Effects of hydrogen peroxide on voltage-dependent K+ currents in human cardiac fibroblasts through protein kinase pathways

Hyemi Bae; Donghee Lee; Young-Won Kim; Jeongyoon Choi; Hong Jun Lee; Sang-Wook Kim; Taeho Kim; Yun-Hee Noh; Jae-Hong Ko; Hyoweon Bang; Inja Lim

doi:10.4196/kjpp.2016.20.3.315

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Bae, Lee, Kim, Choi, Lee, Kim, Kim, Noh, Ko, Bang, and Lim: Effects of hydrogen peroxide on voltage-dependent K+ currents in human cardiac fibroblasts through protein kinase pathways

Original Article

The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology 2016; 20(3): 315-324.

Published online: 26 April 2016

DOI: https://doi.org/10.4196/kjpp.2016.20.3.315

Effects of hydrogen peroxide on voltage-dependent K⁺ currents in human cardiac fibroblasts through protein kinase pathways

Hyemi Bae¹, Donghee Lee¹, Young-Won Kim¹, Jeongyoon Choi¹, Hong Jun Lee², Sang-Wook Kim³, Taeho Kim³, Yun-Hee Noh⁴, Jae-Hong Ko¹, Hyoweon Bang¹, Inja Lim¹

¹Department of Physiology, College of Medicine, Chung-Ang University, Seoul 06974, Korea.

²Biomedical Research Institute, College of Medicine, Chung-Ang University, Seoul 06974, Korea.

³Department of Internal Medicine, College of Medicine, Chung-Ang University, Seoul 06974, Korea.

⁴Department of Biochemistry, School of Medicine, Konkuk University, Seoul 05029, Korea.

Correspondence: Inja Lim. injalim@cau.ac.kr

Received 8 March 2016 Revised 1 April 2016 Accepted 4 April 2016

(open-access, http://creativecommons.org/licenses/by-nc/4.0):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Human cardiac fibroblasts (HCFs) have various voltage-dependent K⁺ channels (VDKCs) that can induce apoptosis. Hydrogen peroxide (H₂O₂) modulates VDKCs and induces oxidative stress, which is the main contributor to cardiac injury and cardiac remodeling. We investigated whether H₂O₂ could modulate VDKCs in HCFs and induce cell injury through this process. In whole-cell mode patch-clamp recordings, application of H₂O₂ stimulated Ca²⁺-activated K⁺ (K_Ca) currents but not delayed rectifier K⁺ or transient outward K⁺ currents, all of which are VDKCs. H₂O₂-stimulated K_Ca currents were blocked by iberiotoxin (IbTX, a large conductance K_Ca blocker). The H₂O₂-stimulating effect on large-conductance K_Ca (BK_Ca) currents was also blocked by KT5823 (a protein kinase G inhibitor) and 1 H-[1, 2, 4] oxadiazolo-[4, 3-a] quinoxalin-1-one (ODQ, a soluble guanylate cyclase inhibitor). In addition, 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP) stimulated BK_Ca currents. In contrast, KT5720 and H-89 (protein kinase A inhibitors) did not block the H₂O₂-stimulating effect on BK_Ca currents. Using RT-PCR and western blot analysis, three subtypes of K_Ca channels were detected in HCFs: BK_Ca channels, small-conductance K_Ca (SK_Ca) channels, and intermediate-conductance K_Ca (IK_Ca) channels. In the annexin V/propidium iodide assay, apoptotic changes in HCFs increased in response to H₂O₂, but IbTX decreased H₂O₂-induced apoptosis. These data suggest that among the VDKCs of HCFs, H₂O₂ only enhances BK_Ca currents through the protein kinase G pathway but not the protein kinase A pathway, and is involved in cell injury through BK_Ca channels.

Keywords: Ca²⁺-activated K⁺ channels, Human cardiac fi broblasts, Hydrogen peroxide, K⁺ currents, Protein kinase

INTRODUCTION

Cardiac fibroblasts (CFs) are the largest population of cells in the heart, and they play a critical role in maintaining its normal function and homeostasis [1]. These cells contribute to the structural, biochemical, mechanical, and electrical characteristics of the heart and are the main source of collagen, which is the most important component of the cardiac extracellular matrix [2]. Excessive extracellular matrix deposition and CFs proliferation lead to heart failure, sudden cardiac death, and other serious complications [3]; therefore, these cells act as mediators of inflammatory and fibrotic myocardial remodeling in injured hearts [4 5].

