Halothane inhibits the 4-aminopyridine-sensitive transient outward K(+) current (I(to)) which in many species, including humans, plays an important role in determining action potential duration. As I(to) is greater in the ventricular subepicardium than subendocardium, halothane may have differential effects on action potential duration and, therefore, contraction in cells isolated from these two regions.
Myocytes were isolated from the subendocardium and subepicardium of the rat left ventricle. Myocytes from each region were electrically stimulated at 1 Hz to measure contractions and action potentials and exposed to 0.6 mm halothane (approximately 2 x minimum alveolar concentration(50) for the rat) for 1 min. The time from the peak of the action potential to repolarization at 0 and -50 mV was measured to assess the effects of halothane on action potential duration.
Halothane inhibited contraction to a significantly (P = 0.002) greater extent in subendocardial myocytes than in subepicardial myocytes: the amplitude of contraction during control conditions was 3.6 +/- 0.4 microm and 3.2 +/- 0.7 microm in subendocardial and subepicardial cells, respectively, and this was reduced to 1.1 +/- 0.2 microm (29 +/- 2% of control, P < 0.0001, n = 10) and 1.4 +/- 0.3 microm (46 +/- 3% of control, P = 0.007, n = 7), respectively, after a 1-min exposure to 0.6 mm halothane. Control action potential duration (at -50 mV) was 67 +/- 10 and 28 +/- 4 ms in subendocardial and subepicardial myocytes, respectively, and these values were reduced to 39 +/- 6 ms (58 +/- 3% of control, P < 0.001) and 20 +/- 3 ms (73 +/- 5% of control, P = 0.009) by halothane, respectively.
Action potential duration was reduced to a greater extent in subendocardial than subepicardial myocytes, which would contribute to the greater negative inotropic effect of halothane in the subendocardium. Furthermore, the transmural difference in action potential duration was reduced by halothane, which could contribute to its arrhythmogenic properties.
THE volatile anesthetic halothane has both positive and negative inotropic effects on isolated ventricular tissue. 1–8It has been proposed that the initial transient positive inotropic effect observed on application of halothane results from a potentiation of the Ca2+-induced Ca2+-release process, 7–10whereas the sustained negative inotropic effect in intact cells is caused by a decrease in myofilament Ca2+sensitivity 8,11,12in conjunction with an inhibition of the electrically evoked Ca2+transient 8,11,13,14(resulting primarily from a decrease in sarcoplasmic reticulum Ca2+content 9,12,15). The inhibitory action of halothane on the L-type Ca2+current (ICa) 16–20and consequent decrease in action potential duration 8would contribute to the decrease in the Ca2+transient (and sarcoplasmic reticulum Ca2+content 12).
Recently, Davies et al. 21reported that halothane inhibited the 4-aminopyridine sensitive transient outward K+current (Ito), which in many species (including humans) is involved in early repolarization of the ventricular action potential. Itodensity is greater in ventricular subepicardium than subendocardium. 22–27As a consequence of the transmural gradient of Itoexpression and its blockade by halothane, halothane may affect action potential duration differentially across the ventricular wall. As contractility is modulated by action potential duration, 28the negative inotropic effect of halothane may be expected to differ in cells isolated from the ventricular subendocardium compared with the subepicardium.
Furthermore, because of the greater expression of Itoin the subepicardium, the action potential duration is shorter than in the subendocardium. This transmural gradient in action potential duration (and therefore refractoriness) is important for the normal spread of repolarization throughout the ventricle (i.e. , repolarization proceeds from subepicardium to subendocardium). As previously alluded to, inhibition of Itoby halothane is likely to affect action potential duration differentially across the ventricular wall, which could result in disturbances in normal transmural action potential duration gradients (with consequent changes in transmural refractoriness). Agents that modify the normal passage of repolarization and refractoriness are potentially arrhythmogenic, 29and it is interesting to note that, in the clinical situation, halothane anesthesia is associated with an increased incidence of arrhythmia. 30,31
The aim of the current experiments was to test the hypothesis that halothane has differential effects on action potential duration and contractility in cells isolated from the ventricular subendocardium and subepicardium, and that halothane affects the transmural gradient of action potential duration, which could contribute to its arrhythmogenic properties.
