HCN212-channel biological pacemakers manifesting ventricular tachyarrhythmias are responsive to treatment with I(f) blockade

Abstract

Background: A potential concern about biological pacemakers is their possible malfunction, which might create ventricular tachycardias (VTs).

Objective: The purpose of this study was to test our hypothesis that should VTs complicate implantation of HCN-channel-based biological pacemakers, they would be suppressed by inhibitors of the pacemaker current, I(f).

Methods: We created a chimeric channel (HCN212) containing the N- and C-termini of mouse HCN2 and the transmembrane region of mouse HCN1 and implanted it in HEK293 cells. Forty-eight hours later, in whole-cell patch clamp recordings, mean steady state block induced by 3 microM ivabradine (IVB) showed HCN1 = HCN212 > HCN2 currents. The HCN212 adenoviral construct was then implanted into the canine left bundle branch in 11 dogs. Complete AV block was created via radiofrequency ablation, and a ventricular demand electronic pacemaker was implanted (VVI 45 bpm). Electrocardiogram, 24-hour Holter monitoring, and pacemaker log record check were performed for 11 days.

Results: All dogs developed rapid VT (>120 bpm, maximum rate = 285 +/- 37 bpm) at 0.9 +/- 0.3 days after implantation that persisted through 5 +/- 1 days. IVB, 1 mg/kg over 5 minutes, was administered during rapid VT, and three dogs received a second dose 24 hours later. While VT terminated with IBV in all instances within 3.4 +/- 0.6 minutes, no effect of IVB on sinus rate was noted.

Conclusion: We conclude that (1) I(f)-associated tachyarrhythmias-if they occur with HCN-based biological pacemakers-can be controlled with I(f)-inhibiting drugs such as IVB; (2) in vitro, IVB appears to have a greater steady state inhibiting effect on HCN1 and HCN212 isoforms than on HCN4; and (3) VT originating from the HCN212 injection site is suppressed more readily than sinus rhythm. This suggests a selectivity of IVB at the concentration attained for ectopic over HCN4-based pacemaker function. This might confer a therapeutic benefit.

Figure 1

Electrophysiological characterization of murine HCN2 and HCN212 channels overexpressed in adult cardiac myocytes. A. Sample I_HCN2 (left) and I_HCN212 (right) normalized current traces recorded during hyperpolarizing steps (from −35 to −95 mV (15 mV step) from a holding potential of −35 mV. B. Percentage of cells patched expressing HCN2 or HCN212 currents. C. HCN2 and HCN212 current density measured at −135 mV. D. Mean steady-state activation curves for HCN2 (n=5, squares) and HCN212 (n=9, circles). Solid lines show fit of Boltzmann relation. E. Mean activation time constants of HCN2 (squares) and HCN212 (circles) obtained between −140 mV and −65 mV. Each point represents the mean of 3–5 values for HCN2 and 7–9 values for HCN212. *P< 0.05 vs. HCN212.

Figure 2

Block of murine HCN1, HCN2 and HCN212 channels by IVB. Time-course of normalized I_HCN1 (A, right), I_HCN2 (B, right) and I_HCN212 (C, right) current amplitude. We used a voltage protocol in which activating/deactivating steps (−100/+5 mV, every 6 s) were applied from a holding potential of −35 mV for tens of seconds. Then, at the time of IVB (3 μM) application membrane voltage was held at −35 mV for 90 s. At this voltage the HCN channels are closed. Then, in the continuous presence of IVB, the pulsing protocol was resumed. Sample normalized current traces recorded at the times indicated are shown on the left side of each panel. D. Mean percentage steady-state block of human HCN4, and murine HCN2, HCN1 and HCN212 currents induced by IVB 3 μM (n=4–6). Values reported for HCN4 current (gray bar) are taken from. *P<0.05 vs. HCN4 and HCN2.

Figure 3

Maximal rate of rhythm that pace-mapped to injection site, recorded during daily 24 h Holter monitoring. The maximal rate was significantly faster during the first week after implantation in animals injected with the murine HCN212 chimera (black bars) compared to animals receiving saline (white bars) and those injected with HNC2 gene (striped bars) on days 2–4 and 5–7.

Figure 4

ECG recorded before, during, and after IVB administration from a dog in complete AV block implanted with HCN212 chimera. Each panel shows continuous traces. The top panel depicts bursts of VT having a rate of 200 bpm and a QRS configuration that pace-mapped to the implantation site. Four min after completing IVB infusion (1 mg/kg IV) over 5 min (middle panel) VT slowed and bursts were shorter. Ten min after IVB infusion (bottom panel) a slow idioventricular rhythm having a wider QRS complex and apparently originating from another site was observed. Atrial rate remained stable during the entire period of observation. ECGs recorded with electronic pacemaker turned off and at paper speed = 25 mm/sec

Figure 5

Atrial and ventricular rates recorded during and after IVB administration. Ventricular rate (white bars) significantly slowed after IVB infusion. In contrast, IVB had no significant effect on atrial rate (black bars) during the period of the experiment.