Mice Display Sex Differences in Halothane-Induced Polymorphic Ventricular Tachycardia
Background— Molecularly engineered mice are extensively used as models of cardiovascular diseases, yet little is known about sex differences in the electrophysiology of mouse hearts.
Methods and Results— This study investigated the influence of sex on drug-induced polymorphic ventricular tachycardia (PVT) in Langendorff-perfused male and female mice hearts (n=54) by injecting a bolus of halothane (1.75 mmol/L) in the perfusate while recording ECGs or optical action potentials (APs). There were no statistically significant differences between male and female hearts (n=54) with respect to mean RR (193±5 ms), PR (47±1 ms), QT intervals (101±3 ms), optical AP durations (APD75=23.11±4.2 ms), dispersion of refractory periods, and conduction velocities (n=5 male and 5 female). Halothane induced PVTs lasting a mean duration of 90 seconds; in female hearts, 55% of PVTs lasted longer than the median, whereas in male hearts 17% exceeded the mean (P<0.05). The total duration of PVTs exposed a marked sex difference, 378±144 seconds in female versus 27±10 seconds in male hearts (P<0.05). In optically mapped male hearts, halothane reduced APD75 (17.61±1.6 ms) and then elicited VTs (n=6 of 6), but in female hearts, halothane elicited PVTs (n=1 of 6) or arrested the hearts (n=5 of 6). Except for KCNE1, Northern blots (KCNQ1, MERG, Kv1.5, connexins 40 and 43, TREK1, and TASK1) did not detect sex differences.
Conclusions— This mouse model reveals sex difference in response to a pharmacological challenge yet does not display sex differences in standard electrophysiological parameters. Differences in KCNE1 may contribute to sex differences uncovered by halothane.
Received February 22, 2002; revision received April 30, 2002; accepted May 1, 2002.
Female sex is associated with two thirds of the cases of drug-induced polymorphic ventricular tachycardia (PVTs), such as Torsades de Pointes (TdP), which reflects how sex differences may impair women’s health.1 Clinical and experimental studies show that female sex is associated with a greater drug-induced QT prolongation, which facilitates the emergence of TdP.2,3⇓ Part of this difference has been ascribed to sex-specific regulation of ionic channel expression by sex steroids. However, a detailed characterization of sex-related alterations of channel expression remains difficult because of spatial heterogeneities in ion channels resulting in dispersions of action potential durations (APDs) and repolarization across the ventricles.4–6⇓⇓ Studies have explored the effects of androgen and estrogen replacement in oophorectomized animals and shed new light on how they influence the QT interval.7,8⇓ Others have investigated the effects of sex on arrhythmia vulnerability, mostly in large animals like the rabbit.9
Mice have become important models for cardiovascular diseases, because they represent the principal mammalian species for the production of molecularly engineered transgenic animals. Surface ECG measurements and in vivo cardiac electrophysiology have been successfully performed in mice,10,11⇓ but fewer studies have investigated the electrophysiology of isolated mouse hearts.12 Larger animals have been used successfully to study sex difference in QT interval,7,13⇓ drug-induced QT lengthening,2,14⇓ and arrhythmia.9 Sex was shown to influence the QT adaptation to heart rate11 in mice, but difficulties in measuring APDs or QT dispersion or eliciting arrhythmia have led to the notion that mice are of limited use in this area.
The present report reveals sex differences in perfused spontaneously beating mouse hearts by exposing the hearts to halothane while recording surface electrograms and optical maps of electrical activity elucidate the nature of halothane-induced arrhythmias.
Sex differences in halothane-induced arrhythmias were studied in mouse hearts perfused in a Langendorff apparatus adapted for ECG or optical mapping.7,12⇓ Briefly, mice (Charles River Laboratories, Wilmington, Mass) were anesthetized with sodium pentobarbital (35 mg/kg IP) plus heparin (400 UI/kg). The heart was removed via a midsternal thoracotomy, cannulated at the aorta, and retrogradely perfused at constant flow rate (2.3 mL/min) with Tyrode’s solution containing (in mmol/L) NaCl 112, KCl 5, NaHCO3 25, KH2PO4 1, MgSO4 1.2, CaCl2 1.8, and glucose 50, gassed with 95% O2 and 5% CO2 at pH 7.38 and 37°C. Pilot experiments (n=5) were performed to determine the stability of the model and showed no signs of rundown for >3 hours with respect to heart rate, ECG, and AP characteristics. ECG studies consisted of 12-week-old male and female OF1 mice (n=54) weighing 23 to 32 g.