Materials and Methods
The experiments described were conducted on Wistar rats (weight, approximately 250 g; provided by Biomedical Services, University of Leeds, Leeds, United Kingdom) that were given access to food and water ad libitum and maintained under a 12-h light–dark cycle. Animals were killed by a blow to the head followed by cervical dislocation (schedule 1 procedure sanctioned by the UK government Home Office, London, United Kingdom, under project license 60/02087), and the heart was rapidly excised into an “isolation solution” (see below for composition), supplemented with 750 μm CaCl2and equilibrated with 100% O2. The heart was flushed of blood by retrograde perfusion via the coronary arteries with the above solution and then perfused for 4 min with the isolation solution, to which 100 μm Na2EGTA was added. 32The heart was then perfused for 7 min with the isolation solution supplemented with 1 mg/ml collagenase (type 1; Worthington Biochemical Corp., Lakewood, NJ) and 0.1 mg/ml protease (type XIV; Sigma, Poole, Dorset, United Kingdom), after which the ventricles were cut from the heart. The left ventricular free wall was then separated from the septum, and tissue was dissected from the subendocardium and subepicardium. Samples from both regions were then finely chopped and shaken in the collected enzyme solution (to which 1% bovine serum albumin was added) for 5-min intervals. Dissociated myocytes were collected by filtration at the end of each 5-min digestion, and any remaining tissue was returned for further enzyme treatment. The dissociated myocytes from each region were centrifuged at 30 g for 1 min, resuspended in the Ca2+-containing isolation solution, and stored at room temperature until required.
Single ventricular myocytes can be readily isolated from different regions of the heart and therefore represent an ideal preparation to investigate regional variations in myocyte properties. Furthermore, individual myocytes are small enough that equilibration of halothane is rapid throughout the entire preparation, which minimizes problems associated with diffusion delays that could occur in multicellular preparations. In addition, single cells appear to respond to inotropic interventions in qualitatively the same way as multicellular preparations. 33,34
The isolation solution was composed of 130 mm NaCl, 5.4 mm KCl, 1.4 mm MgCl2, 0.4 mm NaH2PO4, 5 mm HEPES, 10 mm glucose, 20 mm taurine, 10 mm creatine, pH 7.1 (NaOH) at 37°C. After dissociation, myocytes were perfused with a physiologic salt solution of the following composition: 140 mm NaCl, 5.4 mm KCl, 1.2 mm MgCl2, 0.4 mm NaH2PO4, 5 mm HEPES, 10 mm glucose, 1 mm CaCl2, pH 7.4 (NaOH) at 37°C (contraction experiments) or 30°C (action potential experiments). Halothane (0.6 mm) was added to the above solution from a 0.5-m stock solution made up of dimethyl sulfoxide. Halothane-containing experimental solutions contained 0.12% dimethyl sulfoxide, a concentration that had no significant effect on contractions (not shown).
The tip of each patch pipette was filled with a solution of the following composition: 140 mm KCl, 10 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 10 mm HEPES, pH 7.1 at 30°C. The pipette was then back-filled with the above solution supplemented with 400 μg/ml amphotericin B. Unless stated otherwise, all solution constituents were obtained from Sigma (Poole, Dorset, United Kingdom).
Recording Cell Length
Freshly dissociated myocytes were transferred to a tissue chamber (volume, 0.1 ml) attached to the stage of an inverted microscope (Nikon Diaphot; Nikon UK, Kingston-Upon-Thames, Surrey, United Kingdom). The cells were allowed to settle for several minutes onto the glass bottom of the chamber before being superfused at a rate of approximately 3 ml/min with the physiologic salt solution. Solutions were delivered to the experimental chamber by magnetic drive gear metering pumps (Micropump, Concord, CA), and solution concentration and temperature (37°C) were maintained by feedback circuits. 35
Myocytes were field stimulated at a frequency of 1 Hz (stimulus duration, 2 ms) via two platinum electrodes situated in the sides of the chamber. A charge coupled device camera attached to the side port of the microscope captured the cell image, and cell length was monitored continuously using a system developed by IonOptix (IonOptix Inc., Milton, MA), which is capable of discriminating a 0.01-μm change in cell length. Contractions were digitized at 200 Hz, and their time course was analyzed with IonWizard software (IonOptix Inc.). Contraction amplitude was measured from the baseline to peak and expressed as an absolute change in length and as a percentage of the control contraction amplitude before halothane exposure. Once contractions were stable during control conditions, 0.6 mm halothane was applied for 1 min. After the removal of halothane, if contraction did not return to within ± 10% of control, data were excluded from analysis.
