perfusion of the heart with control solution. Horizontal bars bars show R R, P R and QRS intervals and provide a time scale. Figure 2 The effects of acidosis on heart rate (A) and duration of P R interval and QRS complex (B) before, during and after exposure to acidosis in isolated rat heart. Horizontal bars above curves show period of acid perfusion. Data are means ± s.e.m. (n = 10). **P <0.01,*P <0.05.

Exp. Physiol. 86.1 ECG during acidosis 29 the duration of the P R interval, from 45.0 ± 5.1 ms just before exposure to the acid solution, to 70.9 ± 8.5 ms after 5 min exposure to acidosis (P <0.01, n = 10). Both these changes were completely reversed on returning to control ph. In contrast to these changes, acidosis had no significant effect on the duration of the QRS complex (Fig. 2B). These data suggest that acidosis slows pacemaker activity in the heart, thus decreasing heart rate, but has no significant effect on the time course of the spread of the action potential through the ventricles. The prolongation of the P R interval could be due either to a slower propagation of the action potential through the ventricles, or to slower transmission of the action potential through the AV node. Since there is no apparent effect on the rate of propagation through the ventricles, as measured from the QRS complex it seems most likely that, unless acidosis has different effects on propagation through the ventricles and atria, acidosis is slowing transmission at the AV node. The effect of antagonists of autonomic transmitters on the response of the ECG to acidosis To investigate the possibility that the observed changes in the ECG were due to acidosis-induced changes in the release of endogenous autonomic transmitters from severed nerve terminals within the heart, the response of the ECG to acidosis was investigated in the presence of the â-adrenergic antagonist propranolol and the cholinergic antagonist atropine. Propranolol decreased basal heart rate from 157.8 ± 17.5 to 87.5 ± 12.0 beats min after 10 min (n =5, P < 0.05) (Fig. 3), but had no significant effect on the P R interval or the duration of the QRS complex. The P R interval and duration of the QRS complex in the absence or presence of propranolol were 62.0 ± 11.1 vs. 70.0±8.5ms and64.3±10.7vs. 56.0 ± 65.8 ms, respectively. Exposure to acidosis in the presence of propranolol reduced heart rate further, to 35.7 ± 5.6 beats min after 5 min (P < 0.05). The acidosis-induced decrease in heart rate in the presence of propranolol (to 43.2 ± 8.1% of that at control ph) was not significantly different from that in its absence (to 47.9±4.9% of control) (Fig. 4). Heart rate recovered to 78.9 ± 11.4 beats min 10 min after returning to control ph, still in the presence of propranolol. In the presence of propranolol, acidosis still increased the P R interval, but had no significant effect on the duration of the QRS complex; these changes were not significantly different from those observed in the absence of propranolol. Atropine did not significantly alter basal heart rate (Fig. 3), the P R interval or the duration of the QRS complex. Exposure to acidosis in the presence of atropine reduced heart rate from 122.0 ± 7.6 beats min to 52.8 ± 7.8 beats min after 5 min (n =5, P < 0.05). The acidosis-induced decrease in heart rate in the presence of atropine (to 48.2 ± 8.1% of that at control ph) was not significantly different from that in its absence (to 47.9 ± 4.9% of control) (Fig. 4). Heart rate recovered to 149.6 ± 11.9 beats min 10 min after returning to control ph, still in the presence of atropine. In the presence of atropine, acidosis increased the P R interval, but had no significant effect on the duration of the QRS complex; these changes were not significantly different from those observed in the absence of atropine. Thus atropine appeared to have no significant effect on either basal ECG or on the response of the ECG to acidosis. The effect of 4AP on the ECG The above data suggest that the effect of acidosis on the ECG is due principally to effects on the SA and AV nodes. Because Ito is known to be present in these nodes, to play a role in their function, and to be altered by acidosis at depolarised membrane potentials such as those in the nodes, the effect of blocking Ito using 4AP was investigated. 4AP decreased heart rate at control ph from 179.5 ± 22.6 to 102.5 ± 8.0 beats min after 10 min (P <0.05, n =5)(Fig. 3), consistent with previous suggestions that Ito plays an important role in pacemaking in the heart. 4AP increased the duration of Figure 3 The effect of pharmacological agents on basal heart rate (bpm, beats min ) at control ph. Heart rate was measured during the minute before starting perfusion with the acid solution. The first bar (Con) represents the control rate in the series of experiments in which propranolol (Prop) and atropine (Atrop) were used. The fourth bar (4AP con) represents the control rate for the experiments in which 4AP was used. Data are means ± s.e.m. (n = 5). Figure 4 The effect of acidosis on heart rate (expressed as percentage of heart rate at control ph) in the absence (Con) and presence of propranolol (Prop), atropine (Atrop) and 4AP.

