Oxidative Stress-Induced Afterdepolarizations and Protein Kinase C Signaling

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 (INa,L) (n = 7, p < 0.01) and the L-type calcium current (ICa,L) (n = 5, p < 0.01), which were significantly reversed by Gö 6983 (p < 0.01). H2O2 also increased the transient outward potassium current (Ito) (n = 6, p < 0.05). However, Gö 6983 showed little effect on H2O2-induced enhancement of Ito. Conclusions: H2O2 induced afterdepolarizations via the activation of PKC and the enhancement of ICa,L and INa,L. These results provide evidence of a link between oxidative stress, PKC activation and afterdepolarizations.

Protein kinase C (PKC), a group of serine/threonine protein kinase enzymes, has been identified as an important regulator of a variety of cardiac responses including myocardial fibrosis, cardiomyocyte hypertrophy and inflammation, which are closely associated with heart failure and ischemia [10][11][12]. PKC levels have been found to be significantly elevated in both HF animal models and in patients with HF [13,14], and cardioprotective effects were observed by selective inhibition of PKC isozymes [11,15]. Moreover, PKC is acknowledged to be closely associated with various ion channels and transporters [5]. PKC activation has been shown to facilitate the induction of ventricular fibrillation, which can be reversed by PKC inhibition [16].
Accumulating evidence suggests that increased oxidative stress could serve as an important arrhythmogemic factor in diseased hearts. ROS can persistently activate PKC via direct oxidative modification [17] and increasing the cellular level of inositol-1,4,5-trisphosphate and diacylglycerol [18]. Up until now, there was only scant report on the role of PKC in oxidative stress-induced afterdepolarizations and its ionic mechanisms. In the present study, we tested the hypothesis that oxidative stress may activate PKC and enhance arrhythmogenic activity in cardiomyocytes, and focused on the following issues: (1) whether PKC signaling was involved in oxidative stress-induced afterdepolarizations; (2) the underlying ionic mechanisms. Our study showed that PKC activation plays an important role in oxidative stress-induced afterdepolarizations, and augmentation of I Ca,L and I Na,L seems to be the underlying mechanism.

H 2 O 2 -Induced Afterdepolarizations in Adult Rabbit Ventricular Myocytes
Action potentials (APs) were recorded from isolated rabbit ventricular myocytes using the whole-cell current clamp mode. To elicit EADs stably, the myocytes were paced at basic cycle lengths of 6 s [3]. After the action potential duration (APD) and morphology reached steady state, cells were perfused with bath solution containing 1 mM H 2 O 2 .
As shown in Figure 1, H 2 O 2 perfusion prolonged APD gradually and induced afterdepolarizations and TAs after approximately 7 min, while control experiments in parallel (without H 2 O 2 ) showed that no electrical abnormalities occurred for up to 15 min (n = 9). APD 90 was prolonged from 276.7 ± 77.4 to 585.0 ± 65.0 ms (n = 9, p < 0.01) at 5 min of application of 1 mM H 2 O 2 . Examples of afterdepolarizations and TAs are shown in Figure 1C.
ischemia [10][11][12]. PKC levels have been found to be significantly elevated in both HF animal models and in patients with HF [13,14], and cardioprotective effects were observed by selective inhibition of PKC isozymes [11,15]. Moreover, PKC is acknowledged to be closely associated with various ion channels and transporters [5]. PKC activation has been shown to facilitate the induction of ventricular fibrillation, which can be reversed by PKC inhibition [16].
Accumulating evidence suggests that increased oxidative stress could serve as an important arrhythmogemic factor in diseased hearts. ROS can persistently activate PKC via direct oxidative modification [17] and increasing the cellular level of inositol-1,4,5-trisphosphate and diacylglycerol [18]. Up until now, there was only scant report on the role of PKC in oxidative stress-induced afterdepolarizations and its ionic mechanisms. In the present study, we tested the hypothesis that oxidative stress may activate PKC and enhance arrhythmogenic activity in cardiomyocytes, and focused on the following issues: (1) whether PKC signaling was involved in oxidative stress-induced afterdepolarizations; (2) the underlying ionic mechanisms. Our study showed that PKC activation plays an important role in oxidative stress-induced afterdepolarizations, and augmentation of ICa,L and INa,L seems to be the underlying mechanism.

