The mechanism research of hypoxia-induced increased late sodium current resulting in intracellular Ca2+ overload in ventricular myocytes

Abstract Hypoxia leads to augmentation of late sodium current (INa.L) and cellular Na+ loading, increased reverse Na+-Ca2+ exchange current (reverse INCX) 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 INa.L during hypoxia in ventricular myocytes. Whole-cell and cell-attached patch clamp techniques were used to record INa.L, and the whole-cell mode was also used to record reverse INCX and to study intercellular signal transduction mechanisms that mediate the increased INa.L. Dual excitation fluorescence photomultiplier systems were used to record calcium transient in ventricular myocytes. Hypoxia caused increases of INa.L and reverse INCX. These increases were attenuated by KN-93 (an inhibitor of CaMKII), bisindolylmaleimide VI (BIM, an inhibitor of PKC) and BAPTA/AM (a Ca2+ chelator). KN-93, BIM and BAPTA/AM had no effect on INa.L in normoxia. In studies of KN-93, hypoxia alone increased the density of INa.L from −0.31 ± 0.02 pA/pF to −0.66 ± 0.03 pA/pF (n = 6, P < 0.01 vs control) and the density of reverse INCX from 1.02 ± 0.06 pA/pF to 1.91 ± 0.20 pA/pF (n = 7, P < 0.01 vs control) in rabbit ventricular myocytes. In the presence of 1 μm KN-93, the densities of INa.L and reverse INCX during hypoxia were significantly attenuated to −0.44 ± 0.03 pA/pF (n = 6, P < 0.01 vs hypoxia) and 1.36 ± 0.15 pA/pF (n = 7, P < 0.01 vs hypoxia), respectively. In studies of BIM, hypoxia increased INa.L from −0.30 ± 0.03 pA/pF to −0.60 ± 0.03 pA/pF (n = 6, P < 0.01 vs control), and reverse INCX from 0.91 ± 0.10 pA/pF to 1.71 ± 0.27 pA/pF (n = 6, P < 0.01 vs control). In the presence of 1 μm BIM, the densities of INa.L and reverse INCX during hypoxia were significantly attenuated to −0.48 ± 0.02 pA/pF (n = 6, P < 0.01 vs hypoxia) and 1.33 ± 0.21 pA/pF (n = 6, P < 0.01 vs hypoxia), respectively. In studies of BAPTA/AM, hypoxia increased INa.L from −0.26 ± 0.04 pA/pF to −0.63 ± 0.05 pA/pF (n = 6, P < 0.01 vs control) and reverse INCX from 0.86 ± 0.09 pA/pF to 1.68 ± 0.35 pA/pF (n = 6, P < 0.01 vs control). The effects of hypoxia on INa.L and reverse INCX were significantly attenuated in the presence of 1 mm BAPTA/AM to −0.39 ± 0.02 pA/pF (n = 6, P < 0.01 vs hypoxia) and 1.12 ± 0.27 pA/pF (n = 6, P < 0.01 vs 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 enhanced diastolic Ca2+ concentration, and the attenuated amplitude of [Ca2+]i transient, maximal velocities of Ca2+ increase and Ca2+ decay by hypoxia. In summary, the findings suggest that CaMKII, PKC, and Ca2+ all participate in mediation of the effect of hypoxia to increase INa.L. This article is protected by copyright. All rights reserved