| Objective：Oxidative stress, as a basic pathophysiological process, hasbeen intensively focused on by basic and clinical medicine in cardiovasculardiseases. Mounting studies suggest that oxidative stress is the key factor insuch diseases as atherosclerosis, ischemia-reperfusion, cardiac hypertrophy,congestive heart failure and arrhythmias. Arrhythmia is the common clinicalmanifestation, which makes the disease worse, even leading to cardiac suddendeath. The underlying mechanism involves the ion channels changes inactivity, expression and structure, which is also named electrical remodeling.Therefore, we pay attention to the relationship between oxidative stress andelectrical remodeling in arrhythmias.We investigate the relationship between oxidative stress and electricalremodeling at the molecular level as well as the underlying mechanism,promising it will add theoretical evidence to basic research and put forward anidea for prevention and treatment of arrhythmias. Our previous studies haverevealed that mRNA and ion channel proteins (Kv4.2，Kv4.3,Kv1.4) ofvoltage-gated potassium channel Kv, including Ito and Ik, come downsignificantly in cardiomyocytes of ischemic and failing rat heart. Thiselectrophysiological changes have been thought as the cause of arrhythmiasand heart failure. Studies have showed the increased expression ofhyperpolarization-activated cyclic nucleotide-gated channel (HCN) inoxidative stress, which is associated with the genesis and development ofarrhythmias. However it remains unclear which signal paths participate in theincreased activity/function of HCN in the diseased heart. HCN2is mostlyexpressed in ventricular myocytes. This study aims to identify the effects ofexogenous hydrogen peroxide (H2O2)on the HCN2current(the funny current, If) and its mechanisms in neonatal rat ventricle cardiomyocytes (NRVM), andinvestigate which paths or signal molecules take apart in the changes of H2O2current.Methods：NRVM from1-to3-day-old Wister rats were prepared bycollagenase digestion, and incubated in37℃,95%CO2for patch-clamprecording and western-blotting analysis. Pre-treatment of cells separately withthe following chemicals for one hour, Including H2O2, Genistein or PP2(tyrosine kinase inhibition), auranofin (10mol/L）or13-cis-RA（100mol/L）(Thioredoxin receptor blocking agent), and PTPs(non-specific protein tyrosinephosphatase). Then record If with patch-clamp and record expression of HCNprotein with western-blotting analysis. Compare differences in If and HCNprotein among different groups. Collect and save data using Pulse/PulseFit,V.8.11, HEKA Elektronik. Data is expressed by mean±standard deviation.Analysis of covariance and Tukey–Kramer procedure are used for multiplealignment. Student’s t test is used in comparing two groups of data.Results：Our data showed that exposure (~20min) of NRVM to H2O2(100μmol/L) markedly increased If density (4.7±0.6pA/pF vs.11.7±1.1pA/pF) along increased conductance (Gmax:48.7±5.6pS/pF vs.192.6±64.1pS/pF), a shift in activation voltage (V1/2) to positive potentials (-81.2±1.6mVvs.-64.7±2.0mV) and increased rate of activation (τact)(523.4±24.7ms vs.337.5±24.9ms). Moreover, stimulation by H2O2was largely inhibited by thenon-specific tyrosine kinase blocker genistein (1μmol/L) or the c-Src-specificinhibitor PP2(10μmol/L). Augmented tyrosine phosphorylation of HCN2channels with H2O2treatment was determined by H2O2Western blot using thephosphotyrosine specific antibody4G10. Furthermore, the augmented Ifcurrent was inhibited by pre-treatment with Trx receptor inhibitor (Auranofin10nmol/L;13-cis-retinoic acid1μmol/L). On the other hand, If current ofNRVMs was also increased by treated with non-specific PTP inhibitors,phenylarsine oxide (PAO1μmol/L) or Na-orthovanadate (Na3VO410μmol/L).Conclusion：These data suggest that the c-Src family of tyrosine kinase mediate the augmentation of If density by oxidant agent H2O2via a redoxmechanism involving the Trx system. |
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