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Exploration of human, rat, and rabbit embryonic cardiomyocytes suggests K-channel block as a common teratogenic mechanism

Christian Danielsson, Johan Brask, Anna-Carin Sköld, Rami Genead, Agneta Andersson, Ulf Andersson, Kenneth Stockling, Rickard Pehrson, Karl-Henrik Grinnemo, Sajjad Salari, Heike Hellmold, Bengt Danielsson, Christer Sylvén, Fredrik Elinder
DOI: http://dx.doi.org/10.1093/cvr/cvs296 23-32 First published online: 20 September 2012

Abstract

Aims Several drugs blocking the rapidly activating potassium (Kr) channel cause malformations (including cardiac defects) and embryonic death in animal teratology studies. In humans, these drugs have an established risk for acquired long-QT syndrome and arrhythmia. Recently, associations between cardiac defects and spontaneous abortions have been reported for drugs widely used in pregnancy (e.g. antidepressants), with long-QT syndrome risk. To investigate whether a common embryonic adverse-effect mechanism exists in the human, rat, and rabbit embryos, we made a comparative study of embryonic cardiomyocytes from all three species.

Methods and results Patch-clamp and quantitative-mRNA measurements of Kr and slowly activating K (Ks) channels were performed on human, rat, and rabbit primary cardiomyocytes and cardiac samples from different embryo-foetal stages. The Kr channel was present when the heart started to beat in all species, but was, in contrast to human and rabbit, lost in rats in late organogenesis. The specific Kr-channel blocker E-4031 prolonged the action potential in a species- and development-dependent fashion, consistent with the observed Kr-channel expression pattern and reported sensitive periods of developmental toxicity. E-4031 also increased the QT interval and induced 2:1 atrio-ventricular block in multi-electrode array electrographic recordings of rat embryos. The Ks channel was expressed in human and rat throughout the embryo-foetal period but not in rabbit.

Conclusion This first comparison of mRNA expression, potassium currents, and action-potential characteristics, with and without a specific Kr-channel blocker in human, rat, and rabbit embryos provides evidence of Kr-channel inhibition as a common mechanism for embryonic malformations and death.

  • Arrhythmia
  • Embryo
  • K-channel
  • Long-QT syndrome
  • Teratogenicity

1. Introduction

The delayed-rectifier potassium current (IK) is the major repolarizing current in adult human cardiomyocytes. This current is composed of two components, the rapidly activating IKr and the slowly activating IKs, which are conducted by voltage-gated potassium channels of the KV family. Four pore-forming α-subunits Kv11.1 (originally known as hERG, encoded by the gene KCNH2) form the channel generating IKr (hereafter called the Kr channel). The β-subunit KCNE2 (encoded by KCNE2) regulates Kv11.1 channel function in vitro.1 Four pore-forming α-subunits Kv7.1 (encoded by KCNQ1) form, together with the β-subunit KCNE1 (encoded by KCNE1), the channel generating IKs2 (hereafter called the Ks channel). Two other β-subunits, KCNE3 and KCNE4 (encoded by KCNE3 and KCNE4, respectively), regulate Kv7.1 channel function in vitro but their relevance in vivo has yet to be fully elucidated.3

Genetic mutations in the human subunits forming the Kr and Ks channels are associated with a prolongation of the cardiac action potential (AP).1,4,5 In surface electrocardiographic (ECG) recordings, this is represented by a prolongation of the QT interval, the time between the ventricular contraction and relaxation, and is called the long-QT syndrome (LQTS). LQTS, genetic and/or acquired by drugs, is associated with potentially lethal pro-arrhythmia in the form of Torsades de Pointes (TdP). More than 70 drugs that inhibit IK, especially IKr, can induce acquired LQTS and subsequently TdP in humans6 (www.qtdrugs.org). For new candidate drugs, it is therefore mandatory to investigate the pro-arrythmogenic potential in relevant adult animal models (e.g. dog in vivo and rabbit heart in vitro) before the start of human clinical trials,7 and thoroughly examine signs of LQTS in clinical trials.8