Recording Action Potentials
Action potentials were recorded using the perforated patch clamp technique in current clamp mode (Axo-clamp 200A; Axon Instruments, Inc., Foster City, CA). After the formation of a gigaseal, cells were left for up to 15 min until access resistance had decreased to less than 30 MΩ. Action potentials were evoked in response to current pulses of 1 nA (2 ms in duration) at a frequency of 1 Hz. Voltage traces were filtered at 1 kHz, digitized at 5 kHz, and the time course of action potentials was analyzed using PClamp software (Axon Instruments Inc.). Because of the delay associated with establishing electrical access, these experiments were protracted. To increase the success rate of these experiments, they were conducted at 30°C where the cells were more stable and could tolerate the experimental protocols. However, it should be noted that the resulting action potential configurations recorded at 30°C would differ slightly from those recorded at 37°C because of the temperature sensitivity of ion channel conductance.
Data are presented as mean ± SEM, and statistical comparisons were conducted with the Student paired or unpaired t tests as appropriate or analysis of variance followed by corrected t tests (Tukey) for multiple comparisons, using either SigmaPlot 2000 or SigmaStat 2.0 (Jandel Scientific, Erkrath, Germany). All graphs were prepared using SigmaPlot 2000 (Jandel Scientific).
Effects of Halothane on Contraction of Subendocardial and Subepicardial Cells
Figure 1shows slow time base records of contraction of two representative cells isolated from the subendocardium (fig. 1A) and subepicardium (fig. 1B) of the rat left ventricle. During control conditions, contraction amplitude during 1 Hz stimulation was 3.6 ± 0.4 and 3.2 ± 0.7 μm in subendocardial and subepicardial cells, respectively. In both cell types, application of 0.6 mm halothane led to a similar (P = 0.618) initial increase in contraction amplitude (to 145 ± 7 and 153 ± 16% of control in subendocardial [n = 10] and subepicardial cells [n = 7], respectively), followed by a sustained inhibition of contraction as observed previously. 7,8At the end of a 1-min exposure to halothane, contraction was reduced to 1.1 ± 0.2 μm (P < 0.0001 for control vs. halothane, n = 10; to 29 ± 2% of control) in subendocardial cells, a degree of inhibition that was significantly greater (P = 0.002, analysis of variance) than observed in subepicardial cells (contraction reduced to 1.4 ± 0.3 μm, P = 0.007 for control vs. halothane, n = 7; to 46 ± 3% of control;fig. 1C). On removal of halothane, contractions returned to control values (to 101 ± 2 and 102 ± 1% of control in subendocardial and subepicardial cells, respectively).
The transmural difference in the sustained negative inotropic effect of halothane was qualitatively the same at 23 and 37°C. For example, at 23°C, after a 1-min exposure to 0.6 mm halothane, contractions were inhibited to a greater extent in subendocardial myocytes (to 14 ± 4% of control, n = 5) than in subepicardial myocytes (to 30 ± 6% of control, n = 3).
Figure 2shows fast time base recordings of contractions during control conditions and after a 1-min exposure to 0.6 mm halothane. Neither the time to peak nor the time to half relaxation of contractions differed between subendocardial and subepicardial myocytes during control conditions. However, in subendocardial myocytes, compared with control, halothane significantly prolonged the time to peak of contraction during the initial positive inotropic effect, abbreviated the time to peak of contraction during the sustained negative inotropic effect, and prolonged the time to half relaxation (table 1). These results are similar to those reported by Harrison et al. , 8although those experiments were conducted on cells isolated from general ventricular tissue. In subepicardial myocytes, the only significant difference in time course associated with halothane exposure was prolongation of the time to peak of contraction during the initial positive inotropic effect (table 1).
