Cardiac automaticity is modulated by IKACh in sinoatrial node during pregnancy

Abstract

Aims: Pregnant (P) women have a significantly elevated resting heart rate (HR), which makes cardiac arrhythmias more likely to occur. Although electrical remodelling of the sinoatrial node (SAN) has been documented, the underlying mechanism is not fully understood. The acetylcholine-activated potassium current (IKACh), one of the major repolarizing currents in the SAN, plays a critical role in HR control by hyperpolarizing the maximal diastolic potential (MDP) of the SAN action potential (AP), thereby reducing SAN automaticity and HR. Thus, considering its essential role in cardiac automaticity, this study aims to determine whether changes in IKACh are potentially involved in the increased HR associated with pregnancy.

Methods and results: Experiments were conducted on non-pregnant (NP) and pregnant (P; 17-18 days gestation) female CD-1 mice aged 2 to 4 months. IKACh was recorded on spontaneously beating SAN cells using the muscarinic agonist carbachol (CCh). Voltage-clamp data showed a reduction in IKACh density during pregnancy, which returned to control values shortly after delivery. The reduction in IKACh was explained by a decrease in protein expression of Kir3.1 channel subunit and the muscarinic type 2 receptor. In agreement with these findings, current-clamp data showed that the MDP of SAN cells from P mice were less hyperpolarized following CCh administration. Surface electrocardiograms (ECGs) recorded on anaesthetized mice revealed that the cholinergic antagonist atropine and the selective KACh channel blocker tertiapin-Q increased HR in NP mice and had only a minimal effect on P mice. AP and ECG data also showed that pregnancy is associated with a decrease in beating and HR variability, respectively.

Conclusion: IKACh function and expression are decreased in the mouse SAN during pregnancy, strongly suggesting that, in addition to other electrical remodelling of the SAN, reduced IKACh also plays an important role in the pregnancy-induced increased HR.

Keywords: Acetylcholine-activated K+ current; Heart rate; Mouse model; Pregnancy; Sinoatrial node.

Graphical Abstract

Figure 1

Pregnancy decreases the density of the acetylcholine-activated K⁺ current (I_KACh) in mouse SAN cells. (A) Typical I_KACh recordings in NP and P mice obtained with the ramp protocol shown in inset. (B) Mean IV relationships show a significantly lower I_KACh density in SAN cells from P mice compared with NP mice [NP (n = 14, N = 10) vs. P (n = 11, N = 5): *P < 0.05 from −115 to −85 mV and from −70 to +45 mV]. (C) At −55 mV, mean I_KACh density is decreased in P mice (3.1 ± 0.4 pA/pF) compared wth NP mice (5.7 ± 0.6 pA/pF, P = 0.0026). Two-way ANOVA was used.

Figure 2

Kir3.1 and M2R expressions are decreased during pregnancy in mouse SAN tissue. (A) Mechanism of I_KACh in SAN cell. When CCh binds the M2R, the Gβγ complex binds the heterotetrameric channel composed of both Kir3.1 and Kir3.4, which generates I_KACh. RGS4 and RGS6, two regulators of G protein signalling in SAN tissue, are known to prevent the binding of Gβγ to the Kir channel and inhibit I_KACh. The G protein of the adenosine a1 receptor (A1R) is also known to activate the channel composed of Kir3.1 and Kir3.4. TPQ is a selective KACh channel blocker. (B) qPCR data showing mRNA relative expression of Kcnj3 (Kir3.1/GIRK1; P = 0.037), Kcnj5 (Kir3.4/GIRK4; P = 0.493), Chrm2 (M2R; P = 0.262), Adora1 (A1R; P = 0.713), Rgs4 (RGS4; P = 0.193), and Rgs6 (RGS6; P = 0.110) from NP and P mice (N = 6/group). Only Kcnj3 is decreased in P mice compared with NP mice (NP: 1.00 ± 0.09; P: 0.74 ± 0.05). (C) Western blot of Kir3.1 (left), Kir3.4 (middle), and M2R (right) protein in NP and P mice (N = 4/group). Densitometry analysis shows a reduction of Kir3.1 (NP: 1.00 ± 0.04; P: 0.86 ± 0.03, P = 0.024) and M2R (NP: 1.00 ± 0.08; P: 0.71 ± 0.05, P = 0.021) expression during pregnancy, although Kir3.4 is not statistically different between the two groups (NP: 1.00 ± 0.13; P: 0.61 ± 0.14, P = 0.094). Total protein content on stain-free was used to normalize protein signal (see Supplementary material online, Figure S3). Unpaired Student t-test was used in all figures unless specified otherwise.