It has been reported that extensive networks exist between CFs and cardiomyocytes through numerous anatomical contacts, which suggests potential heterocellular electrical coupling in diseased myocardium in arrhythmogenesis [2 6 7].

The ion channels present in cardiomyocytes and their functions have been well studied. CFs also have multiple ion channels, but their distribution and properties are quite distinct from those in cardiomyocytes [8]. It is well known that K⁺ channel-mediated signals, especially from voltage-dependent K⁺ channels (VDKCs), play an important role in cell death or apoptosis in many cell types [9 10 11 12 13]. There are two types of VDKCs, Ca²⁺-activated K⁺ (K_Ca) channels and voltage-gated K⁺ (K_V) channels. These channels are found in nearly every cell type, which suggests their physiological importance.

K_Ca channels can regulate membrane potential and intracellular K⁺ concentration, and constitute a major link between second messengers and the electrical activity of cells. There are three families of K_Ca channels that are based on differences in their biophysical and pharmacological properties, large-conductance K_Ca (BK_Ca or K_Ca1.1), intermediate-conductance K_Ca (IK_Ca or K_Ca3.1), and small-conductance K_Ca (SK_Ca or K_Ca2.x) channels. The ubiquitous BK_Ca channels are composed of pore-forming α subunits and four auxiliary β subunits, and gating is regulated by Ca²⁺ and membrane voltage [14]. BK_Ca currents have large conductances of 100~250 and 200~300 pS in physiological K⁺ gradients and a symmetrical 140 mM K⁺ solution, respectively [15]. In contrast, IK_Ca channels are voltage-independent and have intermediate single channel conductance values of 20~80 pS in physiological conditions [16]. Finally, SK_Ca channels have a small unitary conductance of 4~14 pS [17].

K_V channels modulate electrical excitability, regulate the repolarization of action potential, and are divided into two subfamilies, delayed rectifier K⁺ (K_DR) or transient outward K⁺ (K_TO). K_DR channels show fast activating kinetics with slow inactivation or no inactivation, whereas K_TO channels show fast activation and inactivation kinetics [18 19].

Hydrogen peroxide (H₂O₂), an oxidative stress inducer, is the main contributor to cardiac injury and remodeling [20]. H₂O₂ has been shown to activate or inhibit ion channels depending on channel type; it inhibits voltage-gated K_TO currents and K_DR currents in rat hippocampal neurons [21], but enhances BK_Ca currents in human dermal fibroblasts [9] and human endothelial cells through the cGMP signaling pathway [22]. In cardiomyocytes, H₂O₂ decreased K_DR currents in the ventricular myocytes of adult guinea pigs and increased apoptosis [23]. In contrast, H₂O₂ decreased K_TO currents but increased K_DR currents in rabbit atrial myocytes [24]. However, in human CFs (HCFs), whether H₂O₂ can modulate K⁺ channels and its mechanism still remain unclear.

In this study, we identified three types of VDKCs in HCFs and investigated the effects of H₂O₂ on these K⁺ channels using a whole-cell patch-clamp technique. The key protein kinase pathways involved in the observed effects of H₂O₂ on the K⁺ currents and H₂O₂-induced injury of HCFs as well as the relationship between H₂O₂-induced injury and K⁺ currents were also investigated.

METHODS

Cell preparation and culture

Human cardiac fibroblasts (adult ventricle, Catalog #6310) were purchased from ScienCell Research Laboratory (San Diego, CA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Welgene, Korea) supplemented with 10% fetal bovine serum (FBS; Welgene) and penicillin-streptomycin solution (GenDEPOT, Barker, TX) in a humidified atmosphere of 5% CO₂ and 95% air at 37℃. Confluent fibroblasts were detached by incubation with 0.25% trypsin (Welgene) and DMEM for a few minutes. The detached cells were pelleted by centrifugation, the supernatant was removed, and the pellet was suspended in 1 mL of bath solution. Cells used in this study were from early passages (3 to 7) to limit possible variation.