Effects of Halothane on Action Potentials in Subendocardial and Subepicardial Cells
Figures 3A and Bshow representative action potentials recorded from subendocardial and subepicardial myocytes during control conditions and after a 1-min exposure to 0.6 mm halothane. During control conditions, action potential duration (time from the peak to repolarization at −50 mV) was 67 ± 10 ms in subendocardial myocytes but was significantly shorter (P = 0.005, unpaired t test) in subepicardial myocytes (28 ± 4 ms). At the end of a 1-min exposure to halothane, action potential duration (at −50 mV) was significantly reduced in both cell types: to 58 ± 3% of control in subendocardial myocytes (i.e. , to 39 ± 6 ms, P < 0.001 vs. control) and to 73 ± 5% of control in subepicardial myocytes (i.e. , to 20 ± 3 ms, P = 0.009 vs. control), with halothane having a greater abbreviating effect in the subendocardium than the subepicardium (P < 0.05, analysis of variance).
Figure 4shows mean data for action potential duration at 0 mV (fig. 4A) and at −50 mV (fig. 4B) in both cell types during control conditions and after a 1-min exposure to halothane. These data illustrate that halothane led to significant reductions in action potential duration during both early and late phases of repolarization. However, it is also important to consider the transmural difference in action potential duration during control conditions and with halothane. Figure 4Billustrates that mean action potential duration (at −50 mV) differed by 39 ms during control conditions between the subendocardium and subepicardium, but this difference was reduced by half (to 19 ms) in the presence of halothane.
Regional Differences in the Negative Inotropic Effect of Halothane
Figure 1illustrates that a clinically relevant dose of halothane (0.6 mm, approximately 2 × minimum alveolar concentration50) reduced contraction to a significantly greater extent in subendocardial than subepicardial myocytes. Halothane is known to affect several targets in cardiac myocytes that could contribute to the observed regional effects of halothane on contraction (see Introduction). For example, halothane reduces the L-type Ca2+current (which results in abbreviation of the action potential), myofilament Ca2+sensitivity, sarcoplasmic reticulum Ca2+content, and Ito, but sensitizes Ca2+-induced Ca2+release (which is likely to contribute to the reduction in sarcoplasmic reticulum Ca2+content). The data presented here suggest that there must be transmural differences in the sensitivity of one or more of these targets to halothane. Cardiac contractility depends crucially on action potential duration, 28and the greater shortening of action potential duration induced by halothane in subendocardial cells would contribute to the greater negative inotropic effect of halothane compared with subepicardial cells (see below).
One interesting additional possibility is that the extent of halothane-induced reduction in myofilament Ca2+sensitivity may differ between the subendocardium and subepicardium, which could contribute to the greater negative inotropic effect of halothane in the subendocardium. Figures 1 and 2show that, during halothane exposure, diastolic cell length increases in both cell types, and the effect appears more marked in subendocardial than subepicardial myocytes. Such an increase in diastolic cell length is indicative of a decrease in myofilament Ca2+sensitivity as it occurs in the absence of a change in diastolic Ca2+. 12Analysis of the extent to which diastolic cell length increased during an exposure to halothane showed that, in subendocardial myocytes, cell length increased significantly from 108. 2 ± 4.8 to 108.9 ± 4.8 μm (P = 0.0016 vs. control, paired t test; by 0.6 ± 0.1% of control cell length) but increased to a lesser extent (from 118.4 ± 9.2 to 118.8 ± 9.3 μm (P = 0.026 vs. control, paired t test; by 0.3 ± 0.1% of control cell length) in subepicardial myocytes. Although there was no significant difference in the magnitude of the increase in cell length between the two cell types, these data suggest that myofilament Ca2+sensitivity may be inhibited to a greater extent in cells from the ventricular subendocardium than the subepicardium, although further experimentation would be required to verify this.
Regional Differences in the Effect of Halothane on the Action Potential
Figure 3illustrates that, during control conditions, the subepicardial action potential is shorter than the subendocardial action potential because of the greater density of Itoin subepicardium versus subendocardium. 22–27In the presence of halothane, action potential duration (at −50 mV) is shortened to a greater extent in the subendocardium than the subepicardium. As previously discussed, this would contribute to the greater negative inotropic effect of halothane observed in the subendocardium versus subepicardium.