Several Kr-channel-blocking drugs (e.g. dofetilide,9 almokalant,9 ibutilide,10 sotalol,9,11 astemizole,12 citalopram,13 and cisapride14) cause malformations or embryonic death in rats and rabbits (the main species used in teratology studies). In the rat, these adverse effects can only be induced during a restricted, sensitive period [gestational days (GD) 9–14].9,15,16 Mechanistically oriented studies by different research groups show that the observed malformations and embryonic death are consequences of embryonic cardiac arrhythmia9,14,17,18 and hypoxia-reoxygenation damage.12,16,19,20 Various cardiac defects, in particular ventricular-septal defects, can be induced by a single dose during the period of cardiac formation.15 Drugs widely used during pregnancy, and known to cause LQTS due to a Kr-channel block in adult humans, such as the antidepressants citalopram,21 clomipramine,22 fluoxetine,23 and paroxetine,24 and the macrolide antibiotic erythromycin,25 have all been associated with a two-fold increased risk for ventricular-septal defects.

Despite the extensive literature on the role of Kr channels for cardiac arrhythmia in the adult heart across species, there is limited data from human, rat, and rabbit embryos, and a direct species comparison is lacking. Only one study is published on rat embryos (GD13), showing that dofetilide and almokalant dose-dependently prolong the cardiac AP, and induce early afterdepolarizations and cardiac arrhythmia.26 Available data on embryo-foetal cardiac electrophysiological development with regard to Kr- and Ks-channel expression and maturation are limited mainly to mouse. The quantification of mRNA encoding ion channels in mouse embryonic and foetal hearts shows that Kr- and Ks-channel subunits are expressed and undergo developmental changes.27 In situ hybridization of α- and β-subunit, mRNA has been used to temporally and spatially localize expression in the embryo-foetal period in mice and in the foetal period in rats.2830 Patch-clamp studies in cultured mouse embryonic and foetal cardiomyocytes found Kr channels to be the dominant K channel, and detected Ks channels solely in ventricular myocytes.31 Regulatory guidelines require animal studies in two species, one rodent (usually rat) and one non-rodent (usually rabbit) to assess the risk for drug-induced teratogenicity. Risk extrapolation from animal studies to humans is based on knowledge of physiological species similarities/differences. However, data from humans as well as from rat and rabbit are scarce; there are only a few studies on APs in human embryonic cardiomyocytes32,33 and none on ion currents. Nor are we aware of any study on ion currents in rabbit embryonic cardiomyocytes. This makes comparison and extrapolation of results across species very difficult.

Therefore, the aims of this study were to (i) electrophysiologically and molecularly quantify the expression of Kr and Ks channels in hearts from humans, rats, and rabbits at different time points of embryo-foetal development, to (ii) explore the pharmacological responses of these cells to the selective Kr-channel blocker E-4031, and to (iii) relate the results to reported stage-specific embryo-foetal adverse effects. The time points studied were chosen to cover the sensitive period for the induction of arrhythmia and teratogenicity in rats (GD9-14), and the corresponding period in humans [gestational weeks (GW) 4.5–10], and rabbits (GD8.5-15). In addition, cardiomyocytes were investigated after the sensitive period in rats (GD15 and 16) and rabbits (GD20), as well as in adult samples from all three species. This study provides, for the first time, explorations of ion currents from human and rabbit embryonic cardiomyocytes, and ECG recording on whole-embryo rat. The study suggests that Kr-channel inhibition is a common mechanism for embryonic malformations and death across species.

2. Methods

A complete Methods section is presented in the Supplementary material online, Methods.

2.1 Ethical permissions

To collect human adult and embryonic heart tissue, individual permission was obtained using a standard informed-consent procedure. Both parts of this study involving human material were approved by the Swedish regional Ethics committee (20040914) and conform to the principles outlined in the Declaration of Helsinki. All parts of this study involving animals were performed under Swedish legislation in accordance with former and current directives from EU (Directive 2010/63/EU of the European Parliament) and were approved by the Swedish local Ethics committee for animal studies (S178-08, S62-09, S19-11). Care and use of the animals also conforms to local regulations of Karolinska Institutet, the NOVUM research facility at Karolinska University Hospital, Stockholm, Sweden, and Safety Assessment, AstraZeneca R&D Södertälje, Södertälje, Sweden, respectively.

