Effect Of Nav1.5-E3 Antibody On Sodium Current In Guinea Pig Ventricular Myocytes

Part 1 Characterization of distribution of cardiac isoform Nav1.5 in guinea pig ventricular myocytesObjective Nav1.5 was localized in cardiomyocytes and accounted for the most part of sodium channel current(INa) in ventricular myocytes. The aim of this part was to observe the characterization of distribution of cardiac isoform Nav1.5 in guinea pig ventricular myocytes by confocal immunofluorescence and to know the combination of Nav1.5 and its antibody.Methods The guinea pig single ventricular myocyte was isolated by enzymolysis and was divided into 3 groups(treated, non-treated and control group), The first group was treated by permealization using TritonX-100 to rupture cells'membrane. The distribution of Nav1.5 was observed in different groups by confocal immunofluorescence.Results Under confocal scanning laser microscope, compared with control group, green fluorescence could be observed on single ventricular myocyte in non-treated and treated group: in non-treated group, the fluorescence distributed on cell membrane and cell junction, whereas in treated group, throughout the whole cell.Conclusion Nav1.5 distributed both on and in ventricular myocyte and combined with its antibody effectively. Nav1.5 participated in electrophysiology activity of membrane of ventricular myocyte as was known; it was possible that it was related to electrophysiology activity of organelle membrane. Part 2 Effect of Nav1.5-E3 Antibody on Sodium Current in Guinea pig Ventricular MyocytesObjective Nav1.5 accounted for the most part of sodium current(INa) in ventricular cardiomyocytes. Nav1.5-E3 antibody, which was prepared by E3 targeting, was subtype-specific inhibitor of Nav1.5. The aim of this part was to investigate the effect of subtype-specific Nav1.5-E3 antibody on sodium channel current in guinea pig ventricular myocytes.Methods The single ventricular myocyte of adult guinea pig was isolated by acute enzymolysis and was divided into four groups. Of these four groups three were treated with Nav1.5 antibody (15μg/ml) of different dilutions: 1:100, 1:50 and 1:25 with the antibody concentrations of 0.15μg/ml, 0.30μg/ml and 0.60μg/ml respectively, and the fourth was control. The sodium channel currents were recorded in these single cells using whole-cell patch-clamp techniques and then current density for each cell was obtained. The data was analyzed by one-way variance analysis, SNK test and Dunnett test.Results Compared with control group, Nav1.5-E3 antibody of 0.15, 0.30 and 0.60μg/ml was shown to inhibit sodium channel current(INa) from (0.026 296±0.004 436) nA/pF to (0.018 41±0.007 161) nA/pF (P﹤0.01, n=10),(0.013 484±0.002 933) nA/pF (P﹤0.01, n=9) and (0.012 738±0.004 554) nA/pF (P﹤0.01, n=8) respectively, and to shift the I-V curves of INa upwardly. But the difference of current density among the antibody-treated groups was not statistically significant (P>0.05).Conclusion Nav1.5-E3 antibody blocked sodium channel current in guinea-pig ventricular myocytes in a voltage-dependent but concentration-independent manner, which implied the third extracellular region (E3) might be related to sodium current of ventricular myocyte.