Involvement of CaMKII in the modulation of IKs under oxidative stress in guinea pig sinoatrial node cells

Abstract

Our previous study found that Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) potentiates the slow delayed rectifier K⁺ current (I_Ks) in sinoatrial node (SAN) pacemaker cells. Recently, oxidative activation of CaMKII has emerged as a major cause of SAN dysfunction; however, its correlation with I_Ks regulation remains unclear. In this study, we investigated the effect of hydrogen peroxide (H₂O₂) on I_Ks in SAN cells isolated from guinea pig heart. Whole-cell patch-clamp recordings were performed using an EGTA (5 mM) pipette solution to stabilize intracellular Ca²⁺ levels (pCa 7). The results showed that 5 min of H₂O₂ (100 μM) perfusion initiated an increase in I_Ks, which gradually increased to saturation (∼60.5 % enhancement from baseline to saturation) after 10 min of H₂O₂ exposure. In contrast, I_Ks remained almost unchanged in the presence of catalase (1000 units mL⁻¹). These observations were replicable in atrial and ventricular cardiomyocytes. H₂O₂ failed to stimulate KCNQ1/KCNE1 currents in HEK and CHO cells expressing low CaMKII levels. In SAN cells, H₂O₂-induced I_Ks enhancement was strongly attenuated by intracellular dialysis with a lower Ca²⁺ concentration (pCa 10) or by pretreatment with KN-93 (1 μM), suggesting that Ca²⁺/calmodulin binding to CaMKII is a prerequisite for CaMKII activation. Autocamtide-2 inhibitory peptide (AIP, 1 μM), an inhibitor of the catalytic domain of CaMKII, almost completely abolished the H₂O₂-induced potentiation of I_Ks. Taken together, these findings imply that H₂O₂ enhances cardiac I_Ks through the oxidative activation of CaMKII.