Electrophysiological recordings

A small aliquot of solution containing HCFs was placed in an open perfusion chamber mounted on the stage of an inverted microscope. Whole-cell currents were recorded with an Axopatch 200B patch clamp amplifier (Axon Instruments, Union City, CA) at room temperature. pCLAMP 9.0 software (Axon Instruments) was used for data acquisition and analysis of whole-cell currents. Activated currents were filtered at 2 kHz and digitized at 10 kHz. Patch pipettes were prepared from filament-containing borosilicate tubes (TW150F-4; World Precision Instruments, Sarasota, FL) using a two-stage microelectrode puller (PC-10; Narishige, Tokyo, Japan), and then fire polished on a microforge (MF-830; Narishige). When filled with pipette solution, the pipettes exhibited a resistance of 2~3 MΩ. The bath solution used for whole-cell recordings contained (in mM): 145 NaCl, 5 KCl, 1 CaCl₂, 1 MgCl₂, 5 glucose, and 5 HEPES (pH adjusted to 7.35 with NaOH). The pipette solution for patch clamping contained (in mM): 145 KCl, 1.652 CaCl₂ (pCa 6.0), 1.013 MgCl₂, 10 HEPES, 2 EGTA, and 2 K-ATP (pH adjusted to 7.3 with KOH). H₂O₂, iberiotoxin (IbTX, a BK_Ca channel blocker), KT5823 (a protein kinase G inhibitor), 1H-[1,2,4]-oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, a soluble guanylyl cyclase inhibitor), 8-bromo-cyclic guanosine 3', 5'-monophosphate (8-Br-cGMP), KT5720 and H-89 (protein kinase A inhibitors), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted from HCFs using the Total RNA Isolation PureLink RNA Mini Kit (Ambion, Carlsbad, CA). First-strand cDNA was prepared with the SuperScript III Cells Direct cDNA Synthesis Kit (Invitrogen, Tokyo, Japan). Reverse transcription was performed in a S1000 Thermal Cycler (Bio-Rad, Hercules, CA), according to the manufacturer's instructions. RT-PCR reaction products (cDNA) were resolved by 1.2% agarose gel electrophoresis, and stained with ethidium bromide for visualization under ultraviolet light. The primer sequences are listed in Table 1.

Western blot analysis

The cells were rinsed twice with ice-cold phosphate buffered saline (PBS) and then lysed in ice-cold lysis buffer. After incubation on ice for 30 min with shaking, the samples were centrifuged at 14,000 rpm for 10 min at 4℃. The protein concentration was measured by the Bradford assay. Extracts containing 20 µg of protein were mixed with 5× sample buffer, boiled for 10 min, and then subjected to continuous electrophoresis using 10~12% SDS-PAGE gels. The separated proteins were transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010; EMD Millipore, Billerica, MA) by electrophoretic transfer and blocked with 5% skim milk in trisbuffered saline containing 0.1% Tween-20 (TBS-T) for 1 h at room temperature. The membranes were rinsed and incubated with primary antibodies at 4℃ overnight. Removal of primary antibodies was carried out by washing for 4×10 min in TBS-T. The secondary antibodies were incubated with membrane in TBS-T with 5% skim milk for 1 h at room temperature. After a final wash with TBS-T for 4×15 min, the bands were detected using the chemiluminescence system (ECL, Thermo Fisher Scientific, Rockford, IL). The results were analyzed with Bio-Rad molecular analysis software. The primary and secondary antibodies used are listen in Table 2.

