Ondansetron Inhibits Voltage-Gated K Current of Ventricular Myocytes from Pregnant Mouse

Background: The Htr3a antagonist, ondansetron, has been reported to prolong the QT interval and induce Torsades de pointes in the treatment of postoperative nausea and vomiting. To explore the mechanisms underlying these findings, we examined the effects of ondansetron on the mouse cardiac voltage-gated K+ (Kv) channel. Methods and Results: Ondansetron increased QT intervals in late pregnant (LP) mice. We measured the Kv channels in freshly isolated left ventricular (LV) myocytes from non-pregnant (NP) and late pregnant (LP) mice, using patch-clamp electrophysiology. Ondansetron blocked Kv current at a dose of 50 μM, and reduced the amplitude of peak current densities in a dose-dependent manner (0, 1, 5, 50 μM), in LP but not in NP mice. In contrast, serotonin and the Htr3 agonist, m-CPBG, increased Kv current densities in NP, but not in LP mice. Interestingly, during pregnancy, serum serotonin levels were markedly increased, suggesting the saturation of the effect of serotonin. Immunostaning data showed that Kv4.3 protein and Htr3a co-localize at the membrane and t-tubule of cardiomyocytes. Moreover, Kv4.3 membrane trafficking was enhanced in response to Htr3a-mediated serotonin stimulation in NP, but not in LP mice. Membrane analysis showed that serotonin enhances Kv4.3 membrane trafficking in NP, but not LP mice. Conclusion: Ondansetron reduced Kv current densities, and reduced the Kv4.3 membrane trafficking in LP mouse ventricular cardiomyocytes. This data suggests that QT prolongation by ondansetron is mediated by the reduction of Kv current densities and Kv4.3 membrane trafficking.


Introduction
Ondansetron (Zofran®) is a serotonin receptor  antagonist, which is used as an antiemetic. It was originally used was for nausea and vomiting after chemotherapy, irradiation, or surgery, but an extended off-label use has arisen, for hyperemesis and also nausea and vomiting in pregnancy (NVP) (Gideon Koren et al., 2012). Htr3 antagonists, granisetron (Kytril) and ondansetron, induce QT prolongation at high concentrations, leaving the patients with an elevated risk of cardiac dysrhythmias. [1][2][3][4] However, the underlying mechanisms of QT prolongation by ondansetron during pregnancy are not well known.
During late pregnancy (LP), the maternal heart needs to adapt to significantly increased circulatory needs. Pregnancy-induced electrocardiogram disturbances are often observed, such as QT interval prolongation accompanied by down-regulation of Kv4.3, one of the key ion channels for repolarization. 5 While all other serotonin receptor genes encode G-protein coupled serotonin receptors, the 5-HT receptor 3a gene (Htr3a) encodes subunits of the ligand-gated cation channels, forming a functional Htr3 channel. [6][7][8] Recently, we reported that Htr3a knockout mice (Htr3a -/-) succumb to sudden death with fatal arrhythmia during pregnancy. 1 In this study, we found that high serotonin levels initiated during pregnancy prevent severe QT prolongation during this period, by increasing the Kv current through Htr3a channels. The molecular mechanism underlying the sudden death and fatal arrhythmia during pregnancy in Htr3a -/animals was associated with blocking the effect of serotonin on Kv current.
To understand the mechanism behind QT prolongation by ondansetron during pregnancy, we directly measured the effect of

Optical mapping
Optical mapping was performed in 6 mice from each group.
After performing a median sternotomy, the hearts were quickly excised. The hearts were stained with RH237 (Invitrogen, California, USA), and were excited with quasi-monochromatic light (520±30 nm) from two green LED lamps. A camera with a 610-nm long pass filter (MiCAM Ultima, BrainVision, Tokyo, Japan) was used to collect the emitted light at 1 ms/frame and 100 x 100 pixels with a spatial resolution of 0.5x0.5 mm 2 /pixel. 8 To determine if the activation of Htr3 could shorten the action potential duration (APD), we measured the APD90 after infusion of serotonin (100 μmol/L), the Htr3 ag onist, 1-(m-Chlorophenyl)-biguanide (m-CPBG, 200 nmol/L), and the Htr3 antagonist, ondansetron (1 μmol/L).