2.2 Animal handling and care

Sprague-Dawley rats (Charles River, Germany) were used at Karolinska Institutet, and Wistar rats (Harlan, The Netherlands) at AstraZeneca R&D Södertälje. New Zeeland White rabbits (Charles River, Germany) were housed at AstraZeneca R&D Södertälje. All animals had free access to food and water.

2.3 Cardiac material

First trimester human aborted material was obtained by using the gentle vacuum aspiration technique. Adult human samples were collected during open heart surgery. Sprague-Dawley rats were sacrificed in a CO2 chamber followed by cervical dislocation. Wistar rats were sacrificed by iv administration of sodium pentobarbital (∼100 mg/kg). Rabbits were sacrificed by boltgun followed by exsanguination.

2.4 Tissue and cell preparation

For patch-clamp measurements, isolated cardiomyocytes were plated on glass coverslips and cultured at 37°C for 1–3 days before the experiments. For the quantitative real-time polymerase chain reaction (qRT–PCR) experiments, heart tissue was kept frozen for later mRNA preparation. The whole-embryo culture method used for ECG experiments was modified from Webster et al.9 and Spence et al.18

2.5 Experimental methods

For the qRT–PCR experiments, embryonic and foetal hearts from rats and rabbits were pooled per litter. Human embryonic and adult hearts were analysed individually. The electrophysiological recordings were carried out using the Axopatch 200B patch-clamp amplifier and pClamp software (both Axon Instruments). ECG recordings from whole-rat embryos were carried out with the multi-electrode array (MEA) method previously used for cells and cardiac tissue specimens.3438

2.6 Statistics

Student's t-test or, when appropriate, one-way ANOVA was used using Graph-Pad Prism 5.0.

3. Results

3.1 Cardiac action potentials at different embryonic time points in humans, rats, and rabbits

Cardiomyocytes from human (GW5 and GW6-9), rat (GD11 and GD16), and rabbit (GD11 and GD20) were investigated with the whole-cell patch-clamp technique. Only spontaneously beating cells were selected for the investigation. Human embryonic cardiomyocytes showed a large variability in resting potential, AP duration, and inter-AP interval. This variability is illustrated in Supplementary material online, Figure S1, but because of the low yield of human cells, the origin to this variability could not be studied in the present investigation. Instead, we present mean values (as for the less variable rat and rabbit cells) and select for the figures cells with properties close to the average cell.

Figure 1A shows typical APs from human, rat, and rabbit embryonic cardiomyocytes, from two different time points. Figure 1B shows a summary of AP characteristics from all investigated cardiomyocytes. The resting potential, measured as the lowest membrane potential between two APs, was significantly more positive in human cells from GW5 than from GW6-9. We found no alterations in the resting potential in rat or rabbit. There were no alterations in the AP peak during the development for the three species suggesting no obvious alterations in Na-channel expression. The AP duration, measured at the 50% level (=APD50), did not change during development in human, while it increased in rat, and decreased in rabbit. The inter-AP interval varied from 600 to 900 ms for the different species but no significant alterations occurred during development. The inter-AP interval strongly correlated with the APD50 for the individual cardiomyocytes; for every millisecond increase in APD50 the inter-AP interval increased with 2.4 ms (data not shown). To explore the differences in more detail and to be able to explore effects of pharmacological compounds on AP, we performed a more detailed analysis on Kr and Ks channels.

Figure 1

Summary of AP characteristics from three different species at two time points. (A) Typical AP recordings from species and time points as indicated. (B) Summary of data. Upper panel, resting potential (open symbols) and peak potential (closed symbols). Middle panel, AP width at the voltage Vresting + (Vpeak − Vresting)/2 (=APD50). Lower panel, inter-AP interval. All data shown as mean ± SEM. n = 9;9;28;7;27;13. *, **, and *** indicate significant differences (P < 0.05, 0.01, 0.001) within each species.

