Cellular And Ionic Mechanisms Of Dauricine And Daurisoline On Acquired Long QT Syndrome

Objective:Daurincine(Dau) and daurinsoline(DS), the bisbenzyl isoquinoline alkaloids, are extracted from the root of Menispermum dauricum DC. Their antiarrhythmic effects have been demonstrated in various experimental models. The excessive prolonged repolarization of action potential duration (APD) is the electrophysiological basis of Long QT syndrome (LQTs).The APD and the morphology of action potential were controlled by the inward currents and outward currents. The main outward currents are the rapidly activated delayed rectifier potassium current(IKr) and the slowly activated delayed rectifier potassium current(IKs).The main outward currents are sodium current(INa) and L-type calcium current(ICa-L)。In this study, using patch-clamp technique and Western blotting, we studied the effects of Dau and DS on ion channels and their proteins which are related to the acquired LQTs, and explored their antiarrhythimic mechanisms.Methods:(1) Single left ventricular myocardial cells of rabbits were isolated by enzymic method. Using patch-clamp technique, we recorded the IK1, IKr, IKs and Ito of single cell and studied the effects of Dau and DS these potassium channels; (2) Using patch-clamp technique, we recorded the hERG current which stably expressed in HEK293 cells and studied the effects of Dau and DS on kinetics of hERG currents; (3) Using Western blotting techinique, we studied the effects of Dau and DS on hERG protein levels. Results:1. The effects of Dau and DS on IK1, IKr, IKs and Ito potassium current(1) Dau:Compared with control, Dau at 1,3,10,30,100μmol·L-1 could inhibit the IK1, but there were no statistical difference and Dau did not affect the reversal potetntials. The inhibiton rates of Dau at 10,30,100μmol·L-1 on IK1 were (5.8±1.8)%,(16.5±2.7)%v and (39.3±8.1)% at-100mV, respectively. The inhibiton rates of Dau at 1,3,10,30μmol·L-1 on IKr were (12.2±8.6)%, (30.4±7.1)%, (37.1±3.7)% and (64.1±5.8)% respectively at-60 mV and the IC50 was 14.0μmol·L-1。Dau 10,30μmol·L-1 made the half activation voltages of IKr change from(-16.4±2.8)mV to (-14.8±2.2) mV, from(-17.8±4.2) mV to(-20.7±1.7) mV, respectively and the slope of the activation curve change from 6.2±0.1 to 6.8±0.4, from 6.2±0.2 to 6.5±0.2, but there were all no statistical differences. The inhibition rates of Dau at 10,30μmol·L-1 on IKs at+60 mV were (28.7±3.3)% and (40.3±5.2)%, respectively。Dau at 10,30μmol·L-1 changed the half activation voltages from (14.1±0.7)mV to (12.8±1.6), from (15.0±1.9) mV to (10.5±1.0) mV, respectively and the slope of activation curve changed from 6.2±0.2 to 6.5±0.2, form 14.7±0.7 to 15.5±1.7, but there were all no statistical differences. Dau at 10,30,100μmol·L-1 inhibted the Ito current. The inhibition rate of Dau 30,100μmol·L-1 on Ito at+30 mV were (26.5±3.6)% and (38.7±0.7)%, respectively. Dau 30,100μmol·L-1 changed the half activation voltages of Ito from (-12.4±4.6) mV to (17.1±1.9) mV, from(-12.5±4.6) mV to (-14.0±3.2) mV, respectively and the activation slope changed from 19.9±1.4 to 20.4±0.2, from 19.9±1.4 to 19.8±0.7, but there were all no statistical differences.