Study On The Effects And Mechanisms Of Dauricine And Daurisoline For 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). It is generally accepted that early afterdepolarizations (EADs) induced by excessive prolonged APD are the electrophysiological mechanisms of LQTS. In this study, using standard microelectrode technique and patch-clamp technique, we developed an in vitro model of LQT2 with dofetilide (Dof), a selective blocker of rapid activating delayed rectifier potassium current, to study the effects of Dau and DS on acquired LQTS, and to explore the antiarrhythmic mechanisms of Dau and DS in celluar and ionic levels.Methods: (1) Coarctation of abdominal aorta were used to induce hypertrophy in rabbits; using standard microelectrode technique, we recorded the transmembrane action potential of papillary muscles of right ventricle in normal and hypotrophied rabbits and studied the effects of Dau and DS on APD and EADs in normal and extracellular hypokalemia. (2) Single left ventricular myocardial cells of rabbits were isolated by enzymic method; using patch-clamp technique, we recorded the transmembrane potential of single cell to study the effects of Dau and DS on APD and EADs; (3) ICa-L of single cell were recored to study the effects of Dau and DS on its kinetics.Results:1. The effects of Dau and DS on early afterdepolarizations of papillary muscles of rabbits(1) Dau: EADs did not occur in papillary muscles of normal rabbits,but in hypokalemia Tyrode's, Dof 1μmol·L-1 prolonged the APD50 and APD90 by 77±6 ms and 72±7 ms, respectively, and the incidence of EADs was 16.7%(1/6). In normal Tyrode's, the incidence of EADs of papillary muscles of hypertrophied rabbits was 33.3%(2/6), Dof 1μmol·L-1 respectively prolonged the APD50,APD90 and APD90-APD30 by 197±9 ms,249±14 ms and 122±5.4 ms and the incidence of EADs was 66.7 %(4/6) in hypokalemia Tyrode's. After EADs induced by Dof, Dau 30μmol·L-1 was administered. We found that Dau 30μmol·L-1 shortened the prolonged APD in EADs and the APD50,APD90 and APD90-APD30 were shortened by 100±8 ms,111±8 ms and 79±14 ms, respectively. Dau 30μmol·L-1 also suppressed the incidence of EAD and the incidence was 0%(0/4) (P<0.05).(2) DS: EADs did not occur in papillary muscles of normal rabbits,but in hypokalemia Tyrode's, Dof 1μmol·L-1 prolonged the APD50 and APD90 by 58±9 ms and 92±3 ms, respectively, and the incidence of EADs was 16.7%(1/6)(P<0.05).In normal Tyrode's, the incidence of EADs of papillary muscles of hypertrophied rabbits was 33.3%(2/6), Dof 1μmol·L-1 respectively prolonged the APD50,APD90 and APD90-APD30 by 174±20 ms,218±19 ms and 129±8.9 ms and the incidence of EADs was 83.3 %(5/6) in hypokalemia Tyrode's. After EADs induced by Dof, DS 15μmol·L-1 was administered. We found that Dau 30μmol·L-1 shortened the prolonged APD in EADs and the APD50,APD90 and APD90-APD30 were shortened by 95±10 ms,113±8 ms and 56±3.8 ms, respectively. DS 15μmol·L-1 also suppressed the incidence of EAD and the incidence was 0%(0/5) (P<0.05). 2. The effects of Dau and DS on early afterdepolarizations of single myocardial cells of left ventrical of rabbits(1) Dau: EADs did not occur in myocardial cells of rabbits, Dof 0.1μmol·L-1 prolonged the APD50,APD90 and APD90-APD30 by 118±9.3 ms,145±13 ms and 73±7.6 ms, respectively, and the incidence of EADs was 16.7%(1/6). In hypokalemia and hypomagnesemia Tyrode's, the incidence of EADs of myocardial cells was 33.3%(2/6), Dof 0.1μmol·L-1 respectively prolonged the APD50,APD90 and APD90-APD30 by 143±13 ms,196±4.6 ms and 59±7.5 ms and the incidence of EADs was 83.3 %(5/6). After EADs induced by Dof, Dau 1, 3, 10μmol·L-1 were administered. We found that Dau suppressed the incidence of EADs(P<0.05), and shortened the prolonged APD in EADs and the APD50,APD90 and APD90-APD30were shortened by 201±22 ms,187±32 ms and 174±15 ms (P<0.05), respectively.