Hyperpolarization activated cation current (I(f)) in cardiac myocytes from pulmonary vein sleeves in the canine with atrial fibrillation

Abstract

Objective: To investigate the characteristics of ectopic automaticity and cation current (I(f)) of cardiac myocytes from pulmonary vein sleeves (PVs) in canines with atrial fibrillation.

Methods: The canines (8-10 years old) were subjected to long-term, rapid atrial pacing (RAP) for 10 weeks, which induced the atrial fibrillation model. Disassociation of PVs of canines yielded single cardiac myocytes from a Landengorff column. Action potential, I(f) and hyperpolarisation activated cyclic nucleotide-gated (HCN) currents were measured with the patch-clamp technique.

Results: Compared with the control group, cardiac myocytes from the RAP canine PVs had spontaneous diastolic depolarization, shorter action potential duration, and larger I(f) densities. In the group of RAP cells, the half maximal activation potential (V(1/2)) was found to be less negative (-105.5 ± 5.2 mV) compared to control cells (-87.3 ± 4.9 mV). Current densities of I(f) were increased significantly by β-adrenergic receptor stimulation with isoproterenol and caused an acceleration of current activation. In contrast, I(f) currents in the RAP were reduced by carvedilol, a selective beta-adrenergic receptor. Another important finding is that HCN4-based channels may make a significant contribution to I(f) in PVs cells, but not HCN2. Meanwhile, HCN4 current significantly increases in canine PVs cardiac myocytes with RAP.

Conclusions: The spontaneous action potential and larger I(f) current were observed in the PVs cardiac myocytes using RAP, which may contribute to more ectopic activity events to trigger and maintain atrial fibrillation.

Keywords: Atrial fibrillation; Canine; Cardiac myocytes; Cation current; β-receptor.

Figure 1.. Cardiac myocytes and tissue from pulmonary vein sleeves in canine.

(A): PVs tissue (yellow arrow); (B): PVs myocytes (100 ×); (C): PVs myocytes (400 ×). PVs: pulmonary vein sleeves.

Figure 2.. APs configurations of PV cardiac myocytes with and without rapidly atrial pacemaking.

(A): APs of control cells with rest potential of –65 ± 3 mV and APD₉₀ of 282 ± 11 ms; (B): Spontaneous AP of PVs cells with maximum diastolic potentials of –63 ± 2 mV and APD₉₀ of 343 ± 14 ms. The spontaneous APs in some PVs cells were recorded directly, while APs in other cells were induced by using test current pulse of 900 pA for 5 ms at a holding potential of 0 mV. APs: action potentilas; PVs: pulmonary vein sleeves; RAP: rapid atrial pacing.

Figure 3.. I_f current characteristics of PVs cardiac myocytes.

(A): I_f current densities were significantly higher in RAP than in control cells at potentials –120 Mv; (B): RAP: –2.66 ± 0.4 pA/pF vs. Ctrl: –0.91 ± 0.2 pA/pF, P < 0.01, n = 10; (C): the current–voltage relationships of the mean I_f for RAP and control cells; (D) the steady-state activation curve showed that V_1/2 was changed from –105.5 ± 5.2 mV in control cells to –87.3 ± 4.9 mV in RAP group and k was changed from 9.5 ± 1.8 mV to 11.1 ± 2.6 mV. The presence of I_f was determined in cells by application of hyperpolarizing voltage steps from –50 to –120 mV in 10 mV increments of 2000 ms duration at the holding potential of –40 mV, and then depolarized to +20 mV for 200 ms to elicit tail current. Current traces obtained in representative cells from Control and RAP. PVs: pulmonary vein sleeves; RAP: rapid atrial pacing.

Figure 4.. Effect of Iso on I_f of PVs cardiac myocytes.

Peak current of I_f was increased from –2.66 ± 0.4 pA/pF to –3.27 ± 0.7 pA/pF to –3.47 ± 0.73 pA/pF by 1.0 µmol/L Iso (P < 0.01, n = 11), while peak currents of I_f of RAP cells were increased from –0.91 ± 0.22 pA/pF to –1.32 ± 0.13 pA/pF by 1.0 µmol/L Iso at –120 mV of test potential (P < 0.01, n = 11, A & B); (C): Iso-induced increase of I_f was tested under the concentration of 0.1–10.0 µmol/L and the EC₅₀ value was 0.7 µmol/L (0.4–1.5 µmol/L, 95% CL); (D): I-V relationships demonstrated I_f densities were markedly enhanced by 1.0 µmol/L Iso more negative –80 mV potentials with a repeated-measures ANOVA. It showed the voltage dependence of I_f activation was significantly affected by the application of Iso; (E): V_1/2 for activation was shifted from –88.3 ± 4.7 mV to 69.3 ± 3.4 mV and k value was changed from 9.5 ± 1.8 mV to 5.2 ± 1.2 mV by 1.0 µmol/L Iso (P < 0.01, n =11); (F): More events of spontaneous diastolic depolarization in PVs cardiac myocytes with Iso were observed. EC₅₀: concentration for 50% of maximal effect; Iso: isoprenaline; PVs: pulmonary vein sleeves; RAP: rapid atrial pacing.

Figure 5.. Effect of Ivabradine on I_f of PVs cardiomyocytes.

(A): peak current densities of I_f in RAP cells were reduced from –2.74 ± 0.52 pA/pF to -1.47 ± 0.26 pA/pF (P < 0.01, n = 12), while densities of I_f in control cells were reduced from –0.93 ± 0.20 pA/pF to –0.61 ± 0.05 pA/pF by 1.0 µmol/L Iva. Both inhibition effects showed significant difference (inhibition percent of 46.4% in RAP cells vs. 33.3% in control cells, P < 0.05); (B): I-V relationships demonstrated that significantly inhibited effects of 1.0 µmol/L Iva on I_f density more negative –90 mV potentials with a repeated-measures ANOVA; (C) Iva-induced inhibition concentration dependence of I_f was tested under the concentration of 0.1–10.0 µmol/L and the IC₅₀ value was 3.2 µmol/L in RAP and control cells respectively; (D): The events of spontaneous diastolic depolarization were reduced by 1.0 µmol/L Iva. Iva: ivabradine; PVs: pulmonary vein sleeves; RAP: rapid atrial pacing.

Figure 6.. mRNA expression of HCN4 and HCN2 from PVs myocytes and current characteristics with HEK cells.

(A) & (B) showed that HCN2 and HCN4 mRNA expression levels of control and RAP groups. Current traces of HCN4 and HCN2 from Control and RAP PVs cells was showed in (C) and (D); (E): peak current densities of HCN4 and HCN2 from PVs in RAP model at test potential of –120 mV; (F): I-V relationship curve showed that HCN4 current densities were significantly higher in RAP than in control cells range of –80 and -120 mV, but not HCN2. HCN: hyperpolarisation activated cyclic nucleotide-gated channels PVs: pulmonary vein sleeves; RAP: rapid atrial pacing.