Abstract: Objective To identify the effects of exogenous hydrogen peroxide on the Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel (HCN) current and its mechanisms in Neonatal Rat Ventricle Cardiomyocytes (NRVM). Methods NRVM from 1- to 3-day-old Wister rats were prepared by collagenase digestion, and incubatd in 37℃, 95%CO2 for patch-clamp recording and western-blotting analysis. Results Our data shown that exposure (~20 min) of NRVM to H2O2 (100 μmol/L) markedly increased If density (4.7±0.6 pA/pF vs. 11.7±1.1 pA/pF) along increased conductance (Gmax: 48.7±5.6 pS/pF vs. 192.6±64.1 pS/pF), a shift in activation voltage (V1/2) to positive potentials (-81.2±1.6 mV vs. -64.7±2.0 mV) and increase rate of activation (tact) (523.4±24.7 ms vs. 337.5±24.9 ms). Moreover, stimulation by H2O2 was largely inhibited by the non-specific tyrosine kinase blocker genistein (1μmol/L) or the c-Src-specific inhibitor PP2 (10 μmol/L). Augmented tyrosine phosphorylation of HCN2 channels with H2O2 treatment by determined by H2O2 Western blot using the phosphotyrosine specific antibody 4G10. Furthermore, the augmented If current was inhibited by pre-treatment with Trx receptor inhibitor (Auranofin 10nmol/L; 13-cis-retinoic acid 1 μmol/L). On the other hand, If current of NRVMs was also increased by treated with non-specific PTP inhibitors, phenylarsine oxide (PAO 1 μmol/L) or Na-orthovanadate (Na3VO4 10 μmol/L). Conclusion These data suggest that the c-Src family of tyrosine kinase mediate the augmentation of If density by oxidant agent H2O2 via a redox mechanism involving the Trx system.

Key words: H2O2, HCN channel current, c-Src, Thioredoxin