Oxidative Stress-Induced Afterdepolarizations and Protein Kinase C Signaling

Abstract

Background: Hydrogen peroxide (H₂O₂)-induced oxidative stress has been demonstrated to induce afterdepolarizations and triggered activities in isolated myocytes, but the underlying mechanisms remain not fully understood. We aimed to explore whether protein kinase C (PKC) activation plays an important role in oxidative stress-induced afterdepolarizations.

Methods: Action potentials and ion currents of isolated rabbit cardiomyocytes were recorded using the patch clamp technique. H₂O₂ (1 mM) was perfused to induce oxidative stress and the specific classical PKC inhibitor, Gö 6983 (1 μM), was applied to test the involvement of PKC.

Results: H₂O₂ perfusion prolonged the action potential duration and induced afterdepolarizations. Pretreatment with Gö 6983 prevented the emergence of H₂O₂-induced afterdepolarizations. Additional application of Gö 6983 with H₂O₂ effectively suppressed H₂O₂-induced afterdepolarizations. H₂O₂ increased the late sodium current (I_Na,L) (n = 7, p < 0.01) and the L-type calcium current (I_Ca,L) (n = 5, p < 0.01), which were significantly reversed by Gö 6983 (p < 0.01). H₂O₂ also increased the transient outward potassium current (I_to) (n = 6, p < 0.05). However, Gö 6983 showed little effect on H₂O₂-induced enhancement of I_to.

Conclusions: H₂O₂ induced afterdepolarizations via the activation of PKC and the enhancement of I_Ca,L and I_Na,L. These results provide evidence of a link between oxidative stress, PKC activation and afterdepolarizations.

Keywords: afterdepolarization; arrhythmia; oxidative stress; protein kinase C; triggered activity.

Figure 1

Afterdepolarizations induced by H₂O₂ perfusion. (A) Action potentials (APs) were elicited consecutively at basic cycle lengths of 6 s and values of action potential durations (APD) 90 were plotted over time. APD 90 was consecutively recorded from a cell perfused with standard Tyrode solution for over 15 min. APs at 1 min (a), 10 min (b), and 15 min (c) are shown below. No early afterdepolarizations (EADs), delayed afterdepolarizations (DADs) or triggered activities (TAs) occurred; (B) H₂O₂ (1 mM) was perfused continuously as indicated by the horizontal bar. APs at the beginning of the perfusion (a), and after perfusion with H₂O₂ for 5 min (b) and 7 min (c) are shown below; (C) Examples of afterdepolarizations and TAs during H₂O₂ exposure, including multiple oscillatory EADs (above), and different electrical abnormalities in a pacing cycle (below).

Figure 1

Afterdepolarizations induced by H₂O₂ perfusion. (A) Action potentials (APs) were elicited consecutively at basic cycle lengths of 6 s and values of action potential durations (APD) 90 were plotted over time. APD 90 was consecutively recorded from a cell perfused with standard Tyrode solution for over 15 min. APs at 1 min (a), 10 min (b), and 15 min (c) are shown below. No early afterdepolarizations (EADs), delayed afterdepolarizations (DADs) or triggered activities (TAs) occurred; (B) H₂O₂ (1 mM) was perfused continuously as indicated by the horizontal bar. APs at the beginning of the perfusion (a), and after perfusion with H₂O₂ for 5 min (b) and 7 min (c) are shown below; (C) Examples of afterdepolarizations and TAs during H₂O₂ exposure, including multiple oscillatory EADs (above), and different electrical abnormalities in a pacing cycle (below).

Figure 2

Prevention of H₂O₂-induced early afterdepolarizations (EADs) by the protein kinase C inhibitor Gö 6983. (A) Time course of action potential duration (APD) 90 in a myocyte treated with Gö 6983 before exposure to 1 mM H₂O₂. Action potentials under control conditions (a), in the presence of Gö 6983 (b); after perfusion of H₂O₂ for 8 min (c) and 14 min (d) are shown below; (B) Incidence of EADs, delayed afterdepolarizations (DADs) or triggered activities (TAs) in the presence of H₂O₂ and pretreated with Gö 6983.

Figure 3

Suppression of H₂O₂-induced early afterdepolarizations (EADs) by the PKC inhibitor Gö 6983. (A) Gö 6983 completely suppressed all H₂O₂-induced EADs and significantly shortened action potential duration (APD). The representative five consecutive action potentials (APs) are shown in each period; (B) Time course of APD 90 in a myocyte treated with Gö 6983 after EADs were induced by H₂O₂. APs under control conditions (a), after perfusion with H₂O₂ for 6 min (b) and 8 min (c), and after application of Gö 6983 (d) are shown below.

Figure 4

Effects of Gö 6983 on H₂O₂-induced L-type calcium current (I_Ca,L) elevation. Representative current-voltage traces (A) and the averaged current-voltage curves (B) showed that H₂O₂ significantly augmented I_Ca,L, which was reversed by Gö 6983. Summary histogram (C) of I_Ca,L at +10 mV under control condition, in the presence of 1 mM H₂O₂ alone or plus 1 μM Gö 6983.

Figure 5

Effects of Gö 6983 on H₂O₂-induced late sodium current (I_Na,L) elevation. Representative current-voltage traces (A) and summary data of I_Na,L at −30 mV under control condition, in the presence of 1 mM H₂O₂ alone or plus 1 μM Gö 6983 (B) are shown.

Figure 6

Effects of Gö 6983 on H₂O₂-induced transient outward potassium current (I_to) elevation. Representative current–voltage traces (A), the averaged current-voltage curves of I_to (B) and summary histogram of I_to at +50 mV (C) under control condition, in the presence of 1 mM H₂O₂ alone or plus 1 μM Gö 6983 are shown.