3.2 Current density and mRNA levels of two different K channels

To understand how the properties of the APs alter during development, we explored the underlying ion currents of the cardiomyocytes by patch voltage-clamp recordings. All of the investigated species displayed large transient inward going Na and Ca currents followed by relatively large outward going K currents (Figure 2A). In the present investigation, we focused on currents and expression of Kr and Ks channels.

Figure 2

Whole-cell patch-clamp recordings of Kr and Ks currents in early embryonic cardiomyocytes. (A and B) Voltage-clamp families from human (GW5), rat (GD11), and rabbit (GD11). Voltage steps from −70 to +50 mV in steps of 10 mV from a resting potential of −70 mV (A). Kr tail currents (i.e. difference between recordings with and without E-4031) at −60 mV, following activation pulses between −60 and +40 mV (B). (CE) Tail current densities following activation pulses to voltages as indicated on the x-axis for Kr (C) and Ks (D), and +40 mV (E). Mean ± SEM. n = 6 (human Kr), 9 (rat Kr), and 8 (rabbit Kr), 5 (human Ks), 7 (rat Ks), and 8 (rabbit Ks).

3.2.1 Kr-channel expression shows large variability between species and stage of development

In all species, a tail current sensitive to the Kr-channel selective blocker E-4031 could be measured after return to −60 mV from different test-step potentials (Figure 2B; for details, see Supplementary material online, Methods). For the quantitative analysis, the peak tail-current amplitudes were adjusted for the cell size (Figure 2C and E). The IKr tail densities, after a step to +40 mV, were of comparable sizes in rats (GD11) and rabbits (GD11), but significantly smaller in humans (GW5-6; ∼50%; P < 0.05). To explore ion-channel expression at a molecular level and to gain increased time resolution, we used qRT–PCR to detect mRNA associated with IKr (Figure 3). mRNA transcripts from the Kr-channel α-subunit-coding gene KCNH2 and the β-subunit-coding gene KCNE2 were measured in cardiac tissue from the human, rat, and rabbit. The mRNA levels of KCNH2 transcripts in humans and rabbits were relatively high and stable throughout the investigated time span, whereas in rat, the mRNA levels decreased >100-fold from GD11 to GD15. Expression in adult rat tissue reappeared to the GD11 level. KCNE2 transcripts were prominent and stable in human throughout the investigated period. In rat, KCNE2 disappeared in adult cells, whereas it was low or even absent in rabbit throughout the investigated period.

Figure 3

mRNA 2−ΔCT values as parts per million of the internal control ribosomal 18s RNA for the Kr-channel-related subunits KCNH2 and KCNE2, and the Ks-channel-related subunits KCNQ1 and KCNE1. Y-axis is logarithmic. Open square indicates that the expression levels were too low to quantify. Number of hearts used in time order; human (n = 1,1,2,1,2,1,1,1,2); rat (n = 2,2,2,2,5); and rabbit (n = 3,3,3,2,4).

3.2.2 Ks-channel expression also shows large variability between species

The second studied K channel conducts IKs. In human and rat cardiomyocytes, a tail-current component sensitive to the Ks-channel selective inhibitor chromanol 298B was measured at −60 mV, following different test-step potentials. IKs were of equal size in the human and the rat, whereas it was not detected in the rabbit (Figure 2D and E). To explore the molecular components of Ks channels, mRNA transcripts for KCNQ1 and KCNE1 were measured in cardiac tissue from the three species (Figure 3). In human, the levels of KCNQ1 were comparable with those of KCNH2 and no major alterations in expression were seen during the studied time period. In the rat, KCNQ1 mRNA levels correlated inversely to those of KCNH2 and peaked at GD15. In the rabbit, however, KCNQ1 transcript levels were low, with prominent levels only in adult ventricular samples. KCNE1 is required for a proper Ks-channel expression and function. mRNA transcript levels were prominent and stable in human throughout the investigated time period. In the rat, KCNE1 was only found in the embryonic and foetal samples, with a peak at GD15. The rabbit was lacking KCNE1. This is in accordance with clear Ks current in the human and the rat and absence in the rabbit in the electrophysiological studies.

