Oxidative-stress-induced afterdepolarizations and calmodulin kinase II signaling

Abstract

In the heart, oxidative stress caused by exogenous H(2)O(2) has been shown to induce early afterdepolarizations (EADs) and triggered activity by impairing Na current (I(Na)) inactivation. Because H(2)O(2) activates Ca(2+)/calmodulin kinase (CaMK)II, which also impairs I(Na) inactivation and promotes EADs, we hypothesized that CaMKII activation may be an important factor in EADs caused by oxidative stress. Using the patch-clamp and intracellular Ca (Ca(i)) imaging in Fluo-4 AM-loaded rabbit ventricular myocytes, we found that exposure to H(2)O(2) (0.2 to 1 mmol/L) for 5 to 15 minutes consistently induced EADs that were suppressed by the I(Na) blocker tetrodotoxin (10 micromol/L), as well as the I(Ca,L) blocker nifedipine. H(2)O(2) enhanced both peak and late I(Ca,L), consistent with CaMKII-mediated facilitation. By prolonging the action potential plateau and increasing Ca influx via I(Ca,L), H(2)O(2)-induced EADs were also frequently followed by DADs in response to spontaneous (ie, non-I(Ca,L)-gated) sarcoplasmic reticulum Ca release after repolarization. The CaMKII inhibitor KN-93 (1 micromol/L; n=4), but not its inactive analog KN-92 (1 micromol/L, n=5), prevented H(2)O(2)-induced EADs and DADs, and the selective CaMKII peptide inhibitor AIP (autocamtide-2-related inhibitory peptide) (2 micromol/L) significantly delayed their onset. In conclusion, H(2)O(2)-induced afterdepolarizations depend on both impaired I(Na) inactivation to reduce repolarization reserve and enhancement of I(Ca,L) to reverse repolarization, which are both facilitated by CaMKII activation. Our observations support a link between increased oxidative stress, CaMKII activation, and afterdepolarizations as triggers of lethal ventricular arrhythmias in diseased hearts.

Fig. 1. Afterdepolarizations and triggered activity during H₂O₂ exposure in isolated rabbit ventricular myocytes

A. Action potentials (APs) were elicited with 2 ms-2nA pulses under current clamp conditions at pacing cycle length (PCL) of 6 sec. Values of consecutive APD₉₀ are plotted over time. H₂O₂ (200 μM) was perfused continuously as indicated by the horizontal bar above the plot. EADs are indicated by the sudden and dramatic increases of APD₉₀ (at ∼ 6 min after H₂O₂ application). Insets show APs under control condition, and after H₂O₂ with an EAD. B. Examples of afterdepolarizations and TA during exposure to 1 mM H₂O₂, including multiple oscillatory EADs (left panel), an EAD followed by a DAD (middle panel), and an EAD followed by a DAD causing TA (right panel). C. Successive APs recorded during H₂O₂ exposure, illustrating the irregular occurrence of EADs (asterisks) and associated DADs (arrows).

Fig. 2. The involvement of both I_Na and I_Ca,L in H₂O₂-induced EAD

A. The specific I_Na blocker TTX (10 μM) reversibly suppressed EADs and shortened APD. B. ATX (30 nM), a selective activator of persistent I_Na, prolonged APD but did not generate cause frank EADs with a distinct upstroke. C. The amplitude of H₂O₂-induced EADs depended on their take-off potentials (arrows). D. The I_Ca,L blocker nifedipine (Nif, 10 μM) suppressed the EAD upstroke, although AP plateau remained prolonged, unless TTX (10 μM) was also added.

Fig. 3. Enhancement of I_Ca,L by H₂O₂

A. Time course of the peak and residual pedestal (ped) I_Ca,L during a 300 ms voltage clamp pulse to 0 mV (voltage protocol shown in B). B. Voltage clamp pulse (above) and superimposed current traces showing I_Ca,L before (black) and ∼5 min after perfusion of 1mM H₂O₂ (red). The difference current is shown in the bottom trace. C. Same as B, but with an AP clamp waveform replacing the square voltage clamp pulse.

Fig. 4. Transient inward current (I_ti) and Ca_i transients during H₂O₂–induced EADs and DADs

A. Similar to Fig. 3B, but with an I_ti following repolarization to -80 mV in the presence of H₂O₂ (expanded in bottom trace). B. AP (top), simultaneous whole-cell Ca_i transient (middle), and a line-scan image along the long axis of the myocyte (bottom) before H₂O₂. C & D. Same following H₂O₂ exposure. In C, two EADs occur, leading to persistent elevation in Ca_i and additional Ca²⁺ release (r) during the second EAD. In D (different cell), two EADs (associated with additional I_Ca,L-gated SR Ca²⁺-release) are followed by a DAD due to a non-voltage gated spontaneous Ca_i wave (w) starting from the middle of the cell (*).

Fig. 5. Dependence of H₂O₂-induced EADs on intact Ca_i cycling

A. Time course of APD₉₀ in a myocyte preloaded with BAPTA-AM (4 μM) for 30 min exposure to 1 mM H₂O₂. Representative APs at points a (control), b (∼10 min in H₂O₂), and c (H₂O₂ + TTX) are shown below. B. Same, but in a myocyte pre-treated with 10 μM Ryanodine (Ry) + 200 nM Thapsigargin (Th) prior to H₂O₂.

Fig. 6. Suppression of H₂O₂–induced EADs by the CaMKII inhibitor KN-93 and AIP

A. Time course of APD₉₀ in a myocyte treated with the CaMKII inhibitor KN-93 prior to exposure to 1 mM H₂O₂. APs under control (a), in the presence of KN-93 and after perfusion with KN93 + H₂O₂ for 9 min (c) and 19 min (d) are shown in the lower panels. B. Same, but with KN-93 applied after EADs were induced by H₂O₂. In both protocols, KN-93 suppressed EADs. C. Bar graph showing the average H₂O₂ (200 μM) perfusion time to cause EAD appearance in the absence and presence of 2 μM AIP.

Fig. 7. Failure of KN-92, an inactive analog of KN-93, to prevent H₂O₂–induced EADs

A. Same protocol as in Fig. 6A, but with KN-92 initially in place of KN-93. KN-92 failed to prevent EADs during exposure to 1 mM H₂O₂, which were subsequently partially suppressed by KN-93. APs at points a (control), b (∼ 2 min after KN-92), c (KN-92 + H₂O₂) and d (KN-93 + H₂O₂) are shown below. B. Same as A, but applying BAPTA-AM to suppress EADs after KN-92 + H₂O₂.