In parallel experiments, hearts were stained with the voltage-sensitive dye di-4-ANEPPS (10 μL of 2 mmol/L in DMSO), as previously described.12 Light from a halogen lamp was collimated, passed through an interference filter (520±30 nm), and focused on the left epicardium by epi-illumination. Fluorescence was collected with a camera lens passed through a long-pass filter (>630 nm), and an image of the heart was focused on a 16×16-element photodiode array to simultaneously monitor APs from 256 diodes. Each diode (0.9×0.9 mm2, 0.11 mm dead space) recorded APs from hundreds of cells (330×330 μm2 area and a depth of ≈70 μm). The photocurrent from each diode was passed through an I/V converter (3-MΩ feedback resistor), amplified, digitized, and stored in computer memory. The perfused hearts were mounted in a chamber designed to abate movement artifacts without the use of chemical uncouplers, which modify the mouse AP.12
Northern Blot Analysis
Ventricle RNAs were isolated from 3-week-old and 2-month-old OF1 male and female littermate mouse hearts. Poly(A)+ RNA (2 μg) were separated by electrophoresis on 1% agarose gel and transferred onto nylon membranes (Hybond N, Amersham). Blots were probed with 32P-labeled specific cDNA fragments of the different cardiac-delayed rectifier K+ channel subunits, including KCNQ1, KCNE1, MERG, and Kv1.5, as well as gap-junction subunits connexins 40 and 43 and the 2P channel TREK1 and TASK1 in Express Hyb solution (Clontech) at 60°C for 16 hours, washed stepwise to a final stringency of 0.2 SSC, 0.3% SDS at 60°C, and exposed to a Fuji Bas-1500 imager for radioactivity quantification. Variations in loading were corrected by β-actin probing for internal quantification. Each channel signal was divided by the corresponding β-actin signal, yielding a ratio that allowed comparison according to sex.
Signals were recorded with 4 Ag+-AgCl electrodes from unpaced hearts, which flanked the epicardium in a simulated Einthoven configuration. Electrodes were connected to an adjustable band-pass differential amplifier (ORTEC Inc). ECG channels were amplified and filtered (1 to 2 kHz), stored, and analyzed with the use of pCLAMP (Axon Instruments). The PR interval was measured from the beginning of the P wave to the beginning of the R wave and the QT from the beginning of the Q (or the base of the R wave if not possible) to the end of the T wave, defined as the point at which the voltage returns to the isoelectric baseline. For each heart, a set of 3 consecutive RR-QT interval pairs was obtained from ECG recordings.
This investigation conformed to the guidelines of the Universities of Nice and Pittsburgh and to the Guide for Care and Use of Laboratory Animals (NIH publication No. 85-23, revised 1996).
Halothane Challenge to the Isolated Heart
A Tyrode solution containing 1.75 mmol/L halothane was prepared by diluting Tyrode saturated with 17.5 mmol/L Halothane (Laboratoires) and verified by gas chromatography. A 200-μL bolus of 17.5 mmol/L halothane was injected in the 1.8-mL reservoir of Tyrode that serves as a compliance chamber. Continuous perfusion with fresh Tyrode washed out the halothane such that the bolus exposed the heart to a maximum concentration of 1.75 mmol/L halothane that decreased rapidly.
Pilot measurements monitored the occurrence of arrhythmia after a 200-μL bolus of halothane; if no arrhythmia occurred, a 300-μL bolus and then a 400-μL bolus were injected after a 5-minute delay. The bolus of 200 μL was chosen because it provoked a ventricular arrhythmia in 5 out of 5 female hearts. In other experiments, an arrhythmia was induced and then propranolol (1 μmol/L) was perfused for 10 minutes before repeating the halothane challenge.