3.2.3 Ancillary subunits KCNE3 and KCNE4

The expression of mRNA coding for the ancillary subunits KCNE3 and KCNE4 was also measured in the cardiac tissue samples (Supplementary material online, Figure S2). In humans, the levels were ∼10 times lower than those of the KCNQ1 with no major alterations over time. In the rat, the KCNE3 mRNA level was slightly lower than that for KCNE4. Both followed the transient pattern of KCNQ1. In rabbit, only KCNE4 was measured, and the levels were low.

3.3 Kr-channel block affects action-potential rate and repolarization in a species- and development-dependent fashion

To further explore the role of the Kr channel, pharmacological experiments with the selective Kr-channel blocker E-4031 were conducted. The effect on AP was studied in cardiomyocytes from all three species with the patch-clamp method. The results showed that E-4031 prolonged the AP in a species- and development-dependent fashion. E-4031 at both 1 and 10 μM was tested for all cell types. However, no difference was seen between the two concentrations, suggesting that the effects saturated at 1 μM. Therefore, for the quantitative analysis, data for the two concentrations were combined.

Figure 4 shows the effect of E-4031 on four different human cardiomyocytes. Surprisingly, the two GW5 cells did not display any AP prolongation, whereas the GW7-7.5 and GW9.5 cells showed prolongations. However, all panels displayed subthreshold oscillations that have been suggested to be associated with cardiac arrhythmia,39 and a less stable rhythmicity. The lower GW5, GW7-7.5, and GW9.5 cells showed increased inter-AP intervals. The GW7 cell also showed a more positive resting potential. However, despite a large response variability, all human cells were investigated in some way affected by E-4031.

Figure 4

Effects of 1 μM E-4031 on human spontaneous APs as indicated.

The rat AP at GD11 was clearly prolonged by E-4031 (Figure 5A), whereas rabbit APD50 at the same GD was essentially unaffected despite a clear effect on the resting potential (Figure 5B). Figure 5CF shows a summary of all electrophysiological E-4031 experiments. E-4031 depolarized the rat GD11 and both rabbit cells, as expected from a K channel blocker. The AP peaks were reduced for both rabbit cells. The APD50 was increased by 60% for both rat GD11 and rabbit GD20, whereas the human cells, rat GD16, and rabbit GD11 were not affected. The prolongation was even larger at the foot of the AP. E-4031 also increased the inter-AP interval for rat GD11 with almost 200%, whereas all other cells were not significantly reduced in frequency. The lack of effects of E-4031 on rat GD16 was expected because KCNH2 expression was almost absent on GD16. Otherwise, the lack of effect can also depend on the amount of other repolarizing K channels (Supplementary material online, Figure S3); Ks channels provide a repolarization reserve that can protect the AP integrity and counteract the prolonging effect of E-4031. This mechanism can explain the lack of AP prolongation in some human cells.

Figure 5

Effects of E-4031 on three different species. (A) Rat GD11. (B) Rabbit GD11. In both panels black line control and red E-4031. (C) Resting potential alteration induced by E-4031. (D) Peak potential alteration induced by E-4031. (E) Relative AP prolongation induced by E-4031. (F) Relative interspike-AP increase induced by E-4031. Data as mean ± SEM. n = 3, 12, 7, 9, 10. *, **, and *** indicate significant differences (P < 0.05, 0.01, 0.001) compared with 0 (C and D) or 1 (E and F).

