Ursodeoxycholic acid prevents ventricular conduction slowing and arrhythmia by restoring T-type calcium current in fetuses during cholestasis

Abstract

Background: Increased maternal serum bile acid concentrations in intrahepatic cholestasis of pregnancy (ICP) are associated with fetal cardiac arrhythmias. Ursodeoxycholic acid (UDCA) has been shown to demonstrate anti-arrhythmic properties via preventing ICP-associated cardiac conduction slowing and development of reentrant arrhythmias, although the cellular mechanism is still being elucidated.

Methods: High-resolution fluorescent optical mapping of electrical activity and electrocardiogram measurements were used to characterize effects of UDCA on one-day-old neonatal and adult female Langendorff-perfused rat hearts. ICP was modelled by perfusion of taurocholic acid (TC, 400μM). Whole-cell calcium currents were recorded from neonatal rat and human fetal cardiomyocytes.

Results: TC significantly prolonged the PR interval by 11.0±3.5% (P<0.05) and slowed ventricular conduction velocity (CV) by 38.9±5.1% (P<0.05) exclusively in neonatal and not in maternal hearts. A similar CV decline was observed with the selective T-type calcium current (ICa,T) blocker mibefradil 1μM (23.0±6.2%, P<0.05), but not with the L-type calcium current (ICa,L) blocker nifedipine 1μM (6.9±6.6%, NS). The sodium channel blocker lidocaine (30μM) reduced CV by 60.4±4.5% (P<0.05). UDCA co-treatment was protective against CV slowing induced by TC and mibefradil, but not against lidocaine. UDCA prevented the TC-induced reduction in the ICa,T density in both isolated human fetal (-10.2±1.5 versus -5.5±0.9 pA/pF, P<0.05) and neonatal rat ventricular myocytes (-22.3±1.1 versus -9.6±0.8 pA/pF, P<0.0001), whereas UDCA had limited efficacy on the ICa,L.

Conclusion: Our findings demonstrate that ICa,T plays a significant role in ICP-associated fetal cardiac conduction slowing and arrhythmogenesis, and is an important component of the fetus-specific anti-arrhythmic activity of UDCA.

Fig 1. Effect of TC on PR interval and heart rate in fetal and maternal hearts.

A and B: Representative ECG trace overlays following baseline and TC 400 μM treatment in fetal (left column; n = 3–7 per group) and maternal (right column; n = 3 per group) models. C and D: Plots of mean percentage change from baseline values in PR interval following treatment with TC 0, 40, 100, 400 μM and TC 400 μM with UDCA 1 μM treatment. E and F: Plots of mean percentage change from baseline values in heart rate following treatment with TC 0, 40, 100, 400 μM and TC 400 μM with UDCA 1 μM treatment. *P<0.05, One-way analysis of variance (ANOVA) and Bonferroni’s post hoc test to determine statistical significance.

Fig 2. Co-treatment with UDCA protects against TC induced conduction velocity slowing in the fetal heart.

A: Representative activation maps from each of the treatment groups in the fetal (FH) and maternal (MH) hearts (dashed arrows indicate direction of impulse propagation from point of stimulation). B: Plots of percentage change from baseline with control, TC 400 μM or TC 400 μM plus UDCA 1 μM treatments on conduction velocity in the fetal heart (n = 4–5 per group) and maternal heart (n = 3–4 per group). TC—taurocholic acid; UDCA—ursodeoxycholic acid. *P<0.05 One-way ANOVA and Bonferroni’s post hoc test to determine statistical significance.

Fig 3. UDCA inhibited conduction velocity slowing induced by a calcium, but not a sodium, channel blocker.

A: Representative activation maps from baseline and drug treatments in fetal hearts (dashed arrows indicate direction of impulse propagation from point of stimulation) B: Plots of percentage change from baseline following control; lidocaine 30 μM; lidocaine plus UDCA 1 μM; verapamil 1 μM and verapamil 1 μM plus UDCA 1 μM in the fetal hearts (n = 4–5 per group). C: Percentage change from baseline plots following control, mibefradil 1 μM, mibefradil 1 μM plus UDCA 1 μM and nifedipine 1 μM in the fetal model (n = 4–5 per group). D: Plots of percentage change from baseline following control and verapamil 1 μM treatment in the maternal hearts (n = 3 per group). *P<0.05 One-way ANOVA and Bonferroni’s post hoc test to determine statistical significance.

Fig 4. Effects of TC and UDCA on L-type whole-cell calcium currents from neonatal rat cardiomyocytes.

A: Representative whole-cell current traces from neonatal rat myocytes for each condition. Inset: test pulse protocol during which currents were elicited from a holding potential of −40 mV to test potentials ranging from −40 to +65 mV over 150 ms in 5 mV increments. Current peaked between 10 and 20 mV. B: Mean peak L-type calcium current density (normalized to cell capacitance) from neonatal cardiomyocytes with vehicle (n = 13), in the acute presence of 100 μM TC (n = 7), 100 nM UDCA combined with TC (n = 12) and with UDCA alone (n = 8). *P<0.05 using One-way ANOVA and Bonferroni’s post hoc test.

Fig 5. Effects of TC and UDCA on T-type calcium currents in neonatal rat and human fetal cardiomyocytes.

A: T-type calcium current density in neonatal rat cardiomyocytes from control (n = 12), TC-treated (n = 14), 100 nM UDCA combined with TC (n = 10) and UDCA only treatments (n = 6). 200 nM nifedipine was included in all solutions to block the L-type current. Data are shown as mean with SEM. B: T-type calcium current density from human fetal cardiomyocytes with vehicle (n = 14), in the presence of TC (n = 12) and with 100 nM UDCA combined with TC (n = 11). C: Voltage-clamp protocol and representative whole-cell current traces from neonatal rat myocytes for each condition. Inset: Test pulse protocol during which currents were elicited from a holding potential of −90 mV to test potentials ranging from −60 to 50 mV over 150 ms, where peak current occurred at -25 mV *P<0.05, ***P<0.0001 using One-way ANOVA and Bonferroni’s post hoc test.