Studies On Molecularbiology And Pharmacology Properties Of I_Na In Cardiomyocytes And The Effect Of Acacetin On SKCa Current

Voltage-gated sodium channel in heart determines the generation and transmission of action potentials, so it is a very important target for treating arrhythmia. Recently, distinctive biophysical and pharmacological properties of sodium current between atrium and ventricle have been reported; however, the molecular basis for these differences is unknown. So, my PhD project was designed to explore the molecular mechanisms of the distinctive biophysical and pharmacological properties sodium current in atrium and ventricle from rat heart. In addition, I am interested to investigate whether TRPC channel blocker SKF-96365can inhibit sodium current in cardiomyocytes and whether the atrial-selective ion channel blocker acacetin has inhibitory effect on SKCa channels using multiple approaches of whole-cell patch voltage-clamp, molecular biology, biochemistry, etc.In the first part of my PhD project, we confirmed that distinctive biophysical and pharmacological properties of sodium current between atrium and ventricle in rat heart:the activation curve is more negative in atrium than ventricle, the inactivation of the current is faster in atrium than ventricle, recovery of INa from inactivation was slower in atrial than in ventricular myocytes, the current in atrium is more sensitive to the anti-arrhythmic drug Dronedarone than that in ventricle (IC50:4.8vs.14.2μM). Molecular biolocal and biochemical approaches revealed that mRNA and protein expression of sodium channel β2(SCN2B) and β4(SCN4B) subunits was much greater in ventricle than in atrium. Imprtantly, the distinctive expression of β2and β4subunit proteins was also detected in human atrial and ventricular tissues.In the second prat of my study, we demonstrated that the so-called specific TRPC channel blocker SKF-96365inhibited INa in cardiomyocytes with an IC50of1.36μM. It slowed down the recovery of sodium channel from inactivation and negatively shifted the activation conductance. The blockade of INa by SKF-96365was frequency-dependent (IC50is1.02μM at1Hz and0.56μat10Hz). However, the selective TRPC3channel blocker Pyr3inhibited INa by only～10%at the concentration (10μM) used to block TRPC3channels.In the third part of my study, we found that the atrial selective ion channel inhibiter Acacetin blocked hSKl, rSK2and hSK3channels expressed in HEK293cells in a dose-dependent manner. The IC50of Acacetin for inhibiting hSKl, rSK2and hSK3current was15.4μM,8.3μM and10.9μM, respectively. Site-directed mutagenesis of hSK3channels revealed that the hSK3mutations H485N, S507T and H516N showed an increase of IC50to>100μM,>100μM and18.2μM respectively, suggesting that Acacetin is interacted with the P-Loop helix of hSK3channels.Collectively, my PhD thesis demonstrates the novel information that1) expression of of sodium channel β2and β4subunits is greater in ventricle, which most likely is the molecular mechanism for the distinctive biophysical and pharmacological properties of INa between atrium and ventricle of the heart;2) so-called TRPC channel blocker SKF-96365significantly inhibits INa in cardiomyocytes, so that cautions should be taken to explain the experimental results when it is used for blocking TRPC channels;3) the atrial selective ion channel blocker Acacetin inhibits hSK channels by interaction with the P-Loop helix, the effect on SK channels may also contribute to its anti-atrail fibrillation.