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

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

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, H₂O₂only 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.