Modulation of late sodium current by Ca²⁺–calmodulin‐dependent protein kinase II, protein kinase C and Ca²⁺ during hypoxia in rabbit ventricular myocytes

Abstract

What is the central question of this study? Hypoxia-induced increase in late sodium current (I_Na,L ) is associated with conditions causing cellular Ca²⁺ overload and contributes to arrhythmogenesis in the ventricular myocardium. The I_Na,L is an important drug target. We investigated intracellular signal transduction pathways involved in modulation of I_Na,L during hypoxia. What is the main finding and its importance? Hypoxia caused increases in I_Na,L , reverse Na⁺ -Ca²⁺ exchange current and diastolic [Ca²⁺ ], which were attenuated by inhibitors of Ca²⁺ -calmodulin-dependent protein kinase II (CaMKII) and protein kinase C and by a Ca²⁺ chelator. The findings suggest that CaMKII, protein kinase C and Ca²⁺ all participate in mediation of the effect of hypoxia to increase I_Na,L . Hypoxia leads to augmentation of the late sodium current (I_Na,L ) and cellular Na⁺ loading, increased reverse Na⁺ -Ca²⁺ exchange current (reverse I_NCX ) and intracellular Ca²⁺ loading in rabbit ventricular myocytes. The purpose of this study was to determine the intracellular signal transduction pathways involved in the modulation of I_Na,L during hypoxia in ventricular myocytes. Whole-cell and cell-attached patch-clamp techniques were used to record I_Na,L , and the whole-cell mode was also used to record reverse I_NCX and to study intercellular signal transduction mechanisms that mediate the increased I_Na,L . Dual excitation fluorescence photomultiplier systems were used to record the calcium transient in ventricular myocytes. Hypoxia caused increases of I_Na,L and reverse I_NCX . These increases were attenuated by KN-93 (an inhibitor of Ca²⁺ -calmodulin-dependent protein kinase II), bisindolylmaleimide VI (BIM; an inhibitor of protein kinase C) and BAPTA AM (a Ca²⁺ chelator). KN-93, BIM and BAPTA AM had no effect on I_Na,L in normoxia. In studies of KN-93, hypoxia alone increased the density of I_Na,L from -0.31 ± 0.02 to -0.66 ± 0.03 pA pF^-1 (n = 6, P < 0.01 versus control) and the density of reverse I_NCX from 1.02 ± 0.06 to 1.91 ± 0.20 pA pF^-1 (n = 7, P < 0.01 versus control) in rabbit ventricular myocytes. In the presence of 1 μm KN-93, the densities of I_Na,L and reverse I_NCX during hypoxia were significantly attenuated to -0.44 ± 0.03 (n = 6, P < 0.01 versus hypoxia) and 1.36 ± 0.15 pA pF^-1 (n = 7, P < 0.01 versus hypoxia), respectively. In studies of BIM, hypoxia increased I_Na,L from -0.30 ± 0.03 to -0.60 ± 0.03 pA pF^-1 (n = 6, P < 0.01 versus control) and reverse I_NCX from 0.91 ± 0.10 to 1.71 ± 0.27 pA pF^-1 (n = 6, P < 0.01 versus control). In the presence of 1 μm BIM, the densities of I_Na,L and reverse I_NCX during hypoxia were significantly attenuated to -0.48 ± 0.02 (n = 6, P < 0.01 versus hypoxia) and 1.33 ± 0.21 pA pF^-1 (n = 6, P < 0.01 versus hypoxia), respectively. In studies of BAPTA AM, hypoxia increased I_Na,L from -0.26 ± 0.04 to -0.63 ± 0.05 pA pF^-1 (n = 6, P < 0.01 versus control) and reverse I_NCX from 0.86 ± 0.09 to 1.68 ± 0.35 pA pF^-1 (n = 6, P < 0.01 versus control). The effects of hypoxia on I_Na,L and reverse I_NCX were significantly attenuated in the presence of 1 mm BAPTA AM to -0.39 ± 0.02 (n = 6, P < 0.01 versus hypoxia) and 1.12 ± 0.27 pA pF^-1 (n = 6, P < 0.01 versus hypoxia), respectively. Results of single-channel studies showed that hypoxia apparently increased the mean open probability and mean open time of sodium channels. These effects were inhibited by either 1 μm KN-93 or 1 mm BAPTA AM. The suppressant effects of drug interventions were reversed upon washout. In addition, KN-93, BIM and BAPTA AM also reversed the hypoxia-enhanced diastolic Ca²⁺ concentration and the attenuated amplitude of the [Ca²⁺ ]_i transient, maximal velocities of Ca²⁺ increase and Ca²⁺ decay. In summary, the findings suggest that Ca²⁺ -calmodulin-dependent protein kinase II, protein kinase C and Ca²⁺ all participate in mediation of the effect of hypoxia to increase I_Na,L .