Annexin V/propidium iodide apoptosis assay

Apoptosis of HCFs was determined using flow cytometric analysis. Cellular apoptosis was observed by annexin V-FITC/PI double staining using the Annexin V/Dead Cell Apoptosis Kit (Invitrogen) according to the manufacturer's instructions. Briefly, cells were seeded at a density of 6×10⁵ cells/mL and cultured in DMEM containing 100 µM H₂O₂ for 6 h, or with IbTX for 24 h, and then 100 µM H₂O₂ was added and incubated for 6 h. Cells were harvested by treatment with trypsin, and then washed twice with cold PBS and centrifuged to collect the cell pellet. Annexin V-FITC and PI were added, and the cells were incubated in the dark at room temperature. Cells were analyzed with a flow cytometer (BD Biosciences) at 530 nm. Data from 10,000 cells were collected for each data file. Annexin V-FITC-positive and PI-negative cells were defined as apoptotic cells. Finally, the number of cells in each category was expressed as a percentage of the total number of stained cells.

Statistical analysis

Data are expressed as the mean±standard error of the mean (S.E.M.). Comparison of measurements between groups was performed using Student's t-test or one way ANOVA, depending on the experimental design. Differences were considered significant at p values less than either 0.05 or 0.01.

RESULTS

Identification of outward K⁺ currents and effect of H₂O₂ in human cardiac fibroblasts

Macroscopic K⁺ currents were generated using a voltage protocol that consisted of depolarizing steps (from −80 mV to +50 mV) in 10 mV increments for 400 ms with a −80 mV holding potential in whole-cell mode patch-clamp recordings. Ca²⁺-activated K⁺ (K_Ca) channels and voltage-gated K⁺ (K_V) currents with typical behavior were recorded in HCFs. K_Ca currents showed strong oscillation in response to strong depolarization, were well maintained throughout the test pulse without marked inactivation during depolarizing voltage increments, and their current-voltage (I~V) relationship showed strong outward rectification (Fig. 1A). These currents were detected in 47% (197 out of 419) of cells. Two types of K_V currents could be distinguished based on their activation and inactivation kinetics: fast activating and slow or non-inactivating currents, called delayed rectifier K⁺ (K_DR) currents (Fig. 1B), and fast activating and inactivating currents, called transient outward K⁺ (K_TO) currents (Fig. 1C). K_DR and K_TO currents were detected in 46.8% and 6.2% of cells, respectively. They did not show the outward rectification in their I~V curves.

To determine the effect of H₂O₂ on outward K⁺ currents in HCFs, H₂O₂ (100 µM) was added to the bath solution. In the presence of H₂O₂, K_Ca currents were significantly increased (Fig. 1A). This increase was 274.6±29.7% of that in the absence of H₂O₂ at +50 mV (n=13, ^*p<0.05 vs. control). The current densities of K_Ca currents at +50 mV in the absence and presence of H₂O₂ were 3.43±0.77 pA/pF and 9.42±2.27 pA/pF, respectively (n=13, p<0.05, Fig. 1D). In contrast, H₂O₂ had no significant effects on K_DR currents (106.7±10.3% of the control, n=12, Fig. 1B) or K_TO currents (101.2±10.1% of the control, n=8; Fig. 1C). At +50 mV, the current densities did not differ (control K_DR, 3.77±0.96 pA/pF vs. with H₂O₂, 4.03±0.99 pA/pF; control K_TO, 3.30±0.91 pA/pF vs. with H₂O₂, 3.34±0.92 pA/pF, Fig. 1D)

Identification of K_Ca channels in human cardiac fibroblasts

To determine which of the K_Ca channels subtypes are present in HCFs, K_Ca channel gene expression was examined by RT-PCR using specific primers for the K_Ca channel families (shown in Table 1). We detected clear amplification of the mRNA encoding the pore-forming α subunit of the K_Ca1.1(BK_Ca) channel along with the auxiliary 1.1β1, 1.1β3, and 1.1β4 subunits (Fig. 2A). The mRNA expression of the K_Ca2.2 and K_Ca2.3 subunits of the SK_Ca channel and the K_Ca3.1 subunit of the IK_Ca channel was also detected by RT-PCR. Expression of the K_Ca1.1β2 subunit of BK_Ca and the K_Ca2.1 subunit of SK_Ca was not detected. To examine whether the ion channels that were detected by RT-PCR were expressed in protein form, a western blot analysis was performed. As shown in Fig. 2B, the expected sizes of immunoreactive protein bands for the BK_Ca channel subunits (K_Ca1.1α and K_Ca1.1β) were detected (molecular weights, 125 kDa and 22 kDa, respectively). Expression of the subunits of the SK_Ca channels K_Ca2.2 and K_Ca2.3 and the IK_Ca channel K_Ca3.1 was also detected as prominent bands of molecular weights 64 kDa, 82 kDa, and 47 kDa, respectively. β-Actin (42 kDa) was used as a control for normalization.

