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 (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 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 CaMKII), bisindolylmaleimide VI (BIM, an inhibitor of PKC) 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 pA/pF to −0.66 ± 0.03 pA/pF (n = 6, P < 0.01 vs control) and the density of reverse I_NCX 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 I_Na.L and reverse I_NCX 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 I_Na.L from −0.30 ± 0.03 pA/pF to −0.60 ± 0.03 pA/pF (n = 6, P < 0.01 vs control), and reverse I_NCX 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 I_Na.L and reverse I_NCX 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 I_Na.L from −0.26 ± 0.04 pA/pF to −0.63 ± 0.05 pA/pF (n = 6, P < 0.01 vs control) and reverse I_NCX 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 I_Na.L and reverse I_NCX 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 I_Na.L.

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