Single-cell electrophysiological recordings and data analysis
All physiological tests were performed during non-pregnant (NP) and late pregnant (LP; G16-18) stages. All procedures were performed after an anesthetic (isoflurane/O2) was administered via inhalation, and the heart was quickly removed after deep anesthesia was established. The heart was cannulated using a 24 G needle and then retrogradely perfused via the aorta on a Langendorff apparatus. During coronary perfusion, all perfusates were maintained at 37°C and equilibrated with 100% O2.
Initially the heart was perfused with normal Tyrode solution for 2-3 min to clear the blood. This was followed by perfusion with a Ca 2+ free solution for 3 min, after which, the heart was perfused with an enzyme solution containing 0.14 mg mL −1 collagenase (Yakult) in Ca 2+ free solution for 12 min. After perfusion with the enzyme solution, the atria were separated from the ventricles, and chopped into small pieces. From these small pieces, single cells were dissociated in a high-K+ and low-Cl − solution using a blunt-tip glass pipette, and stored in the same solution at 4°C until use.
The recording protocols for Kv currents were also performed in ventricular myocytes, which were isolated from adult (8-10 weeks of age) C57BL6 mice as previously described. 2 Briefly, we used pre-pulse to −40 mV for 25 ms to inactivate INa. Kv current was recorded in voltage-clamp mode with 400 ms pulses from a holding potential of −70 mV, with different test potentials increasing from −40 mV to +60 mV in 10 mV steps. The currents were normalized to the cell membrane capacitance (Cm) and averaged. The values were not corrected for the junction potential (pipette offset); this was compensated prior to giga-seal formation.

Cell membrane fractionation assay
Membrane isolation was conducted as explained earlier. 3

Antibodies and immunoblot analysis
The primary antibodies used were anti-Kv4.  After blocking with 5% non-fat dry milk in 1X tris-buffered saline (TBS) containing 1% Tween 20 (for 1h), the membrane was processed for immunoblot analysis with indicated primary antibodies. The primary antibodies were detected with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-goat immunoglobulin G (IgG) secondary antibodies. Detection was performed using an enhanced chemiluminescence detection system (ECL, Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Confocal microscopy
Confocal microscopy was performed on isolated mouse ventricular myocytes as described previously. 4 Briefly, ventricular myocytes were plated on laminin (10 μg/mL)-coated coverslips

Serotonin shortens the QT interval and APD
To evaluate the effect of ondansetron on ventricular repolarization, we examined the effects of ondansetron on APD between NP and LP mice (Figure 1). When the animals were pretreated with an Htr3 antagonist, ondansetron (1 μmol/L), serotonin failed to shorten APD90. These results indicate that serotonin treatment shortens the APD and consequently the QT interval.

The effect of Htr3 antagonist on Kv current
We blocked serotonin receptors by applying ondansetron on freshly isolated LV cardiomyocytes from the NP and LP animals.

Serotonin activates Htr3a-mediated membrane trafficking
We evaluated the interaction between Kv4.3 and Htr3a in NP mice using confocal microscopy. Figure 5 revealed that Htr3a and

Main findings
Ondansetron prolonged the QT interval with a decrease in Kv current densities and reduced the amplitude of peak current densities in a dose-dependent manner in the hearts obtained from pregnant mice, but not non-pregnant mice. During pregnancy, Htr3a-mediated Kv4.3 membrane trafficking was significantly impaired. This data suggests that ondansetron should be used cautiously during pregnancy, which is physiologically related with QT prolongation.

Ondansetron and ion channels
In this study, high concentrations of ondansetron decreased Kv current densities in pregnant animals. Voltage-dependent Na + and K + channels are important determinants of the human action potentials. Yuri et al. reported that the submicromolar affinity of  has not been evaluated.
The maternal heart significantly adapts to the circulatory needs of pregnancy, but the effect of pregnancy on ventricular repolarization is poorly understood. Pregnancy is associated with Eghbali et al. 5,9 reported that Ito,f and IK,slow are down-regulated in

Ondansetron and Kv 4.3 membrane trafficking
Unlike the other serotonin receptor genes, which encode G-protein coupled serotonin receptors, Htr3a encodes subunits of the ligand-gated cation channel, and is essential for the formation of functional Htr3 channels. 6,8,10 The channel is predominantly selective for Na + and K + , and its serotonin-activated opening depolarizes the cell membrane by permeating an inward Na + current. 11,12 The precise role of Htr3 as an ion channel expressed in the heart has not been fully evaluated.