Identification of BK_Ca channels and their role in H₂O₂-induced apoptosis in human cardiac fibroblasts

Previous studies have suggested that BK_Ca channels are the predominant K_Ca channels in HCFs [8 9 25]. Here, we employed IbTX, a specific BK_Ca channel blocker, to examine whether the K⁺ currents modulated by H₂O₂ were BK_Ca currents. IbTX (100 nM) significantly inhibited the strong oscillation of K_Ca currents (44.2±7.4% of the control at 50 mV, n=6, ^*p<0.05 vs. control; Fig. 3A). The current densities of K_Ca currents at +50 mV in the absence and presence of IbTX were 3.38±0.94 pA/pF and 1.49±0.70 pA/pF, respectively (n=6, p<0.05, Fig. 3C).

The H₂O₂-stimulated K_Ca currents were also decreased by IbTX (from 208.1±10.3% to 116.2±5.7%, n=5, ^*p<0.05 vs. control; Fig. 3B). The current densities of K_Ca currents at +50 mV in the control, H₂O₂, and IbTX were 3.11±0.98 pA/pF, 6.48±1.0 pA/pF, and 3.62±0.55 pA/pF, respectively (n=5, ^*p<0.05 vs. control, ^#p<0.05 vs. H₂O₂ ; Fig. 3C).

We next examined whether the H₂O₂-induced increase in BK_Ca currents was associated with H₂O₂-induced cell injury and found that IbTX attenuated the H₂O₂-induced apoptosis of HCFs. The apoptosis patterns of HCFs induced by H₂O₂ were analyzed by flow cytometry. The resulting cytogram shows the percentage of cells with an apoptosis-specific staining pattern (Fig. 4). In total, 28.02% of the cells treated with H₂O₂ showed late apoptotic changes, whereas 7.18% of control cells were late apoptotic. When we pretreated the cells with IbTX for 24 h before adding H₂O₂, only 12.38% of the cells showed the late apoptotic changes induced by H₂O₂. These data demonstrated that the inhibition of K⁺ efflux through BK_Ca channels was able to inhibit late apoptosis and that H₂O₂-induced apoptotic cell death of HCFs is mediated by BK_Ca channels.

Signaling pathway for the effects of H₂O₂ on BK_Ca currents

We studied whether the effects of H₂O₂ on BK_Ca currents was mediated by the cGMP signaling pathway. We added KT5823 (a PKG inhibitor, 1 µM) to the bath solution for 20 min, and then treated the cells with 100 µM H₂O₂. Under these conditions, H₂O₂ failed to increase BK_Ca currents (95.1±8.3%, n=7; Fig. 5A). When we pretreated the cells with ODQ (a soluble guanylate cyclase inhibitor, 10 µM) for 20 min, H₂O₂ also failed to increase BK_Ca currents (89.4±9.9%, n=5; Fig. 5B). We also assessed the effects of cGMP, which is generated by NO binding to soluble guanylate cyclase. Treatment with 8-Br-cGMP (a membranepermeable cGMP analogue, 300 µM) increased the BK_Ca currents to 169.2±14.6% of the control at +50 mV (n=5, p<0.05 vs. control; Fig. 5C). Fig. 5D shows the effect of H₂O₂ on the current density at +50 mV after KT5823 pretreatment (control, 3.20±0.89 pA/pF; +KT5823, 3.02±1.63 pA/pF; +H₂O₂, 3.04±1.57 pA/pF) and ODQ pretreatment (control, 3.07±1.22 pA/pF; +ODQ, 2.70±1.34 pA/pF; +H₂O₂, 2.74±1.21 pA/pF). The effect of 8-Br-cGMP on BK_Ca currents is also shown (from 3.78±0.84 pA/pF to 6.39±1.22 pA/pF, p<0.05).

