Korean J Physiol Pharmacol.  2016 May;20(3):315-324. 10.4196/kjpp.2016.20.3.315.

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

• Bae H
• Lee D
• Kim YW
• Choi J
• Lee HJ
• Kim SW
• Kim T
• Noh YH
• Ko JH
• Bang H
• Lim I

• KMID: 2285604
• DOI: http://doi.org/10.4196/kjpp.2016.20.3.315

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.

Keyword

Ca2+-activated K+ channels; Human cardiac fi broblasts; Hydrogen peroxide; K+ currents; Protein kinase

MeSH Terms

Apoptosis
Blotting, Western
Cyclic AMP-Dependent Protein Kinases
Cyclic GMP-Dependent Protein Kinases
Fibroblasts*
Guanosine
Guanylate Cyclase
Humans*
Hydrogen Peroxide*
Hydrogen*
Oxidative Stress
Phosphotransferases
Potassium Channels, Calcium-Activated
Protein Kinases*
Cyclic AMP-Dependent Protein Kinases
Cyclic GMP-Dependent Protein Kinases
Guanosine
Guanylate Cyclase
Hydrogen
Hydrogen Peroxide
Phosphotransferases
Potassium Channels, Calcium-Activated
Protein Kinases

Figure

• 

  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 KCa currents are stimulated by H2O2. (B) Fast activation with slow or non-inactivation KDR currents are not stimulated by H2O2. (C) Fast activating with rapid inactivation KTO currents are not stimulated by H2O2. (D) Current densities of KCa currents, KDR currents, and KTO 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 KCa channel subtypes in human cardiac fibroblasts.(A) RT-PCR showing strong mRNA expression of KCa1.1α, KCa1.1β1, KCa1.1β3, KCa1.1β4, and KCa2.2 and weak expression of KCa2.3 and KCa3.1. GAPDH was used as control. (B) Western blot analysis showing protein expression of KCa1.1α, KCa1.1β, KCa2.2, KCa2.3, and KCa3.1. 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 KCa currents inhibited by100 nM IbTX. (B) Addition of 100 nM IbTX blocked the H2O2-activated KCa currents (C) Bar graph summarizing the effects of IbTX, and the effect of IbTX on KCa currents after pre-incubation with H2O2. *p<0.05, versus the control; #p<0.05, versus the H2O2.

• 

  Fig. 4 Role of BKCa 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 BKCa current changes.(A, B) Representative current traces showing the effect of 100 µM H2O2 on BKCa currents in the presence of 1 µM KT5823 and 1 µM ODQ. (C) Effect of 300 µM 8-Br-cGMP on BKCa currents. (D) Bar graph summarizing the effects of H2O2 on BKCa currents after pre-incubation with KT5823 or ODQ, and the effect of 8-Br-cGMP on BKCa currents. *p<0.05, versus the control.

• 

  Fig. 6 Effects of PKA pathway modulation on H2O2-induced BKCa current changes.(A, B) Representative current traces showing the effect of 100 µM H2O2 on BKCa currents after pre-incubation with 1 µM KT5720 and 1 µM H-89. (C) Bar graph summarizing the effects of H2O2 on BKCa currents in the presence of KT5720 or H-89. *p<0.05, **p<0.01 versus the control.

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