The Mechanism Research Of Hypoxia-induced Increased Late Sodium Current Resulting In Intracellular Ca²⁺ Over Load In Ventricular Myocytes

Researches showed that hypoxia could augment late sodium current (INa.L)cascade enhancing reverse Na+-Ca2+exchange current (reverse INCX) and leading tointracellular Ca2+overload in rabbit ventricular myocytes. But up to now it wasunknown whether the intracellular signal transduction pathways participated in themodulation of INa.Lduring hypoxia in ventricular myocytes. Whole-cell andcell-attached patch clamp technique were adopted to record INa.Land reverse INCXaccounted for intercellular signal transduction mechanism of increased INa.Lin thehypoxia and the relationship of augmented INa.Lto cytoplasm Ca2+overload. Theexperimental results revealed that hypoxia-induced increased INa.Land reversed INCXcould be apparently inhibited by KN-93(inhibitor of CaMK Ⅱ)、BisindolyImaleimideⅥ (BIM, inhibitor of PKC) and BAPTA/AM (Ca2+-chelator). Infirst experiment group, hypoxia augmented the density of INa.Lfrom-0.31±0.02pA/pFto-0.66±0.03pA/pF (n=6, P<0.01versus control) in rabbit ventricular myocytes，andapplying1μM KN-93in the presence of hypoxia, the increased INa.Lwas reduced to-0.44±0.03pA/pF(n=6, P<0.01versus hypoxia). At the same time, the reverse INCXdensity increased from1.02±0.06pA/pF to1.91±0.20pA/pF(n=7, P<0.01versuscontrol) in the hypoxia and1μM KN-93could make it decrease to1.36±0.15pA/pF inthe presence of hypoxia(n=7, P<0.01versus hypoxia). In second group, hypoxiaaugmented the density of INa.Lfrom-0.30±0.03pA/pF to-0.60±0.03pA/pF(n=6, P<0.01versus control) and1μM BIM decreased the increased INa.Lto-0.48±0.02pA/pF(n=6,P<0.01versus hypoxia). At the same time, the reverse INCXdensity increased from0.91±0.10pA/pF to1.71±0.27pA/pF(n=7, P<0.01versus control) in the hypoxia and1μM BIM made it decrease to1.33±0.21pA/pF(n=7, P<0.01versus hypoxia). In thirdgroup, hypoxia-induced increased INa.L(from-0.26±0.04pA/pF to-0.63±0.05pA/pF,n=6, P<0.01versus control) and reverse INCX(from0.86±0.09pA/pF to1.68±0.35pA/pF, n=7, P<0.01versus control) were declined to-0.39±0.02pA/pF (n=6, P<0.01 versus hypoxia) and1.12±0.27pA/pF(n=7, P<0.01versus hypoxia), respectively, afteradding1mM BAPTA/AM. In addition, single channel results showed hypoxiaapparently increased the mean open probability and mean open time of persistentsodium channel, and the impacts on single sodium channel were inhibited by1μMKN-93or1mM BAPTA/AM. Meanwhile, the suppressant effects of drugs could beabolished via washing them out. In summary, we considered that both Ca/CaMKⅡ、PKC and Ca2+possibly participate in the adjustment of hypoxia-evoked increasedINa.L. Researches showed that hypoxia could augment late sodium current (INa.L)cascade enhancing reverse Na+-Ca2+exchange current (reverse INCX) and leading tointracellular Ca2+overload in rabbit ventricular myocytes. But up to now it wasunknown whether the intracellular signal transduction pathways participated in themodulation of INa.Lduring hypoxia in ventricular myocytes. Whole-cell andcell-attached patch clamp technique were adopted to record INa.Land reverse INCXaccounted for intercellular signal transduction mechanism of increased INa.Lin thehypoxia and the relationship of augmented INa.Lto cytoplasm Ca2+overload. Theexperimental results revealed that hypoxia-induced increased INa.Land reversed INCXcould be apparently inhibited by KN-93(inhibitor of CaMK Ⅱ)、BisindolyImaleimideⅥ (BIM, inhibitor of PKC) and BAPTA/AM (Ca2+-chelator). Infirst experiment group, hypoxia augmented the density of INa.Lfrom-0.31±0.02pA/pFto-0.66±0.03pA/pF (n=6, P<0.01versus control) in rabbit ventricular myocytes，andapplying1μM KN-93in the presence of hypoxia, the increased INa.Lwas reduced to-0.44±0.03pA/pF(n=6, P<0.01versus hypoxia). At the same time, the reverse INCXdensity increased from1.02±0.06pA/pF to1.91±0.20pA/pF(n=7, P<0.01versuscontrol) in the hypoxia and1μM KN-93could make it decrease to1.36±0.15pA/pF inthe presence of hypoxia(n=7, P<0.01versus hypoxia). In second group, hypoxiaaugmented the density of INa.Lfrom-0.30±0.03pA/pF to-0.60±0.03pA/pF(n=6,P<0.01versus control) and1μM BIM decreased the increased INa.Lto-0.48±0.02pA/pF(n=6, P<0.01versus hypoxia). At the same time, the reverse INCXdensityincreased from0.91±0.10pA/pF to1.71±0.27pA/pF(n=7, P<0.01versus control) in the hypoxia and1μM BIM made it decrease to1.33±0.21pA/pF(n=7, P<0.01versushypoxia). In third group, hypoxia-induced increased INa.L(from-0.26±0.04pA/pF to-0.63±0.05pA/pF, n=6, P<0.01versus control) and reverse INCX(from0.86±0.09pA/pF to1.68±0.35pA/pF, n=7, P<0.01versus control) were declined to-0.39±0.02pA/pF (n=6, P<0.01versus hypoxia) and1.12±0.27pA/pF(n=7, P<0.01versushypoxia), respectively, after adding1mM BAPTA/AM. In addition, single channelresults showed hypoxia apparently increased the mean open probability and mean opentime of persistent sodium channel, and the impacts on single sodium channel wereinhibited by1μM KN-93or1mM BAPTA/AM. Meanwhile, the suppressant effects ofdrugs could be abolished via washing them out. In summary, we considered that bothCa/CaMKⅡ、PKC and Ca2+possibly participate in the adjustment of hypoxia-evokedincreased INa.L.