Effects Of Neferine On Kv4.3 And Nav1.5 Channels Expressed In HEK 293 Cells And Exvivo Electrophysiology Of Rabbit Hearts

Part Ⅰ Effects of neferine on Kv4.3 channels expressed in HEK 293 cellsObjective:The present study was designed to determine the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (Ito) in human heart.Methods:Effects of neferine on human cardiac Kv4.3 channels expressed in HEK 293 cells were examined using whole-cell patch-clamp techniques.Results:(1) Neferine potently and reversibly inhibited Kv4.3 currents in a concentration-dependent manner (the IC50 value was 8.44 μmol/L); (2) The inhibitory effects of neferine on Kv4.3 currents were similar to that of quinidine (10 μmol/L neferine reduced the peak currents about 25.71% ±1.23%, whereas 10 μmol/L quinidine reduced about 30.10% ±4.01%); (3) Neferine significantly shifted the steady-state activation curve of Kv4.3 currents in a positive direction (V1/2 shifted from 2.52 ±0.76 mV to 15.07 ± 1.99 mV, n= 8, P<0.05) while shifting the steady-state inactivation curve in a negative direction (V1/2 shifted from -30.18 ± 7.13 mV to-37.85 ± 7.92 mV, n= 5, P<0.05); (4) Neferine accelerates the inactivation and decelerates the recovery from the inactivation of Kv4.3 currents; (5) Neferine-induced block of Kv4.3 currents was frequency-dependent.Conclusion:Neferine is a potent direct block er of Kv4.3 channels likely by blocking the open state and inactivating state channels. The inhibitory effects of neferine on Kv4.3 currents were similar to that of quinidine.Part Ⅱ Effects of neferine on Nav1.5 channels expressed in HEK 293 cellsObjective:The present study was designed to determine the effects of neferine on Nav1.5 channels, the major voltage-gated channel isoform in adult heart, which playing a key role in generating rhythm of heartbeat.Methods:Human cardiac Nav1.5 channels were stably expressed in the HEK 293 cells, and the effects of neferine on Nav1.5 channels were studied using whole-cell patch-clamp technique.Results:(1) Neferine potently and reversibly inhibits Navl.5 currents in a concentration-dependen manner (the IC50 value was 26.15 μmol/L); (2) The inhibitory effects of neferine on Nav1.5 currents were weaker than that of quinidine under the same concentration (the currents were maximally diminished about 43.19% with 300 μmol/L neferine, while 80.29% with 1000 μmol/L quinidine); (3) Neferine significantly shifts the steady-state inactivation curve of Navl.5 currents in a negative direction (the V1/2 value was-75.32 ±1.26 mV with control and-80.34 ± 1.96 mV with 30 μmol/L neferine) while not shifting the steady-state activation curve; (4) Neferine prolonged the time-to-peak of activation and the time constants of inactivation, it also slowed markedly the recovery from inactivation of Nav1.5 currents. (5) Neferine-induced block of Navl.5 currents was frequency-dependent.Conclusion:Neferine may be also a potent blocker of Nav1.5 channels by blocking the open state and inactivating state channels. The inhibitory effects of neferine on Nav1.5 currents were weaker than that of quinidine.Part Ⅲ Effects of neferine on electrophysiology of rabbit left ventricles in vitroObjective:Using arterially perfused wedges of rabbit left ventricles to explore effects of neferine on electrophysiology of rabbit hearts in vitro.Methods:Arterially perfused wedges of rabbit left ventricles (LV) were prepared using male New Zealand white rabbits, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG).Results:In arterially perfused wedges of rabbit LV, neferine (1,3, and 10 μmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, APD10 was dramatically extended at Epi sites (the APD10 at Epi sites dramatically increased from 8.67 ± 2.28 ms of the control to 19.97±4.51 ms,27.96±5.41 ms and 49.95 ± 6.57 ms at 1,3 and 10 μmol/L neferine), while APD50 and APD90 at both Epi and Endo sites was just slightly lengthened. In addition, neferine slightly decreased the action potential amplitude (APA).Conclusion:Neferine dramatically extended APDio at Epi sites while just slightly lengthened APD50 and APD90 at both Epi and Endo sites, and slightly decreased the action potential amplitude in arterially perfused wedges of rabbit LV.ConclusionNeferine is a potent inhibitor of Kv4.3 channels, with blockade probably due to blocking of open state and inactivated state channels. This action may contribute to the ability of neferine in dramatically extending cardiac Epi sites APD10. Cause the inhibitory effects of neferine on Nav1.5 currents were weaker than that of quinidine, while the inhibitory effects on Kv4.3 currents were similar to that of quinidine, neferine may be a potential drug for the treatment of Brugada syndrome with fewer side effects if compared to quinidine.