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

New Findings What is the central question of this study? Hypoxia‐induced increase in late sodium current ( I Na,L ) is associated with conditions causing cellular Ca 2+ 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 + –Ca 2+ exchange current and diastolic [Ca 2+ ], which were attenuated by inhibitors of Ca 2+ –calmodulin‐dependent protein kinase II (CaMKII) and protein kinase C and by a Ca 2+ chelator. The findings suggest that CaMKII, protein kinase C and Ca 2+ 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 + –Ca 2+ exchange current (reverse I NCX ) and intracellular Ca 2+ 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 Ca 2+ –calmodulin‐dependent protein kinase II), bisindolylmaleimide VI (BIM; an inhibitor of protein kinase C) and BAPTA AM (a Ca 2+ 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 μ m 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 μ m 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 m m 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 μ m KN‐93 or 1 m m 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 Ca 2+ concentration and the attenuated amplitude of the [Ca 2+ ] i transient, maximal velocities of Ca 2+ increase and Ca 2+ decay. In summary, the findings suggest that Ca 2+ –calmodulin‐dependent protein kinase II, protein kinase C and Ca 2+ all participate in mediation of the effect of hypoxia to increase I Na,L .