Revisiting the ionic mechanisms of early afterdepolarizations in cardiomyocytes: predominant by Ca waves or Ca currents?

Abstract

Early afterdepolarizations (EADs) have been implicated in severe cardiac arrhythmias and sudden cardiac deaths. However, the mechanism(s) for EAD genesis, especially regarding the relative contribution of Ca(2+) wave (CaW) vs. L-type Ca current (I(Ca,L)), still remains controversial. In the present study, we simultaneously recorded action potentials (APs) and intracellular Ca(2+) images in isolated rabbit ventricular myocytes and systematically compared the properties of EADs in the following two pharmacological models: 1) hydrogen peroxide (H(2)O(2); 200 μM); and 2) isoproterenol (100 nM) and BayK 8644 (50 nM) (Iso + BayK). We assessed the rate dependency of EADs, the temporal relationship between EADs and corresponding CaWs, the distribution of EADs over voltage, and the effects of blockers of I(Ca,L), Na/Ca exchangers, and ryanodine receptors. The most convincing evidence came from the AP-clamp experiment, in which the cell membrane clamp was switched from current clamp to voltage clamp using a normal AP waveform without EAD; CaWs disappeared in the H(2)O(2) model, but persisted in the Iso + BayK model. We postulate that, although CaWs and reactivation of I(Ca,L) may act synergistically in either case, reactivation of I(Ca,L) plays a predominant role in EAD genesis under oxidative stress (H(2)O(2) model), while spontaneous CaWs are a predominant cause for EADs under Ca(2+) overload condition (Iso + BayK model).

Fig. 1.

Pacing cycle length (PCL) dependence of early afterdepolarization (EAD) induction in different models. A: action potentials (APs) recorded from rabbit ventricular myocytes treated with 200 μM H₂O₂ (for 8 min in this case). Representative APs at PCL of 1 s (top) and 6 s (bottom) are shown. EADs were observed at long PCL of 6 s (arrows) but not at PCL of 1 s. B: APs recorded from myocytes treated with 100 nM isoproterenol and 50 nM BayK 8644 (Iso + BayK). EADs (arrows), delayed afterdepolarizations (DADs; d), and triggered AP (T) were induced at PCL of 1 s (top) but not at PCL of 6 s (bottom). C: summarized bar graphs showing the incidence of EADs within 20 APs at various PCLs in the 2 models (n ≥ 4 cells). The EAD incidence rate was higher at a low pacing rate (or long PCL) in the H₂O₂ model but at fast pacing rate (or short PCL) in Iso + BayK model. ***P < 0.0001, Fisher's exact test vs. no incidence of EADs.

Fig. 2.

Formation process of EADs in different models. A: simultaneous recordings of whole cell (global) Ca²⁺ transients (CaTs) and APs at 5 min after treatment with 200 μM H₂O₂. Note the increase of CaT amplitude (F/F₀) following the emergence of EADs. B: same as A, except that the myocyte was treated with Iso + BayK (10 s after treatment). Note the Ca²⁺ accumulation before the emergence of EADs. Spontaneous Ca²⁺ transients or Ca²⁺ waves (SCaTs/CaWs) are indicated by ○ and *, which correspond to APD prolongation and EADs (arrows), respectively. Spontaneous CaT (S) and triggered APs (T) are also shown.

Fig. 3.

Temporal relationship between EAD and DAD corresponding SCaTs/CaWs in different models. Whole cell CaTs and APs were simultaneously recorded to compare the initiation time of an EAD upstroke (arrows) and corresponding SCaT/CaW (dashed lines). A: representative recording from H₂O₂ model. EAD was 130 ms ahead of corresponding SCaT. B: in Iso + BayK model, the start of a CaW preceded the upstroke of the EAD with a time difference (ΔT) of 50 ms in this representative cell. C and D: temporal relationship between DAD and CaW in H₂O₂ and Iso + BayK models, respectively. While the temporal relationship between EAD and SCaT remained the same as A and B, the DADs (d) and DAD-triggered APs (T) in both models seemed to always occur (as indicated by ▴) behind the initiation of their corresponding CaW (#), suggesting Ca overload and Na/Ca exchange current (I_NCX) as a common mechanism for the DADs in both models.

Fig. 4.

Distribution of take-off potentials (TOPs) of EADs in H₂O₂ and Iso + BayK models. A: representative recording of H₂O₂-induced EADs (top; stimulation marks are indicated below the trace) and a histogram graph showing distribution of their TOPs. Width of each bar is 3 mV. Data showed in the histogram were from 9 cells. B: same as A, except for Iso + BayK-induced EADs. Data showed in the histogram were from 7 cells.

Fig. 5.

Behaviors of SCaTs/CaWs under AP-clamp condition. After EADs were induced under current-clamp configuration (left), the recording from the same cell were switched to voltage-clamp configuration under an AP morphology without EAD (right) in H₂O₂ (A) and Iso + BayK (B) model. AP was recorded previously from a control cell. Note the SCaTs/CaWs were completely eliminated under AP-clamp condition in H₂O₂ model, while they persisted in Iso + BayK model.

Fig. 6.

Effects of I_NCX inhibition on EADs and corresponding SCaTs/CaWs. A: effect of SEA0400, a selective I_NCX blocker, on H₂O₂-induced EADs (arrows) and CaTs (*). B: Iso + BayK-induced CaWs (* and #, corresponding to EAD and DAD, respectively), EADs (arrows), and DAD-triggered action potential (T; left) and the effect of SEA0400 (right). DADs (d) are indicated at right.

Fig. 7.

Effects of ryanodine (10 μM), a ryanodine receptor inhibitor, on EADs and corresponding SCaTs/CaWs. A: CaT and AP recording under control condition (left), after H₂O₂ treatment (middle) and the effect of ryanodine (Rya) on H₂O₂-induced EADs (arrows) and CaTs (*; right). B: CaT and AP recording under control condition (left), after Iso + BayK treatment (middle), and the effect of ryanodine (right). Iso + BayK-induced CaWs (* and #, corresponding to EAD and DAD, respectively), EADs (arrows), and DAD-triggered action potential (T) are indicated.