Study Of Mutations And Deletion Of SCN5A Gene Responsible For Sinus Node Disorder

PartⅠStudy of Molecular Mechanism of Sick Sinus Syndrome Caused by Point Mutation of SCN5A GeneObjective: Point mutations of SCN5A gene(E161K,T187I and L212P) and their effects on kenetics of Nav1.5 channel and transportation of channel protein.Method: Site-directed mutagenesis method was used to construct plasmids, then transfected into HEK-293 cell. Sodium channel kenetics were studied by patch clamp technique between the wild type channel and mutant channels. The flag gene was inserted into the genes encoding the ecto-linker between segament I and II in homologous domain I of Nav1.5 sodium channel and co-expressed with Nav1.5. Flag was detected by immunofluorescence.Result: At -20mv, Normal wild type sodium channel showed maximal current, the peak current was 800pA/pF, V1/2 and K of activation curve were -33.8±0.7 mV and 6.7±0.2 mV respectively, V1/2 and K of inactivation curve were -80.3±0.9 mV and 6.2±0.1 mV respectively. The peak current of T187I was reduced 100%; The peak current of E161K was reduced to 70%, stable activation curve showed obvious right shift, V1/2 and K of activation curve were -14.9±0.6mV and 8.8±0.1mV respectively (P<0.01 ); The peak current of L212P had no change, but stable activation curve showed obvious left shift, V1/2 and K of activation curve were -14.9±0.6mV and 8.4±0.2mV respectively (P<0.01); stable inactivation curve showed obvious left shift, V1/2 mV and K of inactivation curve were -90.7±1.1mV and -5.2±0.1mV respectively (P<0.01). E161K and L212P mutations did not affect the recovery of the Nav1.5 channel. Flags in three mutational channels could express on the cell membrance normally.Conclusion:T187I showed non-function of Nav1.5 channel; E161K and L212P showed loss of function. Three mutations did not affect the transport of the Nav1.5 channel.PartⅡEstablishment and Identification of Mouse Model of Targeted Disruption of Gene Scn5aObjective: To establish the mouse model of targeted disruption of gene Scn5a.Method: Scn5a gene targeted disruption mouse model was generated by gene targeting and homologous recombination of DNA. One group of Scn5a +/- mice and one group of wild type normal mice as control with the same matched age of 5 months were chosen. The template of PCR was got from DNA extracted from fresh mice tails. Single mouse ventricular cell was enzymatically isolated to be identified by anti-αactin antibody. Sodium currents from two different groups were recorded by patch clamp technique. Kenetics of sodium channels between cells from two different groups were compared by activation and inactivation.Results: PCR showed in wild type mouse group, there was a band at size of 980 base pair, however, in Scn5a +/- mouse group, there was a band at size of 980 and 650 base pair respectively. At -20mv, showed the maximum current density, compared with WT mice cells, the current density from heterozygous mice cells was reduced to 45%(16±6PA/PF vs 34±8PA/PF,n=5,P<0.05). But there was no significant difference in V1/2 and K of activation and inactivation between these two groups. Conclusion:Scn5a +/- mouse is a good animal model to investigate the disease related to abnormalities of Nav1.5 channel.PartⅢElectrophysiological study of cardiac conduction system of Scn5a +/- mouseObjective: To explore the electrophysiological disturbance of cardiac conduction systems of Scn5a +/- mouse.Method: ECG was recorded in the condition of langendorff perfusion system, heart rate and rhythm were observed. Impulse conduction time from sinus node to coronary sinus was measured by electrical mapping recording. The current and function of Nav1.5 channel of sinus node cell were detected by patch clamp thechique. The expression of Nav1.5 protein in SAN area was detected in tissue frozen section by immunoflurescence.Result: Compared with WT mouse, heart rates in the Scn5a +/- mouse were slowed (259±27 bpm VS 336±8 bpm,P<0.05,n=6)and discrepancy accompanied with 2:1 atrial ventricle block; the durations of P wave (12.91±4.17ms VS 22.35±6.44ms, P<0.05,n=6) and PR intervals (44.22±5.98ms VS 66.81±8.24ms, P<0.01,n=6) in ECG were prolonged; the conduction time from SAN to cornarary sinus was also prolonged(8.16±1.36ms VS 3.56±0.26ms P<0.05, n=6). The maximal current density of Nav1.5 channel in SAN cells was reduced (36±1PA/PF VS 57±4PA/PF, P<0.05, n=5) but without functional change. Expression of Nav1.5 protein in SAN area was reduced.Conclusion: The trafficking and expression abnormalities of Nav1.5 protein contribute predominantly to the decrease of Nav1.5 currents resulting in sick sinus syndrome.