The regional difference in the effects of halothane on action potential duration can be explained in terms of the inhibitory effects of halothane on ICaand Ito. Considering the expected electrophysiologic effects of halothane on ICain isolation, inhibition of an inward current (e.g. , ICaby halothane) would induce a shortening of the ventricular action potential. It is clear that there is no difference in the density of ICaacross the ventricular wall, 23,27,36,37and one would therefore expect similar action potential–abbreviating effects resulting from halothane-induced inhibition of ICain the subendocardium and subepicardium.
For illustrative purposes, figure 5shows representative records of Itofrom subendocardial and subepicardial myocytes (measured during conditions to block ICa21) in the absence and presence of 0.6 mm halothane. Itodisplays rapid activation (and thus overlaps in time with the activation of ICa) and is greater in subepicardial than subendocardial myocytes. Halothane induced a robust inhibition of Itoin subepicardial myocytes but had little effect on subendocardial Ito. Inhibition of this outward current alone by halothane 21would be expected to prolong action potential duration and, as Itois expressed to a greater extent in the subepicardium than subendocardium, 22–27then the effect of blocking this current on the action potential duration would be expected to be greater in the subepicardium than the subendocardium. However, the net effect of halothane on action potential duration will be the result of the combined effects of inhibition of both ICaand Ito. In subepicardial myocytes, where Itodensity is high (fig. 5B), simultaneous inhibition of an outward current (Ito) and an inward current (ICa) would result in only a small net change in the balance of inward and outward currents, and consequently only a modest effect on the duration of the action potential. That action potential duration is reduced in subepicardial cells suggests that inward current is inhibited to a greater extent than outward current (i.e. , the inhibitory effect of 0.6 mm halothane on ICais greater than Itoduring these experimental conditions).
Considering subendocardial myocytes, where Itodensity is much lower (fig. 5A), exposure to halothane would lead to only a small inhibition of outward current (because of the limited blockade of Ito), whereas the inward ICawould be inhibited to the same degree as in subendocardial cells. As such, inward current (ICa) would be inhibited to a greater degree than outward current (Ito), with the result that action potential duration would be abbreviated to a greater extent (figs. 3 and 4).
Transmural Gradient in Action Potential Duration Is Reduced by Halothane
One consequence of the regional effects of halothane is that the transmural difference in action potential duration is reduced. Figure 4illustrates that, during control conditions, the difference between the mean subendocardial and subepicardial action potential duration (at −50 mV) is 39 ms, but this difference is reduced to 19 ms by halothane. It should be noted, however, that action potential duration was measured in isolated cells and that, in vivo , the transmural gradient in action potential duration will be smaller because of the electronic coupling of cells (see Wolk et al. 38for review). The small transmural gradient in action potential duration that exists in vivo is important for normal transmural dispersion of repolarization–refractoriness, which helps prevent reentrant arrhythmias. However, agents that affect the normal passage of repolarization or transmural dispersion of refractoriness are known to predispose to reentrant arrhythmias. 29Our data suggest that, in vivo , the transmural dispersion of repolarization is reduced by halothane, which generally would be considered to be antiarrhythmic, not proarrhythmic. However, it is possible that in vivo , where the transmural gradient in action potential duration is smaller than recorded in isolated cells (see above), that the greater action potential–shortening effect of halothane on the subendocardium could potentially reduce the normal transmural gradient of repolarization. Given the complex architecture of the heart in terms of fiber orientation and impulse propagation, a potential consequence of altered repolarization would be an enhanced prospect of collision of repolarization wavefronts that would induce local conduction block. 38Therefore, the inhibitory influence of halothane on membrane currents, by altering normal repolarization, may modulate the vulnerability of the heart to arrhythmias induced by additional stimuli such as a premature ventricular excitation 39and could contribute to the increased incidence of arrhythmias observed in the clinical situation. 30,31
These data show for the first time that halothane has differential effects on contractility and action potential duration in subendocardial and subepicardial myocytes. The greater action potential shortening observed in the subendocardium versus the subepicardium may contribute to the greater negative inotropic effect of halothane observed in the subendocardium. However, further experiments examining halothane-induced changes in myofilament Ca2+sensitivity and cytosolic Ca2+regulation in the two cell types are required to further elucidate the differential effects of halothane on the subendocardium and subepicardium.
The authors thank Luke Blumler and Andy O’Brien (School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom) for expert technical assistance.