To determine whether PKA is also involved in the effect of H₂O₂ on BK_Ca currents, we pretreated fibroblasts with KT5720 and H-89, well-known PKA inhibitors. KT5720 (1 µM) was added to the bath solution for 20 min, and then H₂O₂ (100 µM) was added. KT5720 increased BK_Ca currents (116.9±15.6% of the control at +50 mV, n=6, Fig. 6A); however, this difference was not significant. BK_Ca currents were still increased by 100 µM H₂O₂ in the presence of KT5720 (224.1±22.2%, n=6, p<0.01 vs. control; Fig. 6A). H-89 (1 µM) also increased BK_Ca currents, and the difference was significant (131.2±12.9%, n=5, p<0.05 vs. control; Fig. 6B). However, pretreatment with H-89 did not inhibit the stimulatory effect of H₂O₂ on BK_Ca currents because 100 µM H₂O₂ increased the BK_Ca currents (174.7±12.6% of the control at +50 mV). Fig. 6C shows the effects of H₂O₂ on the current density at +50 mV after KT5720 pretreatment (control, 3.21±0.31 pA/pF; +KT5720, 3.69±0.48 pA/pF; +H₂O₂, 7.21±0.66 pA/pF) and H-89 pretreatment (control, 3.35±0.41 pA/pF; +H-89, 4.56±0.41 pA/pF; +H₂O₂, 5.85±0.50 pA/pF).

DISCUSSION

The main finding of this study is that, among the VDKCs in HCFs, H₂O₂ stimulates BK_Ca currents but not K_DR or K_TO currents and that this H₂O₂-stimulating effect on BK_Ca currents is mediated by activation of protein kinase G (PKG) pathways but not the protein kinase A (PKA) pathway. In addition, H₂O₂-induced apoptosis of HCFs is mediated by BK_Ca channels.

The presence of VDKCs has been previously reported. These currents are thought regulate resting membrane potential, proliferation of ventricular fibroblasts [26], the functional expression of BK_Ca channels, and electrical coupling between cardiomyocytes and fibroblasts [25]. Li et al. [8] also reported the presence of three types of VDKCs in HCFs. However, we found some discrepancies between their results and ours. In their report, BK_Ca currents were present in most HCFs (88%), whereas K_DR and K_TO currents were present in a small population of cells (15 and 14%, respectively). In contrast, our results showed 47% of cells with K_Ca currents, 46.8% of cells with K_DR currents, and 6.2% of cells with K_TO currents. This difference may be due to some confusion in their report. We think that they may have miscounted K_DR currents as K_Ca currents. Their I-V curve of K_Ca currents did not show the strong outward rectification that is characteristic of K_Ca currents, even for currents inhibited by paxilline, this may explain why the K_DR current is co-present with the BK_Ca current in these cells. We also detected the coexistence of these three VDKCs in HCFs. To prevent confusion in identifying the types of K⁺ current, we defined only strongly oscillating outward K⁺ currents that had strong outward rectification in the I-V curve as K_Ca currents.

Furthermore, this earlier study [8] also showed significant mRNA expression of the K_Ca1.1 subunit of BK_Ca channels, but no mRNA expression of the 2.1 subunit of SK_Ca channels or the K_Ca3.1 subunit of IK_Ca was detected. In contrast, we detected mRNA expression of channel genes corresponding to the three subtypes of K_Ca, including clear mRNA amplification of the poreforming α subunit of K_Ca1.1 (BK_Ca) channels and the auxiliary 1.1β1, 1.1β3, and 1.1β4 subunits, the K_Ca2.2 and K_Ca2.3 subunits of SK_Ca channels, and the K_Ca3.1 subunit of IK_Ca channels by RT-PCR. The protein expression of BK_Ca, SK_Ca, and IK_Ca channels (K_Ca1.1, K_Ca2.2, K_Ca2.3, and K_Ca3.1) was also detected as prominent bands in the western blot analysis. Even though these ion channels contribute to the electrical coupling between cardiomyocytes and fibroblasts, BK_Ca currents have not been detected in human cardiomyocytes [8 25].