30 A. Aberra, K. Komukai, F. C. Howarth and C. H. Orchard Exp. Physiol. 86.1 the QRS complex by a small, but insignificant amount, and had no significant effect on the P R interval, suggesting that Ito is not a major determinant of the rate of propagation of the action potential through the heart. In the presence of 4AP, acidosis still caused a significant decrease in heart rate (from 102.5 ± 8.0 just before perfusion with the acid solution to 41.0 ± 7.4 beats min after 5 min exposure to acidosis, P <0.01, n = 5). The acidosis-induced decrease in heart rate in the presence of 4AP (to 39.6 ± 5.2% of that at control ph) (Fig. 4) was not significantly different from that in the absence of 4AP (to 38.4 ± 11.8% of control). Acidosis increased the P R interval by the same amount in the presence as in the absence of 4AP (by 150.5 ± 21.9% and 146.2 ± 7.4% (P > 0.05), respectively), and had no significant effect on the duration of the QRS complex in the presence of 4AP. These data suggest, therefore, that the effects of acidosis on heart rate and on the P R interval are not due to acidosisinduced changes in Ito. DISCUSSION The present data show that acidosis has marked effects on the electrical activity of the heart, in particular causing a decrease in heart rate and an increase in P R interval, although there was no significant effect on the duration of the QRS complex. These effects of acidosis did not appear to be due to acidosisinduced changes in transmitter release from severed autonomic nerve terminals within the heart, because they persisted in the presence of the antagonists propranolol and atropine. Because the duration of the QRS complex reflects the propagation of the action potential through the ventricles, the observation that acidosis has no effect on the duration of the QRS complex suggests that acidosis has little effect on the rate of propagation of the action potential. This is in apparent contrast to previous studies which have reported an increase in intercellular resistance (Reber & Weingart, 1982; Pressler, 1987) and a decrease in the Na current, and hence a decrease in the rate of upstroke of the action potential during acidosis (Kagiyama et al. 1982), and a decrease in the rate of conduction of the action potential (Kagiyama et al. 1982). However, these effects were reported at more acid ph than those used in the present study. This suggests that such effects might only be important at more acid ph values than those found either physiologically or pathophysiologically and that more modest acidosis has little effect on the rate of propagation. If acidosis is not altering the rate of propagation of the action potential then the increase in the P R interval could be due to an increased delay in the AV node, rather than a decrease in the rate of conduction of the action potential through the atria. Similarly, the marked decrease in heart rate observed during acidosis could be explained by a direct inhibitory effect of acidosis on the SA node. Acidosis has previously been reported to alter the rate of contraction of isolated atria in the presence of propranolol and atropine (Gende et al. 1978), consistent with an effect of acidosis on the SA node. The mechanism whereby acidosis alters the function of the SA and AV nodes is less clear. Because acidosis has been reported to alter Ito when the diastolic membrane potential is depolarised (as in the nodes; Hulme & Orchard, 2000), one possible explanation is that the effects of acidosis on the nodes is mediated by a change in Ito (see Introduction). However, the same changes were observed in the presence of 4AP, an inhibitor of Ito, making this unlikely. It appears possible, therefore, that acidosis is altering one of the currents that underlie the pacemaker potential in the nodes. It has previously been suggested that an acidosis-induced decrease in resting potential (Orchard et al. 1987) might contribute to the T-Q segment depression observed during ischaemia (Orchard & Cingolani, 