Analysis of Optical Data
Simultaneously recorded optical APs were used to determine the spatial heterogeneities of APDs, activation, repolarization patterns, and refractory periods. The activation time point at each diode was taken as the maximum first derivative of the fluorescence upstroke (dF/dtmax), and APD75 was calculated from the activation minus the repolarization time point (ie, the time when the downstroke recovers 75% to baseline).12,15⇓ Hearts were under sinus rhythm or paced with electrodes on the right or left ventricles at the apex or base of the free wall. Mean APD75 (mean±SD) was averaged from 9 diodes on the center of the left ventricle and compared for statistical significance between male and female hearts (CL=200 ms). Refractory periods were measured by pacing the heart (S1 to S1=200 ms) for 10 beats and then applying a premature impulse (S2) at decreasing S1 to S2 intervals from 200 to 100 ms in 5-ms steps, from 100 to 50 ms in 2-ms steps, and then from 50 ms until refractory period in 1-ms steps.12 Isochronal maps of activation were generated from local activation time points and conduction velocities from the sum of local velocity vectors.12,15⇓ APs with S/N ratios <10/1 or excessive movement artifact were not included in the analysis. Isochronal maps of activation were generated with the Delaunay’s triangulation algorithm.12 Fast Fourier Transforms (FFTs) were calculated to characterize the frequency distribution and spatial organization of VT.
Analysis of ECG waveforms was reported as mean±SEM. Global descriptive statistics were performed. Statistical analysis according to sex included the proportion of drug-induced arrhythmia that lasted ≥90 seconds (median value of the sample) (analyzed with Fisher’s exact test) and the duration of the anesthetic-induced arrhythmia (analyzed with 1-way ANOVA). Because the variability of the data increased with the duration of the arrhythmia and in some hearts an occasional zero value arose (absence of arrhythmia), a logarithmic transformation of the variable was performed16 according to the formula y=log(1+x), where x is the total duration of the anesthetic-induced ventricular arrhythmia (in seconds). Continuous variables, such as APD75, RR, QT, and PR interval values and their increase from baseline, were analyzed with 1-way ANOVA, Student’s t test, or Mann-Whitney-Wilcoxon rank-sum test when applicable (all tests performed with Statview 4.5 and SuperAnova 1.11, Abacus Corp). A value of P<0.05 was considered statistically significant.
Sex Differences in ECG Waveform
ECG recordings of Langendorff-perfused hearts began 10 minutes after the start of each experiment. The excellent stability of ECG signals made it possible to reliably measure the P, PR, and QT intervals in a total of 54 hearts. As illustrated in Figure 1, isolated hearts in sinus rhythm had the following mean values (in ms): RR, 193±25; P-wave duration, 21±1; PR, 47±1; and QRS, 15±0.5 (n=54). The average QT interval was 101±3 ms, yielding a corrected QT (Fridericia) of 175±4 ms (Figure 1A). Females tended to have a slower heart rate than males and a longer QT and QTc, but none of those differences were statistically significant (see Table 1).
Effects of Halothane
Halothane produced within 1 minute (39±2 seconds) a statistically significant prolongation of RR, PR, and QRS intervals (Figure 1B, Table 2). This was rapidly followed by complex ventricular arrhythmia resembling TdP (Figure 1C) in all but 5 hearts (n=49 of 54). When measurable, the average CL during VT was 57±2 ms (n=44 hearts). The mean duration of these arrhythmias (median, 90 seconds) was approximately equal to the time taken for the bolus of halothane to pass and wash out through the coronary circulation. After washout, arrhythmias ceased and the heart returned to sinus rhythm (Figure 1D) at a slower rate (RR=232±8 ms) and with longer PR intervals (75±3 ms, P<0.0001), which gradually returned to control in ≈3 minutes. Table 2 summarizes the changes in electrophysiological parameters caused by halothane.
Sex and Arrhythmias
Even though the time elapsed from the bolus injection to the onset of arrhythmia did not differ between sexes (male, 38±3 seconds versus female, 40±2 seconds, P<0.7), the occurrence and the duration of PVTs did vary with sex (Figure 2A). Male hearts were more resistant (25%) to halothane-induced PVTs than were their female counterparts (0%, P<0.05), and the durations of arrhythmias markedly differed according to sex (Figure 2B). Male hearts (17%) had PVTs lasting longer than the median value of 90 seconds, whereas most female hearts (55%) had arrhythmias exceeding it (P<0.05). The duration of PVTs was longer in female (378±44 seconds) than in male (27±10 seconds) hearts (n=49, P<0.05) (Figures 2, B and C). The cycle length of arrhythmias tended to be longer in females (60±3 ms) than in males (51±5 ms), but the difference fell within experimental error (P=0.07). Propranolol (1 μmol/L) was remarkably effective at protecting the hearts from halothane-induced arrhythmias in both sexes (n=5).