The effect of E-4031 was also studied at the whole-embryo level of the rat. To our knowledge, whole-embryo culture with ECG recordings has not been performed before. ECG from GD11 rat embryos was obtained before (Figure 6A–C) and after (Figure 6D) exposure to 50–1000 nM E-4031. Treatment with the vehicle decreased the QT interval by 32 ± 5 ms (Figure 6E) and the RR interval by 24 ± 12 ms. There was no relationship (r2 = 0.03) between changes in the RR and QT intervals, eliminating the need for the heart rate QT interval correction. E-4031 (50 nM) prolonged the QT interval by 46 ± 10 ms (78 ms relative vehicle, P < 0.001, Figure 6E) and the RR interval by 56 ± 13 ms (80 ms relative vehicle, P < 0.01). Higher concentrations of E-4031 reduced the complex amplitudes and induced arrhythmias, resulting in inability to automatically detect and measure QT and RR intervals. We found bradycardia and AV block of 2:1 and higher grade (Figure 6D). An amount of 200 nM provoked a AV block in 4/10 embryos, and 1000 nM in all embryos (15/15).

Figure 6

MEA ECG recordings of rat GD11. (A) Typical ECG recording. (B) Average signal. Another recording than (A). (C) ECG recording in untreated control. (D) ECG recording with 2:1 AV-block induced by 1 µM E-4031. (E) QT-time difference induced by 50 nM E-4031 (n = 10) compared with vehicle (n = 40). Data as mean ± SEM.

4. Discussion

In this study, we provide information from human, rat, and rabbit cardiomyocytes on AP characteristics, molecular and functional expression of Kr- and Ks-channels, and effects of a Kr-channel block at multiple embryo-foetal time points. This study describes for the first time Kr- and Ks-channel expression and ion currents in the human heart during embryonic development. Below, our data will be discussed in relation to reported embryo-foetal adverse effects in pregnant rats and rabbits, and in human pregnancy studies.

4.1 Rat

The Kr channel (KCNH2) is expressed only during a restricted period (Figure 3). This period, taking the 16 h half-life of the Kr-channel protein in consideration,40 is identical to the susceptible period on GD9-14 for the induction of developmental toxicity by Kr-channel blockers.9,15,16 The link between Kr and developmental toxicity is supported by functional and in vivo data: (i) The AP is shorter on GD11 compared with GD16 (Figure 1). (ii) Kr-channel blockers depolarize the cell, prolong the AP, and increase the inter-AP interval on GD11 (E-4031; Figure 5) and on GD13, respectively (almokalant and dofetilide26). In contrast, there is no effect on the resting membrane potential, the AP duration, or the inter-AP interval on GD16 after the susceptible period (E-4031; Figure 5). (iii) The Kr-channel blocker E-4031 increases the QT interval and induces bradycardia in whole-embryo MEA ECG recordings on GD11 (Figure 6). (iv) A teratogenic dose of the Kr-channel blockers almokalant,19 astemizole,12 and cisapride14 induces cardiac arrhythmia and hypoxia in embryos exposed in uteri on GD13. It is well established that transient hypoxia by temporary clamping of uterine vessels in rats cause embryonic death and similar malformations as Kr-channel blockers during the susceptible period.20,41,42 In contrast, these Kr-channel blockers do not induce embryo-foetal adverse effects, arrhythmia, or hypoxia on GD16, even at much higher doses (up to 30-fold) than those inducing developmental toxicity on GD13.12,14,19

The rapid decline in Kr-channel expression in late embryonic life occurs at the same time as the channel carrying the transient outward K current (Kto) takes over as the dominating repolarizing channel in rodents.28 This makes the adult rat inappropriate to use for the evaluation of pro-arrhythmic potential7 and explains why the mother, but not the embryo, tolerates high doses of Kr-channel blockers. No decline was observed in Ks-channel expression. Altogether, available data from rats indicate that the extensive embryonic death in teratology studies with repeated daily dosing GD6-15 by dofetilide, almokalant, ibutilide, cisapride, citalopram, and astemizole9,10,1214 is related to Kr-channel inhibition, and repeated episodes of cardiac arrhythmia and hypoxia. In contrast single escalating dosing on different days in the sensitive period in rats, which better mimic what may happen if a human embryo is suffering from one episode of drug-induced arrhythmia, produces stage-specific malformations at doses where the majority of the embryos survive.13 Ventricular-septal defects are the most ‘easy’ to induce; these could be induced after a single dose in high incidences on GD10, 11, or 12.15