In our experiment, the H₂O₂-increased, strongly oscillating, outward rectifying K_Ca currents were considered BK_Ca currents since they were inhibited by IbTX, a BK_Ca- specific channel blocker. In addition, these currents were activated by high Ca²⁺ levels introduced using an intra-pipette solution and a high-stimulation voltage, which is the cause of strong outward rectification.

Our results showing H₂O₂-enhanced BK_Ca currents in whole-cell recordings are consistent with previous reports on vascular smooth muscle cells, human endothelial cells, and human dermal fibroblasts [9 22 27]. In contrast, in this study, H₂O₂ did not stimulate voltage-gated K⁺ currents (K_DR and K_TO currents) in HCFs. These results are not consistent with previous studies in cardiomyocytes [23 24] and coronary vascular smooth muscle cells [28]. These differences in the distribution of K⁺ currents and H₂O₂ effects imply variations in the functions of these channels in these two types of heart cells.

To the best of our knowledge, this is the first report showing that H₂O₂ enhances the BK_Ca currents of HCFs through PKG pathways but not through the PKA pathway. Several studies have reported that H₂O₂ stimulates BK_Ca channels through the PKG pathway in cultured human endothelial cells [22] and human coronary arterioles [29]. However, the effect of H₂O₂ on BK_Ca channels in HCFs and the underlying mechanism were not completely clear. In addition, inhibition of PKG by KT5823 and inhibition of soluble guanylate cyclase by ODQ attenuated the increase in BK_Ca currents following H₂O₂ exposure. 8-Br-cGMP, a membrane permeable analogue of cGMP, also increased BK_Ca currents. These results suggest that the PKG pathway is involved in the modulation of BK_Ca currents by H₂O₂ in HCFs.

Activation of BK_Ca channels likely elevates the intracellular Ca²⁺ level, which further enhances the release of NO in endothelial cells [30]. It has also been reported that NO increases BK_Ca currents in human dermal fibroblasts via the cGMP/PKG pathway [31] and cross-activation of the PKA pathway that stimulates currents through cGMP [32]. However, in this study, the H₂O₂-stimulating effects on BK_Ca currents were not decreased by pretreatment with KT5720 or H-89, suggesting that the PKA pathway is not involved in the modulation of BK_Ca currents by H₂O₂ in human cardiac fibroblasts. The stimulating effect of H-89 on BK_Ca currents was reported previously [33] and could explain the direct modulation of the currents observed here.

It is well known that K⁺ channel-mediated signals play an important role in cell death or apoptosis and that increased K⁺ efflux is one of the earliest indicators of apoptosis [10 12]. The K_Ca currents that are associated with apoptosis have been reported in a wide variety of cells [34 35]. When we pretreated cells with IbTX for 24 h before the addition of H₂O₂, the H₂O₂-induced early apoptotic changes decreased, which means that BK_Ca channels mediate early apoptotic cell death induced by H₂O₂ in HCFs. Similar results were also reported in human dermal fibroblasts [9]. We have also reported that activation of BK_Ca channels by its agonist, NS1619, mediates early apoptotic cell death in human dermal fibroblasts [36].

One limitation of the present study is that we cannot rule out the existence of other mechanisms for H₂O₂-induced cell injury in HCFs, especially the involvement of IK_Ca channels, since their expression was detected by RT-PCR and western blot analysis, and they also play important roles in cell proliferation [37 38].

ACKNOWLEDGEMENT

This work was supported by National Research Foundation of Korea Grant funded by the Korean Government (2010-0023712).

Notes

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

Author contributions: H.B., D.L., Y.K., and J.C. performed experiments. H.B., I.L., J.K., and H.B. analyzed and interpreted data. H.B. and I.L. wrote the manuscript. H.J.L., S.K., T.K., Y.N. and H.B. revised the manuscript. I.L. supervised and coordinated the study.

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Fig. 1

Identification of outward K⁺ currents in human cardiac fibroblasts and effect of H₂O₂.