Sex Differences in APDs and Refractory Periods
Optical recordings detected a tendency for shorter APD75 in male compared with female hearts, although the difference was not statistically significant. The mean spontaneous CL was 225±12 ms with an APD75=23.11±4.2 ms (n=5 male and 5 female, 9 diodes/heart) in the absence of halothane (Figure 3A). When paced at a CL of 200 ms, ventricular APD75 of male and female hearts (n=5 hearts each) were not statistically different. Refractory periods were heterogeneous across the epicardium, being short at the apex and progressively longer toward the base. The gradient of refractoriness (difference between apex and base) had a tendency of being greater in female than in male hearts (10.8±2.5 versus 9.6±0.89 ms), but without statistical significance (Table 1). Halothane (200-μL bolus) elicited a tachycardia (CL=65 ms) and a shortening of APD75 to 17.61±1.6 ms (n=5) (Figure 3C). An activation pattern of an arrhythmic beat (Figure 3D) shows that activation propagated from right to left (bright to dark) in 5 to 6 ms. The stability of activation patterns and ECG from the opposite side of the heart indicated that the arrhythmia was a reentrant monomorphic circuit around the perimeter of the heart.12
The nature of arrhythmias elicited by halothane was investigated by mapping optical voltage oscillations during VTs. Halothane elicited more severe arrhythmias in female than male hearts, resulting in complete arrest of female hearts but brief MVTs (n=4 of 6) or PVTs (n=2 of 6) in male hearts. MVTs (960 beats/min) had a dominant frequency (mean 13.9±3.3 ms Hz, n=4) in their FFT spectra (Figure 3E) and Poincaré maps of interactivation intervals with stable CL (65.0±3.24, n=9 beats), with all points falling in a tight cluster indicative of stable reentry (Figure 3F). Hearts in halothane-induced PVTs exhibited regions with MVTs (Figure 4A) with a dominant frequency (Figure 4B) and regions with more complex voltage oscillations (Figure 4C) and FFT spectra (n=2 of 6) (Figure 4D). These results indicate that on the anterior surface of the heart, there are regions with out-of-phase monomorphic VTs and other regions with polymorphic VTs.
As in previous work, the level of KCNE1 mRNA was high in 3-week-old mouse hearts and decreased with age, reaching a low but detectable level at 12 weeks.11 The signal was sex-balanced in 3-week-old mice (ratio of male to female, 1.1) but more pronounced in adult female mice by 2- to 3-fold. The amount of messenger RNA for the other delayed rectifier subunits, including KCNQ1, Kv1.5 (encoding IKsus), MERG (the mouse counterpart of HERG), and connexin 40 and 43, TREK1, and TASK1 were not influenced by sex.
The manipulation of genes in the mouse has yielded important animal models to study how specific gene products modify the phenotype of complex physiological systems. Transgenic and gene-targeted mice have been developed to study cardiac arrhythmias; some are engineered to model human arrhythmias or human diseases in which arrhythmias occur.17 Other models are made to manipulate steroid hormone receptors to investigate how sex differences influence the cardiac phenotype.18 Once a molecularly engineered mouse is made, its phenotype must be characterized and correlated to an aspect of human physiology or pathophysiology. There are striking differences between mice and men with respect to heart rate, ventricular AP shape and durations, ionic currents involved in cardiac repolarization, and size of hearts.17 Despite these differences, there are striking parallels in mice and larger mammals with regard to gradients of repolarization and refractoriness, the spatial distribution of ionic channels, and the induction of reentry, although different ion channels are involved.12
Sex Differences Exposed by Halothane
Halothane has long been known to exert variable effects on cardiac rhythm19 and was shown to decrease heart rate and increase PR and QT intervals in dogs.20 Several cardiac channel currents are altered by halothane, namely a ubiquitous blockade of INa, ICaL,21 potassium channels, including IK22,23⇓ and derangements of gap junctions that could contribute to arrhythmia.24 In mammalian species, gap junctions are formed by isoforms of connexins where connexin 43 is expressed throughout the heart, connexin 40 in atrial muscles, and connexin 45 in the conduction system.25 Connexin 43 has been implicated in the pathogenesis of ventricular arrhythmia in mice.24 It is unlikely that sex differences in connexin explain these PVTs because no differences in connexin mRNAs were observed and normal P waves were recorded throughout such arrhythmia. Similarly, an eventual role of IKr, the rapid component of the delayed rectifier IK+, is not obvious, because specific IKr blockers (dofetilide and E4031) do not modify the mouse ECG. 2P channels (TREK 1 and 2 and TASK 1) could be targeted by halothane but would be protective rather than arrhythmogenic because of their tendency to induce cell hyperpolarization. Finally, with the exception of KCNE1 mRNA, which is expressed at lower levels in males (1 of 3) than in females, no other sex differences were seen in Northern blots. The duration of halothane-induced PVTs in male hearts (≈1 minute, maximum 3 minutes) roughly equals the time that halothane lasts in the coronary circulation. This is markedly different in female hearts where arrhythmias last far longer (ie, 14 to 44 minutes), emphasizing the importance of this observation, even if the mechanism of such a difference remains unclear.
