Intracellular signalling mechanism responsible for modulation of sarcolemmal ATP-sensitive potassium channels by nitric oxide in ventricular cardiomyocytes

Key points Both the ATP-sensitive potassium (K-ATP) channel and the gaseous messenger nitric oxide (NO) play fundamental roles in protecting the heart from injuries related to ischaemia. NO has previously been suggested to modulate cardiac K-ATP channels; however, the underlying mechanism remains largely unknown. In this study, by performing electrophysiological and biochemical assays, we demonstrate that NO potentiation of K-ATP channel activity in ventricular cardiomyocytes is prevented by pharmacological inhibition of soluble guanylyl cyclase (sGC), cGMP-dependent protein kinase (PKG), Ca2+/calmodulin-dependent protein kinaseII (CaMKII) and extracellular signal-regulated protein kinase1/2 (ERK1/2), by removal of reactive oxygen species and by genetic disruption of CaMKII. These results suggest that NO modulates cardiac K-ATP channels via a novel cGMP-sGC-cGMP-PKG-ROS-ERK1/2-calmodulin-CaMKII ( isoform in particular) signalling cascade. This novel intracellular signalling pathway may regulate the excitability of heart cells and provide protection against ischaemic or hypoxic injury, by opening the cardioprotective K-ATP channels. Abstract The ATP-sensitive potassium (K-ATP) channels are crucial for stress adaptation in the heart. It has previously been suggested that the function of K-ATP channels is modulated by nitric oxide (NO), a gaseous messenger known to be cytoprotective; however, the underlying mechanism remains poorly understood. Here we sought to delineate the intracellular signalling mechanism responsible for NO modulation of sarcolemmal K-ATP (sarcK(ATP)) channels in ventricular cardiomyocytes. Cell-attached patch recordings were performed in transfected human embryonic kidney (HEK)293 cells and ventricular cardiomyocytes freshly isolated from adult rabbits or genetically modified mice, in combination with pharmacological and biochemical approaches. Bath application of the NO donor NOC-18 increased the single-channel activity of Kir6.2/SUR2A (i.e. the principal ventricular-type K-ATP) channels in HEK293 cells, whereas the increase was abated by KT5823 [a selective cGMP-dependent protein kinase (PKG) inhibitor], mercaptopropionyl glycine [MPG; a reactive oxygen species (ROS) scavenger], catalase (an H2O2-degrading enzyme), myristoylated autocamtide-2 related inhibitory peptide (mAIP) selective for Ca2+/calmodulin-dependent protein kinaseII (CaMKII) and U0126 [an extracellular signal-regulated protein kinase1/2 (ERK1/2) inhibitor], respectively. The NO donors NOC-18 and N-(2-deoxy-,-d-glucopyranose-2-)-N-2-acetyl-S-nitroso-d,l-penicillaminamide (glycol-SNAP-2) were also capable of stimulating native sarcK(ATP) channels preactivated by the channel opener pinacidil in rabbit ventricular myocytes, through reducing the occurrence and the dwelling time of the long closed states whilst increasing the frequency of channel opening; in contrast, all these changes were reversed in the presence of inhibitors selective for soluble guanylyl cyclase (sGC), PKG, calmodulin, CaMKII or ERK1/2. Mimicking the action of NO donors, exogenous H2O2 potentiated pinacidil-preactivated sarcK(ATP) channel activity in intact cardiomyocytes, but the H2O2-induced K-ATP channel stimulation was obliterated when ERK1/2 or CaMKII activity was suppressed, implying that H2O2 is positioned upstream of ERK1/2 and CaMKII for K-ATP channel modulation. Furthermore, genetic ablation (i.e. knockout) of CaMKII, the predominant cardiac CaMKII isoform, diminished ventricular sarcK(ATP) channel stimulation elicited by activation of PKG, unveiling CaMKII as a crucial player. Additionally, evidence from kinase activity and Western blot analyses revealed that activation of NO-PKG signalling augmented CaMKII activity in rabbit ventricular myocytes and, importantly, CaMKII activation by PKG occurred in an ERK1/2-dependent manner, placing ERK1/2 upstream of CaMKII. Taken together, these findings suggest that NO modulates ventricular sarcK(ATP) channels via a novel sGC-cGMP-PKG-ROS(H2O2)-ERK1/2-calmodulin-CaMKII ( isoform in particular) signalling cascade, which heightens K-ATP channel activity by destabilizing the long closed states while facilitating closed-to-open state transitions. This pathway may contribute to regulation of cardiac excitability and cytoprotection against ischaemia-reperfusion injury, in part, by opening myocardial sarcK(ATP) channels.