1994). However, such depression would only be expected during regional acidosis, whereas in the present study the entire heart was perfused with acid solution. The present data suggest, therefore, that acidosis alters node function, but has little other effect on the ECG. This suggests either that regional differences in the response of cardiac cells to acidosis are too small to be detected by the ECG, or that electrotonic current spread in the intact heart minimises such regional differences in the intact heart (Li et al. 1999) when all of the heart is made acid. There are, however, a number of differences between the isolated heart used in the present study and the heart in vivo. In particular, exogenous influences such as changes in plasma potassium, and chemoreceptor-mediated reflexes, will influence the ECG in vivo. In addition, in the present study the heart was not working against a load, and was not, therefore, stretched. Since stretch of the heart is known to alter its electrical activity, it is possible that the response of the ECG to acidosis might be altered in the working heart. Antzelevitch, C., Sicouri, S., Liovsky, S. H., Lukas, A., Krishnan, S. C., Di Diego, J. M., Gintant, G. A. & Liu, D. W. (1991). Heterogeneity within the ventricular wall. Electrophysiology and pharmacology of epicardial, endocardial and M cells. Circulation Research 69, 1427 1449. Antzelevitch, C., Sicouri, S., Lukas, A., Di Diego, J. M., Nesterenko, V. V., Liu, D. W., Roubache, J. F., Zygmunt, A. C., Zhang, Z. Q. & Iodice, A. (1994). Clinical implications of electrical heterogeneity in the heart. The electrophysiology and pharmacology of epicardial, M and endocardial cell. 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Exp. Physiol. 86.1 ECG during acidosis 31 Gende, O. A., Camilion de Hurtado, M. C. & Cingolani, H. E. (1978). Chronotropic response of isolated atria to acid base alterations. Archives internationales de physiologie et de biochimie 86, 997 1009. Hulme, J. T. & Orchard, C. H. (1998). Effect of acidosis on Ca uptake and release by the sarcoplasmic reticulum of intact rat ventricular myocytes. American Journal of Physiology 275, H977 987. Hulme, J. T. & Orchard, C. H. (2000). Effect of acidosis on transient outward potassium current in isolated rat ventricular myocytes. American Journal of Physiology 278, H50 59. Irisawa, H. & Sato, R. (1986). Intracellular and extracellular actions of proton on the calcium current of isolated guinea-pig ventricular cells. Circulation Research 59, 348 355. Kagiyama, Y., Hill, J. L. & Gettes, L. S. (1982). Interaction of acidosis and increased extracellular potassium on action potential characteristics and conduction in guinea pig ventricular muscle. Circulation Research 51, 614 623. Kurachi, Y. (1982). The effects of intracellular protons on electrical activity of single ventricular cells. Pfl ugers Archiv 394, 264 270. Li, Z., Zhang, H., Holden, A. V. & Orchard, C. H. (1999). Computer simulation of electrical activity of guinea-pig ventricular cell: regional differences and propagation. Journal of Physiology 521.P, 30P. Orchard, C. H. & Cingolani, H. E. (1994). Acidosis and arrhythmias in cardiac muscle. Cardiovascular Research 28, 1312 1319. Orchard, C. H., Houser, S. R., Kort, A. A., Bahinski, A., Capogrossi, M. C. & Lakatta, E. G. (1987). Acidosis facilitates spontaneous sarcoplasmic reticulum Ca release in rat myocardium. Journal of General Physiology 90, 145 165. Orchard, C. H. & Kentish, J. C. (1990). The effects of changes of ph on the contractile function of cardiac muscle. American Journal of Physiology 258, C967 981. Pressler, M. L. (1987). Effects of pcai and phé on cell-to-cell coupling. Experientia 43, 1084 1091. Reber, W. R. & Weingart, R. (1982). Ungulate cardiac Purkinje fibres: the influence of intracellular ph on the electrical cell-to-cell coupling. Journal of Physiology 328, 87 104. Acknowledgements This project was supported by a grant from the British Council, Addis Ababa, Ethiopia.