(2) DS: EADs did not occur in myocardial cells of rabbits, Dof 0.1μmol·L-1 prolonged the APD50,APD90 and APD90-APD30 by 91±7.9 ms,132±6.5 ms and 77±7.6 ms, respectively, and the incidence of EADs was 16.7%(1/6). In hypokalemia and hypomagnesemia Tyrode's, the incidence of EADs of myocardial cells was 33.3%(2/6), Dof 0.1μmol·L-1 respectively prolonged the APD50,APD90 and APD90-APD30 by 95±2.3 ms,139±9.9 ms and 74±6.4 ms and the incidence of EADs was 83.3 % (5/6). After EADs induced by Dof, DS 1.5, 1.5, 5μmol·L-1 were administered. We found that DS suppressed the incidence of EADs(P<0.05), and shortened the prolonged APD in EADs and the APD50,APD90 and APD90-APD30were shortened by 243±12 ms,206±17 ms and 184±12 ms (P<0.05), respectively.3. The effects of Dau and DS on L-type calcium current(1) Dau: Dau could inhibit the ICa-L. The peak current of ICa-L of control and Dau 1, 3, 10μmol·L-1 were -1.8±0.3 nA,-1.3±0.3 nA,-1.1±0.2 nA and -1.0±0.2 nA,respectively. The maximal activation voltage in control and Dau groups all were 0 mV. Dau made the I-V curve shift upward. Dau 1, 3, 10μmol·L-1 increased the half activation voltage (V1/2) from the predrug of -20.9±1.5 mV to -16.4±0.6 mV (P<0.05),from -20.8±1.6 mV to -15.6±1.7 mV (P<0.05) and from -20.5±1.4 mV to -12.8±1.2 mV (P<0.01), respectively. The slopes of activation curve was increased from the predrug of 2.2±0.5 to 3.2±0.8,2.2±0.5 to 3.2±0.1 (P<0.05) and 1.8±0.5 to 3.7±0.2 (P<0.05) , respectively. The half inactivation voltage were decreased (V1/2) from the predrug of -23.0±5.0 mV to -27.7±4.0 mV,-20.5±5.3 mV to -25.6±4.3 mV and -21.8±5.4 mV to -32.3±2.5 mV(P<0.05), respectively. The slopes of inactivation curve were decreased from -6.4±0.8 to -8.1±1.0,-5.8±0.7 to -7.7±1.2 and -6.6±0.7 to -10.2±1.6(P<0.05), respectively. The recovery time from inactivation (τ)of ICa-L were prolonged from the predrug of 150±35 ms to 216±36 ms,209±29 ms to 327±61 ms(P<0.05) and 275±16 ms to 361±11 ms(P<0.05) , respectively.(2) DS: DS could inhibit the ICa-L. The peak current of ICa-L of control and DS 0.5, 1.5, 5μmol·L-1 were -1.6±0.4 nA,-1.1±0.1 nA,-1.0±0.2 nA and -0.9±0.1 nA, respectively. The maximal activation voltage in control and Dau groups all were 0 mV. Dau made the I-V curve shift upward. DS 1.5, 5μmol·L-1 increased the half activation voltage (V1/2) from the predrug of–12.8±1.7 mV to -10.4±1.7 mV and from–12.8±1.3 mV to–9.7±1.1 mV (P<0.05), respectively. The slope of activation curve were increased from the predrug of 3.5±0.7 to 5.1±0.8 and 5.0±0.6 to 6.9±0.8 (P<0.05), respectively. The half inactivation voltage were decreased (V1/2) from the predrug of -23.5±2.0 mV to–25.3±1.2 mV (P<0.05) and–24.3±1.8 mV to–27.8±3.4 mV(P<0.05), respectively. The slope of inactivation curve was decreased from–5.6±0.7 to -7.1±0.9(P<0.05) and–5.9±0.8 to–8.1±1.3(P<0.05), respectively. The recovery time from inactivation (τ) of ICa-L were prolonged from the predrug of 245±49 ms to 290±38 ms and 222±60 ms to 363±46 ms(P<0.05), respectively.Conclusion:1. In normal,hypokalemia-hypomagnesemia and hypertrophy conditions, Dau and DS could inhibit the EADs induced by Dof in papillary muscles and single myocardial cells of rabbits, shorten the APD50 and APD90 prolonged by Dof after EADs occurred, and decreased the APD90-APD30, which may be associated with the antiarrhythmic mechanisms of Dau and DS.2. Dau and DS could reduce the peak current of ICa-L, make the I-V curve shift upward, the steady state activation curve shift right and the steady state inactivation curve shift left. The half -activation voltage was increased and the half-inactivation voltage was decreased, and the effects of both make the"window current"of ICa-L decreas. Dau and DS could also prolong the recovery time of ICa-L. These results showed that the activation of ICa-L was stepped down, the inactivation was accelerated and the recovery time from inactivation was prolonged, which demonstrated that Dau and DS exerted inhibitory effects on ICa-L. These effects of Dau and DS on ICa-L may be one of their antiarrythmic mechanisms.