4.2 Rabbit

Our data show that Kr-channel expression (KCNH2) is substantial in the embryonic (GD11-15), foetal (GD20), and the adult rabbit heart, whereas Ks-channel expression (KCNQ1 in combination with KCNE1) was absent. The high Kr- and low Ks-channel activity makes the adult rabbit heart very sensitive to assess arrhythmogenic potential of drug candidates,43 and our results fit well with reported maternal and extensive embryonic death in teratology studies with daily dosing during embryogenesis, at much lower doses than in rats,13 by the Kr-channel blockers cisapride, astemizole, or cimipramil.13 Although the specific Kr-channel blocker E-4031 depolarized the cells and reduced the AP peak (probably because of more inactivated Na and Ca channels as a consequence of the depolarized resting potential) at both embryonic (GD11) and foetal (GD20) time points (Figure 5), there were also indications of a larger importance of Kr channels during the foetal than the embryonic period: (i) The AP was shorter on GD20 than on GD11 (Figure 1). (ii) E-4031 prolonged the AP on GD20 but not on GD11. (iii) A fixed high single dose of the Kr-channel blocker sotalol caused more extensive embryo-foetal death at later time points11; the death was 15% on GD8-11 in the early embryo, 55% on GD12-14 in the ‘mid’ embryo and 90% on GD15-17 in the late embryo/early foetal period. Only 9% of the mothers died. Altogether, these results suggest that the embryo-foetal rabbit heart during the mid-to-late embryonic and early foetal period is much more susceptible to react with Kr-channel-related arrhythmia than the adult rabbit heart.

4.3 Human

In human, Kr- (KCNH2) and Ks-channel expression (KCNQ1 + KCNE1) was substantial and stable in both the embryonic and in the adult heart (Figure 3). E-4031 caused AP prolongation (Figure 4) in a similar way as in cardiomyocytes derived from human embryonic stem cells.44 However, there was large response variability (Figure 4), probably reflecting the inhomogeneity in the tested cells (Supplementary material online, Figure S1). Despite this variability, all cells responded to E-4031 with disturbances in the rhythm generation (Figure 4). Recent epidemiological studies associate widely used Kr-channel blockers with LQTS liability with foetal adverse effects. A two-fold increased risk for cardiac-septal defects is reported for the antidepressants cimipramil,21 paroxetine,24 and clomipramine22 and the macrolide antibiotic erythromycin.25 Antidepressants with QT risk, such as paroexetine and venlafaxine45,46 and the macrolide antibiotic clarithromycin,47 are associated with a two-fold increased risk for spontaneous abortions. Animal studies show that septal defects and embryonic death were the most ‘easy’ to induce by Kr-channel-blocking drugs, and that embryonic heart seems to be more susceptible to react with arrhythmia, than the adult heart. Altogether, these data suggest that Kr-channel inhibition is a relevant mechanism for developmental toxicity also in humans. The risk may be higher in the case of exposure to more than one risk factor for LQTS (e.g. high plasma concentrations due to interactions in metabolism, fever, genetic susceptibility, or simultaneous exposure to two Kr-channel-blocking drugs). New studies strengthen this hypothesis; the risk for ventricular-septal defects was four times increased21 and the risk for abortions was 3.5 times increased46 when pregnant women were using two or more antidepressants.

4.4 Conclusion

Our study provides new insights into the human, rat, and rabbit cardiac electrophysiological development, it provides further evidence of a Kr-channel block as an important mechanism for embryo lethality and malformations in animal teratology studies, and it suggests that the Kr-channel block-mediated teratogenic mechanism is of human relevance.

Funding

This work was supported by grants from the Swedish Governmental Agency for Innovation systems (Vinnova) (grant number P3349-1); the Swedish Research Council; the Swedish Heart-Lung Foundation; the County Council of Östergötland; King Gustaf V and Queen Victoriás Freemasons Foundation.

Acknowledgements

We would like to thank Eva Wärdell, Malin Larsson, Anette Wallstedt, Elisabeth Jalkesten, and Magdalena Grebius for excellent technical assistance.

Conflict of interest: none declared.

References

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