Representative K⁺ current traces were obtained by voltage step pulses from −80 mV to +50 mV for 400 ms in whole-cell mode patch clamp recordings (holding potential, −80 mV). (A) Strongly oscillating, noninactivating K_Ca currents are stimulated by H₂O₂. (B) Fast activation with slow or non-inactivation K_DR currents are not stimulated by H₂O₂. (C) Fast activating with rapid inactivation K_TO currents are not stimulated by H₂O₂. (D) Current densities of K_Ca currents, K_DR currents, and K_TO currents, at +50 mV from a holding potential of −80 mV, in control cells and after the addition of H₂O₂. ^*p<0.05, versus the control.

Fig. 2

Expression of various K_Ca channel subtypes in human cardiac fibroblasts.

(A) RT-PCR showing strong mRNA expression of K_Ca1.1α, K_Ca1.1β1, K_Ca1.1β3, K_Ca1.1β4, and K_Ca2.2 and weak expression of K_Ca2.3 and K_Ca3.1. GAPDH was used as control. (B) Western blot analysis showing protein expression of K_Ca1.1α, K_Ca1.1β, K_Ca2.2, K_Ca2.3, and K_Ca3.1. β-actin was used as a loading control.

Fig. 3

Effect of iberiotoxin on outward K⁺ currents and H₂O₂–activated K⁺ currents in human cardiac fibroblasts.

(A) Strongly oscillating K_Ca currents inhibited by100 nM IbTX. (B) Addition of 100 nM IbTX blocked the H₂O₂-activated K_Ca currents (C) Bar graph summarizing the effects of IbTX, and the effect of IbTX on K_Ca currents after pre-incubation with H₂O₂. ^*p<0.05, versus the control; ^#p<0.05, versus the H₂O₂.

Fig. 4

Role of BK_Ca channels in H₂O₂-induced apoptosis.

Monolayer of human cardiac fibroblasts was treated by 100 µM H₂O₂ for 24 h. The H₂O₂-induced apoptosis was inhibited in the presence of 100 nM IbTX. The cytogram of the flow cytometry analysis showing the percentages of cells with specific staining patterns; In the lower left-hand quadrant (annexin V negative, PI negative) are viable cells, in the lower right-hand quadrant (annexin V positive, PI negative) are early apoptotic cells, in the upper left-hand quadrant (annexin V negative, PI positive) are dead cells, and in the upper right-hand quadrant (annexin V positive, PI positive) are late apoptotic/necrotic cells.

Fig. 5

Effects of PKG pathway modulation on H₂O₂-induced BK_Ca current changes.

(A, B) Representative current traces showing the effect of 100 µM H₂O₂ on BK_Ca currents in the presence of 1 µM KT5823 and 1 µM ODQ. (C) Effect of 300 µM 8-Br-cGMP on BK_Ca currents. (D) Bar graph summarizing the effects of H₂O₂ on BK_Ca currents after pre-incubation with KT5823 or ODQ, and the effect of 8-Br-cGMP on BK_Ca currents. ^*p<0.05, versus the control.

Fig. 6

Effects of PKA pathway modulation on H₂O₂-induced BK_Ca current changes.

(A, B) Representative current traces showing the effect of 100 µM H₂O₂ on BK_Ca currents after pre-incubation with 1 µM KT5720 and 1 µM H-89. (C) Bar graph summarizing the effects of H₂O₂ on BK_Ca currents in the presence of KT5720 or H-89. ^*p<0.05, ^**p<0.01 versus the control.

Table 1

Primers used for RT-PCR

K_Ca1.1, large conductance type of K_Ca (BK_Ca) channels; K_Ca2.1~2.3, small conductance type of K_Ca channel (SK_Ca1~3); K_Ca3.1, intermediate conductance type of K_Ca (IK_Ca) channel.

Table 2

List of primary and secondary antibodies

Maxi K, large conductance calcium-activated potassium channels (K_Ca1.1, BK_Ca); KCNN2, small conductance calcium-activated potassium channels (K_Ca2.2, SK_Ca2); KCNN3, small conductance calcium-activated potassium channels (K_Ca2.3, SK_Ca3); KCNN4, intermediate conductance calcium-activated potassium channels (K_Ca3.1, IK_Ca).