H2O2-Induced Afterdepolarizations in Adult Rabbit Ventricular Myocytes
Action potentials (APs) were recorded from isolated rabbit ventricular myocytes using the whole-cell current clamp mode. To elicit EADs stably, the myocytes were paced at basic cycle lengths of 6 s [3]. After the action potential duration (APD) and morphology reached steady state, cells were perfused with bath solution containing 1 mM H2O2.
As shown in Figure 1, H2O2 perfusion prolonged APD gradually and induced afterdepolarizations and TAs after approximately 7 min, while control experiments in parallel (without H2O2) showed that no electrical abnormalities occurred for up to 15 min (n = 9). APD 90 was prolonged from 276.7 ± 77.4 to 585.0 ± 65.0 ms (n = 9, p < 0.01) at 5 min of application of 1 mM H2O2. Examples of afterdepolarizations and TAs are shown in Figure 1C.

The Role of PKC Signaling in H2O2-Induced Afterdepolarizations
Next we tested whether PKC activation was involved in H2O2-induced afterdepolarizations by using the specific classical PKC inhibitor Gö 6983. Unlike those myocytes consistently presenting EADs approximately 7 min after exposure to 1 mM H2O2 ( Figure 1A), pretreatment with Gö 6983 (1 μM) prevented the emergence of H2O2-induced EADs for up to 15 min ( Figure 2A). As shown in Figure 2B, the incidence of EADs induced by H2O2 was significantly reduced by pretreatment with Gö 6983 (100% vs. 0%, n = 8). To further confirm the effect of PKC inhibition on H2O2-induced EADs, we applied another widely used selective PKC inhibitor, Bisindolylmaleimide (BIM). As expected, pretreatment with BIM (1 μM) prevented the emergence of H2O2-induced afterdepolarizations in six of six ventricular myocytes ( Figure S1A).
In another series of experiments, after EADs were induced by H2O2 perfusion, myocytes were perfused with bath solution containing both Gö 6983 and H2O2. Gö 6983 effectively suppressed H2O2-induced EADs, DADs and TAs in five out of five myocytes. Five consecutive APs under control conditions, in the presence of H2O2 and after the addition of Gö 6983, are shown in Figure 3A. Values of APD 90 are plotted over time in Figure 3B. In another group of myocytes, BIM was applied after EADs were induced by H2O2 perfusion, and BIM also effectively reversed EADs in five of five myocytes ( Figure S1B).

The Role of PKC Signaling in H 2 O 2 -Induced Afterdepolarizations
Next we tested whether PKC activation was involved in H 2 O 2 -induced afterdepolarizations by using the specific classical PKC inhibitor Gö 6983. Unlike those myocytes consistently presenting EADs approximately 7 min after exposure to 1 mM H 2 O 2 ( Figure 1A), pretreatment with Gö 6983 (1 µM) prevented the emergence of H 2 O 2 -induced EADs for up to 15 min ( Figure 2A). As shown in Figure 2B, the incidence of EADs induced by H 2 O 2 was significantly reduced by pretreatment with Gö 6983 (100% vs. 0%, n = 8). To further confirm the effect of PKC inhibition on H 2 O 2 -induced EADs, we applied another widely used selective PKC inhibitor, Bisindolylmaleimide (BIM). As expected, pretreatment with BIM (1 µM) prevented the emergence of H 2 O 2 -induced afterdepolarizations in six of six ventricular myocytes ( Figure S1A).
In  Figure 3A. Values of APD 90 are plotted over time in Figure 3B. In another group of myocytes, BIM was applied after EADs were induced by H 2 O 2 perfusion, and BIM also effectively reversed EADs in five of five myocytes ( Figure S1B).

PKC Mediates I Ca,L Enhancement in H 2 O 2 -Induced Afterdepolarizations
The effects of H 2 O 2 and the PKC inhibitor on the major membrane currents were analyzed using a voltage clamp. The representative current-voltage traces of I Ca,L are displayed in Figure 4A, and the averaged I-V curves (n = 5) are shown in Figure 4B. Our results show that 1 mM H 2 O 2 significantly enhanced I Ca,L , which was effectively attenuated by Gö 6983. The peak amplitudes of I Ca,L at +10 mV were measured for data analysis. As shown in Figure 4C, I Ca,L was elevated from −6.11 ± 0.47 to −8.82 ± 0.61 pA/pF after H 2 O 2 perfusion (p < 0.01, control vs. H 2 O 2 , n = 5), and decreased to −6.21 ± 0.45 pA/pF after the addition of Gö 6983 (p < 0.01, H 2 O 2 vs. Gö 6983, n = 5). In the control experiments, the run-down of I Ca,L was less than 5% [19].

Less Involvement of I to in PKC Signaling in H 2 O 2 -Induced Afterdepolarizations
The effects of H 2 O 2 and the PKC inhibitor on I to were also analyzed. The representative current-voltage traces of I to are displayed in Figure 6A, and the averaged I-V curves (n = 6) are shown in Figure 6B. The peak amplitudes of I to at +50 mV were measured and are summarized in Figure 6C. I to was significantly elevated from 3.07 ± 0.62 to 3.88 ± 0.50 pA/pF after H 2 O 2 perfusion (p < 0.05, control vs. H 2 O 2 , n = 6), and decreased to 3.58 ± 0.46 pA/pF after the addition of Gö 6983 (p > 0.05, H 2 O 2 vs. Gö 6983, n = 6).