Relevant Model of Sex Differences
Several models have been used for evaluating sex differences in QT interval and arrhythmia, including dogs26 and rabbits.8 Mice display a sex-dependent QT adaptation to heart rate with a steeper slope in females resulting in a longer QT during bradycardia in female mice.11 The present study sheds new light on the relevance of mouse hearts as a model of sex differences by revealing sex differences in the occurrence of PVTs in this species. Differences in the expression cardiac ionic channels or their modulation can account for sex-dependent PVTs, yet Northern blot experiments do not suggest so, with the exception of KCNE1.
Supported by National Institutes of Health grants HL57929 and HL59614 to Dr Salama, a postdoctoral fellowship from the American Heart Association to Dr Baker, and the Délégation à la Recherche Clinique (DRC) of the Centre Hospitalier Universitaire de Nice to Dr Drici.
- ↵Brahmajothi MV, Campbell DL, Rasmusson RL, et al. Distinct transient outward potassium current (Ito) phenotypes and distribution of fast-inactivating potassium channel alpha subunits in ferret left ventricular myocytes. J Gen Physiol. 1999; 113: 581–600.
- ↵Brahmajothi MV, Morales MJ, Reimer KA, et al. Regional localization of ERG, the channel protein responsible for the rapid component of the delayed rectifier, K+ current in the ferret heart. Circ Res. 1997; 81: 128–135.
- ↵Drici MD, Burklow TR, Haridasse V, et al. Sex hormones prolong the QT interval and downregulate potassium channel expression in the rabbit heart. Circulation. 1996; 94: 1471–1474.
- ↵Pham TV, Sosunov EA, Gainullin RZ, et al. Impact of sex and gonadal steroids on prolongation of ventricular repolarization and arrhythmias induced by IK-blocking drugs. Circulation. 2001; 103: 2207–2212.
- ↵Berul CI, Aronovitz MJ, Wang PJ, et al. In vivo cardiac electrophysiology studies in the mouse. Circulation. 1996; 94: 2641–2648.
- ↵Drici MD, Arrighi I, Chouabe C, et al. Involvment of IsK associated K+ channel in heart rate control of repolarization in a murine engineered model of Jervell and Lange-Nielsen syndrome. Circ Res. 1998; 83: 95–102.
- ↵Baker LC, London B, Choi BR, et al. Enhanced dispersion of repolarization and refractoriness in transgenic mouse hearts promotes reentrant ventricular tachycardia. Circ Res. 2000; 86: 396–407.
- ↵Liu XK, Katchman A, Drici MD, et al. Gender difference in the cycle length-dependent QT and potassium currents in rabbits. J Pharmacol Exp Ther. 1998; 285: 672–679.
- ↵Salama G, Kanai A, Efimov IR. Subthreshold stimulation of Purkinje fibers interrupts ventricular tachycardia in intact hearts: experimental study with voltage-sensitive dyes and imaging techniques. Circ Res. 1994; 74: 604–619.
- ↵Woolson RF. Transformation of Data to Equalize Variance. New York, NY: Wiley; 1987: 170–171.
- ↵Johnson BD, Zheng W, Korach KS, et al. Increased expression of the cardiac L-type calcium channel in estrogen receptor-deficient mice. J Gen Physiol. 1997; 110: 135–140.
- ↵Jongsma HJ, Wilders R. Gap junctions in cardiovascular disease. Circ Res. 2000; 86: 1193–1197.
- ↵Coppen SR, Dupont E, Rothery S, et al. Connexin45 expression is preferentially associated with the ventricular conduction system in mouse and rat heart. Circ Res. 1998; 82: 232–243.