(2) DS:Compared with control, DS 10,30,100μmol·L-1 could inhibit the IK1, but there was no statistical difference and DS did not affect the reversal potetntials. The inhibiton rates of DS at 10,30,100μmol·L-1 on IK1 were (8.0±2.8)%,(16.0±3.4)% and (26.1±8.5)% at-100 mV, respectively. The inhibiton rate of DS at 1,3,10, 30μmol·L-1 on IKr were (6.7±1.9)%,(26.1±6.0)%,(31.1±5.0)% and (55.4±6.9)% at-60 mV and the IC50 was 19.9μmol·L-1. DS 10,30μmol·L-1 made the half activation voltages of IKr-change from(-22.5±1.1) mV to (-25.2±1.5) mV, from(-21.5±1.6) mV to (-26.5±3.2) mV, respectively and the slope of the activation curve change from 6.6±0.2 to 5.6±0.4, from 6.7±0.4 to 4.7±1.1, but there were all no statistical difference. The inhibition rates of DS at 10, 30μmol·L-1 on IKs at +60mV were (25.7±3.8)% and (38.0±3.8) %, respectively。DS at 10μmol·L-1, 30μmol·L-1changed the half activation voltages from (12.0±3.8) mV to (11.5±1.7) mV, from (12:2±3.0)mV to (7.3±3.3) mV, respectively and the slope of activation curve changed from 13.5±0.8 to 12.3±1.3, form 13.9±0.9 to 13.9±1.9, but there were all no statistical difference. DS 10, 30μmol·L-1 inhibted the Ito current. The inhibition rate of DS at 10, 30μmol·L-1 on Ito at +30 mV were (6.1±9.2)% and (36.5±11.4)%, respectively. DS 10, 30μmol·L-1 changed the half activation voltages of Ito from (-10.9±4.8) mV to (-9.0±4.3) mV, from(-19.9±1.9) mV to (-15.7±1.5) mV, respectively and the activation slope changed from 17.0±1.5 to 18.0±1.5, from 18.4±1.5 to 17.6±1.8, but there were all no statistical difference.2. The effects of Dau and DS on kinetics of hERG current, which stably expressed in HEK293 cells(1) Dau: At +20mV, the inhibition rates of Dau at 1, 3, 10, 30μmol·L-1 on hERG step current were (16.1±5.5)%, (28.4±6.7)% (P<0.05,n=8), (59.7±8.9)% (P<0.05,n=10), (79.7±4.7)% (P<0.05,n=7), respectively and the IC50 was 6.9μmol·L-1; At +60mV, the inhibition rates of Dau 1, 3, 10, 30μmol·L-1 on hERG tail current were (6.1±5.2)%, (26.5±3.6)% (P<0.05,n=4), (57.6±8.8)%(P<0.05,n=9), (85.3±2.2)% (P<0.05,n=6), respectively, and the IC50 was 8.4μmol·L-1. Dau 1,3, 10, 30μmol·L-1 changed the half activation voltages from (4.5±3.0)mV to (3.3±3.3) mV, from (5.1±2.9) mV to (2.6±4.0) mV, from (0.7±2.8) mV to (-6.2±5.8)mV, from (1.1±2.7) mV to (3.7±4.1) mV, respectively, but there were no statistical differences. The slopes of activation curve were changed from 10.6±0.6 to 11.0±0.7, from 10.7±0.6 to 11.2±0.8, from 9.9±0.4 to 13.6±1.6, from 9.6±0.4 to 17.1±1.9 (p<0.05) . Dau 10μmol·L-1 decreased the half inactivation voltage from -52.7±3.7 to -57.8±3.5 (P<0.05, n=11) and changed the slope of inactivation curve from -21.7±0.6 to -21.0+0.5(n=9)( P>0.05). Dau had no effects on steady-state inactivation curve, the onset of inactivation curve and the recovery time constant. Dau exerted the characteristics of an open channel blocker of hERG current. (2) DS:At+20 mV, the inhibition rates of DS at 1,3,10,30μmol·L-1 on hERG step current were (32.2±4.2)%(n=6, P<0.05), (41.6±2.6)%(n=4, P<0.05), (62.1±5.9)%(n=12,P<0.05), (74.8±9.8)%(n=5, P<0.05), respectively and the IC50 was 9.1μmol·L-1; At+60 mV, the inhibition rates of DS 1,3,10,30μmol·L-1 on hERG tail current were(16.7±5.8)%(n=6), (31.1±4.5)%(n=7, P<0.05), (55.1±11.2)% (n=13,P<0.05), (81.2±7.0) (P<0.05,n=6), respectively, and the IC50 was 9.6μmol·L-1. DS 1,3,10,30μmol·L-1 changed the half activation voltages of hERG current from (8.2±2.0) mV to (7.6±3.2) mV, from (4.7±3.4) mV to (6.5±3.1) mV, from (2.8±3.3) mV to (-2.6±4.2) mV, from (0.4±4.5) mV to (2.5±4.9) mV, respectively, but there were no statisitical differences. The slopes of activation curve were changed from 10.0±0.6 