Less Involvement of Ito in PKC Signaling in H2O2-Induced Afterdepolarizations
The effects of H2O2 and the PKC inhibitor on Ito were also analyzed. The representative current-voltage traces of Ito are displayed in Figure 6A, and the averaged I-V curves (n = 6) are shown in Figure 6B. The peak amplitudes of Ito at +50 mV were measured and are summarized in Figure 6C. Ito was significantly elevated from 3.07 ± 0.62 to 3.88 ± 0.50 pA/pF after H2O2 perfusion (p < 0.05, control vs. H2O2, n = 6), and decreased to 3.58 ± 0.46 pA/pF after the addition of Gö 6983 (p > 0.05, H2O2 vs. Gö 6983, n = 6).

Discussion
The present study provided the following novel findings: (1) the specific classical PKC inhibitor Gö 6983 both prevented and reversed the emergence of H 2 O 2 -induced afterdepolarizations effectively, indicating PKC activation mediates oxidative stress-induced afterdepolarizations; (2) PKC inhibition by Gö 6983 attenuated H 2 O 2 -induced enhancement of I Ca,L and I Na,L , but not I to .
PKC, a classical second messenger, is acknowledged to be involved in several pathological progressions including hypertrophy, fibrosis, inflammation, ischemic preconditioning and atherosclerosis. Thus, PKC serves as a potential therapeutic target for heart failure, hypertension and coronary heart diseases [10,22]. Previous studies have investigated the effect of PKC activation by endothelin-1 and a PKC agonist on arrhythmogenesis [16,23]. However, the relationship between PKC and arrhythmias in different pathological conditions, including oxidative stress, remains unclear.
Oxidative stress is involved in a variety of cardiovascular diseases, including heart failure and ischemic heart disease. Cardiac arrhythmias are prevalent in patients with these conditions. Afterdepolarization-mediated TAs play an important role in ROS-induced arrhythmias [24], but the underlying molecular mechanisms remain not fully elucidated. It is well known that classical PKC isozymes are activated by Ca 2+ and diacylglycerol [25]. When inactive, the regulatory domain of PKC is bound to the catalytic domain, inhibiting the enzyme activity, and when PKC is activated, this autoinhibition is released [26]. It is noteworthy that, besides Ca 2+ and diacylglycerol, mild oxidative stress can activate PKC by selectively oxidizing the regulatory domain [17]. These findings motivated us to explore the role of PKC signaling in oxidative stress-induced afterdepolarizations, and the present study provided direct evidence showing that PKC is indeed involved. In order to confirm that the results were not the side effects of PKC inhibitors, two widely used selective PKC inhibitors (Gö 6983 and BIM) were both tested, and the results demonstrated that it was the PKC inhibitory effect that affected H 2 O 2 -induced afterdepolarizations.
H 2 O 2 may affect I Ca,L , I Na,L and I to via direct oxidative modification, intracellular signaling pathways or transcription and translation regulations [5]. Our results show that acute application of H 2 O 2 is capable of enhancing these major membrane currents significantly. In our study, adding the PKC inhibitor along with H 2 O 2 for several minutes effectively reversed the H 2 O 2 -induced alterations on I Ca,L and I Na,L , suggesting the mechanistic role of PKC on the H 2 O 2 -induced intracellular signaling cascade. However, acute application of the PKC inhibitor failed to reverse the enhancement of I to , indicating a minor role of I to on H 2 O 2 -induced effects.
When activated, PKC can phosphorylate downstream proteins including the L-type Ca 2+ channel [27] and voltage-gated Na + channel [28], resulting in I Ca,L facilitation and I Na,L augmentation. Previous studies have demonstrated that the elevation of not only I Na,L but also I Ca,L was involved in the ROS-induced afterdepolarization formation [3]; thus, elevation of I Na,L can reduce the repolarization reserve, while activation of I Ca,L can generate EAD upstroke. It is known that augmentation of I Ca,L can increase Ca 2+ influx and enhance cellular Ca 2+ loading. In addition, APD prolongation can also enhance cellular Ca 2+ loading, and further promotes PKC activation. In our study, the inhibition of PKC activation significantly reversed H 2 O 2 -induced enhancement of both I Ca,L and I Na,L , these might result in reduced cellular Ca 2+ loading by attenuating Ca 2+ influx via I Ca,L and shortened APD.
The results collectively suggested that PKC signaling plays a central role in oxidative stress-induced afterdepolarizations, although activation of other kinases such as protein kinase A and Ca/calmodulin-dependent protein kinase II (CaMKII) cannot be ruled out, and it is beyond the present study protocol. Previous studies have demonstrated that CaMKII activation played a role in H 2 O 2 -induced afterdepolarizations [3]. As PKC and CaMKII are both kinases that can be activated by the oxidative modification and elevated Ca 2+ level, they may share similar ionic mechanisms in oxidative stress-induced afterdepolarizations. Another study demonstrated similar inhibitory effects on intracellular Ca 2+ -induced I Na,L by both inhibitors of PKC and CaMKII [29]. This evidence together with our results indicates that PKC activation is essential in oxidative stress-induced afterdepolarizations.
It has already been demonstrated that ROS levels are elevated in a number of cardiovascular diseases and predispose the heart to arrhythmias. Free radical scavengers were supposed to be a potential therapy strategy for alleviating arrhythmias [30], but this proposal was found to be not effective due to some limitations [31]. Thus, downstream second messengers, such as PKC, may be potential novel therapeutic targets for arrhythmias. Regulators of PKC are already in clinical trials for different indications [32][33][34][35][36], and systemic delivery of activators and inhibitors of PKC has been proven to be well tolerated [32,33]. Future studies are warranted to test the safety and feasibility of PKC inhibitors to treat arrhythmias in experimental settings and in patients.

