Effects of nitric oxide on large‐conductance Ca²⁺‐activated K⁺ currents in human cardiac fibroblasts through PKA and PKG‐related pathways

Abstract

The human cardiac fibroblast (HCF) is the most abundant cell type in the myocardium, and HCFs play critical roles in maintaining normal cardiac function. However, unlike cardiomyocytes, the electrophysiology of HCFs is not well established. In the cardiovascular system, Ca -activated K (KCa) channels have distinct physiological and pathological functions, and nitric oxide (NO) plays a key role. In this study, we investigated the potential effects of NO on KCa channels in HCFs. We recorded strong oscillating, well-maintained outward K currents without marked inactivation throughout the test pulse period and detected outward rectification in the I-V curve; these are all characteristics that are typical of KCa currents. These currents were blocked with iberiotoxin (IBTX, a BKCa blocker) but not with TRAM-34 (an IKCa blocker). The amplitudes of the currents were increased with SNAP (an NO donor), and these increases were inhibited with IBTX. The SNAP-stimulating effect on the BKCa currents was blocked by pretreatment with KT5823 (a protein kinase G [PKG] inhibitor) or 1 H-[1,-2, -4] oxadiazolo-[4,-3-a] quinoxalin-1-one (ODQ; a soluble guanylate cyclase inhibitor). Additionally, 8-bromo-cyclic guanosine 3',5'-monophosphate (8-Br-cGMP) stimulated the BKCa currents, and pretreatment with KT5720 (a protein kinase A [PKA] inhibitor) and SQ22536 (an adenylyl cyclase inhibitor) blocked the NO-stimulating effect on the BKCa currents. Furthermore, 8-bromo-cyclic adenosine 3',5'-monophosphate (8-Br-cAMP) activated the BKCa currents. These data suggest that BKCa current is the main subtype of the KCa current in HCFs and that NO enhances these currents through the PKG and PKA pathways.

Effects of Nitric Oxide on Voltage-Gated K+ Currents in Human Cardiac Fibroblasts through the Protein Kinase G and Protein Kinase A Pathways but Not through S-Nitrosylation

“…These results indicate that NO (100 µM SNAP) increased I K of HCFs through AC/cAMP/PKA signaling mechanisms. It has been reported that NO also modulates cardiac IBK Ca channels, which are also important K + channels in HCFs [ 13 ], and Na + channels in guinea pig and mouse ventricular myocytes through the cAMP and cGMP pathways [ 53 ].…”

“…Commercially available primary adult human ventricular cardiac fibroblasts (HCF-av, Catalogue #6310 from ScienCell, Carisbad, CA, USA) were used. These cells were used for many bimolecular and electrophysiological experiments [ 3 , 13 , 40 , 56 , 57 , 58 ] and confirmed as fibroblasts by discoidin domain receptor 2 staining [ 58 ]. The cells were cultured in Dulbecco’s modified eagle’s medium (DMEM; Welgene, Gyeongsan, Gyeongsangbuk-do, Korea) that was supplemented with fetal bovine serum (10%, Welgene) and a penicillin-streptomycin solution (100×; GenDEPOT, Barker, TX, USA) in a humidified atmosphere of 5% CO 2 and 95% air at 37 °C.…”

“…Voltage-gated K + (K V ) currents are activated on membrane depolarization, regulation of resting membrane potential, influence the amplitudes and durations of myocardial action potentials, and the electrical coupling between the cells and cardiac fibroblasts [ 11 , 12 ]. Mainly, two classes of K V currents have been distinguished based primarily on differences in time- and voltage-dependent properties: slowly inactivating delayed rectifier K + current, referred to as I K and rapidly inactivating transient outward K + current, I to , which can be differentiated by electrophysiological and pharmacological studies [ 3 , 13 , 14 , 15 ]. The two types of K V currents are also present in HCFs [ 3 ] but the molecular determinants underlying I K and I to have not been extensively studied.…”

Carbon monoxide activates large-conductance calcium-activated potassium channels of human cardiac fibroblasts through various mechanisms

“…Binding of NO to the heme group of sGC leads to increased conversion of GTP to cGMP, which in turn activates PKG. The cGMP signaling pathway are the main mechanisms for mediating the effects of NO on IBK in HCFs [25]. Therefore, we assessed the effects of cGMP on IBK of HCFs.…”

“…The large-conductance Ca 2+ -activated K + (BK) channel is the main K + channel in HCFs [22,25]. The BK channel contributes to the resting membrane potential of cardiac fibroblasts [26] and the electrical coupling of cardiomyocytes-fibroblasts [23].…”