to 11.1±0.8, from 10.5±0.7 to 11.1±1.1, from 10.5±0.7 to 13.2±2.2, from 10.0±0.7 to 12.7±1.6, respectively, and there were no statistical differences. DS 10μmol·L-1 decreased the half inactivation voltages from (-48.7±7.6) mV to (-64.6±5.2) mV(P<0.05) and changed the slope of inactivation curve from-21.9±0.7 to-22.5±1.0 (P>0.05). DS 10μmol·L-1 increased the inactivation at the test potential from-120mV to-100 mV(P<0.05), shifted the onset of inactivation curve at the test potential from-50 mV to+10 mV(P<0.05), and decreased the time constant of the onset of inactivation at test potential from-20 mV to+10 mV(P<0.05).DS had no effect on the recovery time constant. DS exerted the characteristics of an open channel blocker of hERG current.3. The effects of Dau and DS on protein levels of hERG current, which stably expressed in HEK293 cells(1)Dau:After the HEK293-hERG cells were incubated for 24 hours with 10μmol·L-1 Dau, the step and tail hERG currents were increased(P>0.05). The capacitance of control and Dau were (32.4±4.4) pF(n=17) and (32.4±4.4) pF(n=17), respectively(P>0.05).The HEK293-hERG cells were incubated with Dau at 3,10,30μmol·L-1 for 24 hours and the protein were tested by using Western blotting technique. Dau 30μmol·L-1 could decrease the hERG protein expression(P<0.05).(1)DS:After the HEK293-hERG cells were incubated for 24 hours with 10 μmol·L-1 DS, the step and tail hERG currents were decreased at the test potential from-60 mV to 0 mV(P<0.05). The capacitance of control and DS were (16.5±3.1) pF(n=12) and (45.7±9.4)(n=6), respectively(P<0.05).The HEK293-hERG cells were incubated with DS at 3,10,30μmol·L-1 for 24 hours and the protein were tested by using Western blotting technique. DS 30μmol·L-1 could decrease the hERG protein expression(P<0.05).Conclusion:1. Dau and DS could inhibit IK1 and Ito, but the inhibiton rate did not reach 50% at 100μmol·L-1.The IC50 of Dau and DS on IKr tail current were 14.0μmol·L-1 and 19.9μmol·L-1, respectively. Dau and DS had no effects on the activation curves of IKr.The inhibitory effects of Dau and DS on IKs were weaker than their effects on IKr, the inhibiton rate did not reach 50% at 30μmol·L-1.2. Dau and DS had inhibitory effects on hERG currents and it was voltage-dependent. As the depolarization of membrane potential, the inhibitory effects became more and more strong. But Dau and DS had no effects on activaton curves of hERG current. Dau and DS could decrease the half inactivation voltage and the inactivation time constant of hERG current and could speed up the inactivaton. Dau did not affect the onset of inactivaton and the recovery time constant of hERG current. DS shifted the onset of inactivaton curve to left and markedly decreased the time constant of the onset of inactivation at test potential from-20mV to+10mV and had no effects on recovery time constant.3. After the HEK293-hERG cells were incubated with Dau 10μmol·L-1 for 24 hours, the step hERG currents were decreased, the tail hERG currents were increased and the hERG protein expression were decreased, but there were no significant difference. Dau 30μmol·L-1 couled markedly decreased the hERG protein expression. After the HEK293-hERG cells were incubated with DS 10μmol·L-1 for 24 hours, the step and tail hERG currents were significantly decreased, but DS 10μmol·L-1 had no effects on hERG protein expression. DS 30μmol·L-1 could markedly decrease the hERG protein expression.