Solutions and Drugs
The

Cell Isolation
The use and care of animals conformed to the Guide for the Care and Use of Laboratory Animals of Shanghai, China and was approved by the Institutional Animal Care and Use Committee of Xinhua Hospital (Approval No.: XHEC-F-2016-009, Date: 22 March 2016). Hearts were removed from 23 adult New Zealand White rabbits (2 to 3 kg) anesthetized with intravenous pentobarbital, and were then perfused retrogradely in Langendorff apparatus at 37 • C with Ca 2+ -free Tyrode's solution containing 1.4 mg/mL collagenase (Type II, Worthington, Lakewood, NJ, USA) and 0.1 mg/mL protease (type XIV, Sigma, St. Louis, MO, USA) for 15 to 30 min. After washing out the enzyme solution, the hearts were removed from the apparatus and swirled in a culture dish in KB solution. The Ca 2+ concentration was gradually increased to 1.8 mM, and the cells were then kept at 4 • C until use.
For I Ca,L recording, the cells were superfused with a modified Tyrode's solution in which KCl was replaced by CsCl, and the patch pipettes (resistance 1 to 2 MΩ) were filled with an internal solution containing (in mM): 110 Cs-Aspartate, 30 CsCl, 5 NaCl, 10 HEPES, 5 MgATP, 5 creatine phosphate, 0.1 EGTA, 0.05 cAMP, pH 7.2 with CsOH. I Ca,L was recorded using a series of 200 ms steps from −40 to +50 mV with increments of 10 mV from a holding potential of −80 mV prior to a 100 ms prepulse to −40 mV.
To isolate I Na,L , the pipette solution contained (in mM) 110 Cs-aspartate, 30 CsCl, 10 HEPES, 0.5 EGTA, 0.2 Na 3 -GTP, 5 Na 2 -phosphocreatine, 5 MgATP, pH 7.2 with CsOH. The bath solution was the same as that used for I Ca,L . Nifedipine (30 µM) was added to the bath solution to block calcium currents. I Na,L was elicited by 300 ms pulses from −120 to −30 mV, and the amplitude was measured at 200 ms.
For transient outward potassium current (I to ) recording, the pipette and bath solutions were the same as those for AP recording. TTX (10 µM) and Cd 3+ (0.3 mM) were added into the Tyrode's solution to inhibit I Na,L and I Ca,L . I to was elicited at basic cycle lengths of 10 s by a series of 300 ms steps from −40 to +70 mV with increments of 10 mV from a holding potential of −70 mV prior to a prepulse to −40 mV.

Data Acquisition and Analysis
Voltage and current signals were measured with a MultiClamp 700B patch-clamp amplifier (Axon Instruments, Sunnyvale, CA, USA) controlled by a personal computer using a Digidata 1440A acquisition board driven by pCLAMP 10 software (Molecular Devices, Sunnyvale, CA, USA). Data are presented as means ± standard error of means unless indicated. Statistical significance was assessed using unpaired Student's t tests, and p < 0.05 was considered significant.

Conclusions
PKC activation mediates oxidative stress-induced afterdepolarizations via enhancement of I Ca,L and I Na,L , and PKC might be a potential antiarrhythmic therapeutic target.