Figure 3

The CCh sensitivity of SAN cells is reduced during pregnancy. (A) Typical recordings of spontaneous AP of SAN cells from NP mice in baseline condition and after application of 0.1 μM of CCh. (B) Bar graph shows that MDP is not altered by the application of 0.1–0.5 μM CCh (ΔMDP: −0.4 ± 0.9 mV; n = 6, N = 3) and is hyperpolarized after the application of 1 μM of CCh (ΔMDP: −10.5 ± 1.4 mV; n = 10, N = 6, P = 0.00014). (ΔMDP compared with respective controls, NP vs. NP + 0.1–0.5 μM CCh, P = 0.708; NP vs. NP + 1 μM CCh, P = 0.00003, *paired Student t-test). (C) Typical example of continuous recordings of spontaneous APs of SAN cells from NP mice under baseline conditions, in the presence of CCh (1 μM), and washout. (D) Application of CCh hyperpolarizes the MDP of spontaneous AP of both (left) NP (−55.8 ± 1.4 mV, +CCh: −66.3 ± 1.4 mV; n = 10, N = 6, P = 0.00003) and (middle) P (−56.8 ± 1.1 mV, +CCh: −60.9 ± 0.6 mV; n = 11, N = 4, P = 0.0019) mice. Paired Student t-test was used. (right) Bar graph shows the ΔMDP of NP and P SAN cells. The MDP is significantly less sensitive to CCh during pregnancy (ΔMDP: NP: −10.5 ± 1.4 mV, P: −4.1 ± 1.0 mV; P = 0.0012).

Figure 4

Pregnancy modifies the HR response to parasympathetic antagonist atropine. (A) Typical ECG recordings in NP (upper panel) and P (lower panel) mice before (baseline, left) and after (right) intravenous injection of atropine solution (0.5 mg/kg). (B) Atropine (left) increased HR of NP mice (461 ± 17 bpm, +Atropine: 527 ± 10 bpm, N = 8, P < 0.0001), (middle) whereas atropine has no effect on HR of P mice (535 ± 12 bpm, +atropine: 540 ± 11 bpm, N = 9, P = 0.145). Paired Student t-test was used. (right) Bar graph reports the ΔHR of NP and P mice after atropine administration. HR is significantly more responsive to atropine in NP compared with P mice (ΔHR: NP: 66 ± 8 bpm; P: 5 ± 3 bpm, P < 0.0001). (C) Typical ECG recordings in NP (upper panel) and P (lower panel) mice before (baseline, left) and after (right) intra-peritoneal injection of TPQ (5 mg/kg). (D) (left) TPQ increased HR in NP mice (475 ± 18 bpm, +TPQ: 537 ± 25 bpm, N = 7, P = 0.0003) and, to a much lesser extent, HR in P mice (593 ± 11 bpm, +TPQ: 605 ± 12 bpm, N = 6, P = 0.024). Paired Student t-test was used. (right) The bar graph shows the ΔHR of NP and P mice after TPQ administration. HR is significantly more sensitive to TPQ in NP than in P mice (ΔHR: NP: 62 ± 8 bpm; P: 12 ± 4 bpm, P = 0.0003).

Figure 5

HRV and BRV are both reduced in pregnancy. (A) Typical surface ECG recordings in anaesthetized NP and P mice. In vivo, pregnancy reduces (B) (left) RR intervals (NP: 128 ± 2 ms, N = 45; P: 110 ± 1 ms, N = 43, P < 0.00001), (middle) RMSSD (NP: 2.76 ± 0.25 ms; P: 2.06 ± 0.16 ms, P = 0.0186), and (right) SDNN (NP: 2.54 ± 0.21 ms; P: 1.67 ± 0.13 ms, P = 0.00066). (C, D) Atropine (left) decreased RMSSD (2.73 ± 0.48 ms, +atropine: 1.21 ± 0.18 ms, P = 0.023) and SDNN (2.53 ± 0.47 ms, +atropine: 1.28 ± 0.21 ms, P = 0.033) in NP mice (N = 8) but (right) has no effect on RMSSD (1.88 ± 0.27 ms, +atropine: 1.91 ± 0.29 ms, P = 0.818) nor SDNN (1.50 ± 0.20 ms, +atropine: 1.41 ± 0.19 ms, P = 0.571) in P mice (N = 9). Paired Student t-test was used. (E, F) TPQ (left) decreased RMSSD (2.70 ± 0.51 ms, +TPQ: 1.17 ± 0.44 ms, P = 0.0014) and SDNN (2.15 ± 0.34 ms, +TPQ: 0.94 ± 0.25 ms, P = 0.0027) in NP mice (N = 7) but (right) has no effect on RMSSD (1.66 ± 0.30 ms, +TPQ: 1.36 ± 0.14 ms, P = 0.392) nor SDNN (1.46 ± 0.18 ms, +TPQ: 1.35 ± 0.11 ms, P = 0.694) in P mice (N = 6). Paired Student t-test was used. (G) Typical spontaneous AP recordings of SAN cells from NP and P mice. (H) At cellular level, there is a decrease in (left) inter-beat intervals (NP: 212 ± 10 ms, n = 17, N = 10; P: 182 ± 6 ms, n = 12, N = 10, P = 0.020), (middle) RMSSD (NP: 20.5 ± 1.9 ms; P: 14.3 ± 1.0 ms, P = 0.0071), and (right) SDNN (NP: 16.0 ± 1.3 ms; P: 11.9 ± 0.9 ms, P = 0.020).

Figure 6

Reduction of I_KACh density is reversed following delivery. (A) Typical example of I_KACh recordings obtained in NP and PP mice. Inset shows ramp protocol. (B) Mean IV curves show nearly identical current of I_KACh density in SAN cells from NP (n = 14, N = 10) and PP (n = 12, N = 6) mice (P > 0.05). (C) Mean I_KACh current at −55 mV is similar in both NP and PP mice (NP: 5.7 ± 0.6 pA/pF; PP: 5.6 ± 0.5 pA/pF; P = 0.8639). Two-way ANOVA was used.