| Objective: To investigate the effect of puerarin on action potential of singleisolated rat ventricular myocyte and currents of HEK293cells, which expressedKCNQ1(KvLQT1),KCNQ1/KCNE1(IKs), KCNJ2(Kir2.1) and KCNJ4(Kir2.3-) channels, by electrophysiological method. Methods: In this study, we usedLangendoff systerm and reversedly perfused CollagenaseⅡthrough the aorta ofacutely isolated rat heart, resulted in single isolated ventricular myocyte; then,by using patch clamp technique, action potential was observed in rat ventricularmyocyte before and after administration of different concentrations of (0.01,0.1,1mM) puerarin; HEK293cells were transiently transfected KCNQ1(KvLQT1),KCNQ1/KCNE1(IKs), KCNJ2(Kir2.1) or KCNJ4(Kir2.3) plasmidby lipsomes, respectively. After24-48h, transfected channel currents wereinvestigated before and after administration of different concentrations of(0.01-10mM) puerarin by electrophysiological recording of Whole-cell andInside-out model. Moreover, after the inhibited effect of pretreated1mMpuerarin, the effect of β receptor activator Isoproterenol (100nM), proteinkinase A activator8-Br-cAMP(100uM), adenylate cyclase activatorForskolin(10μM), PI3K inhibitor LY294002(10μM) and PLC inhibitorU73122(10μM) on KvLQT1channel current were detected. Results:(0.01,0.1, 1mM) puerarin altered RMP of rat ventricular myocyte from (-80.1±2.7) mV to(-78.5±4.2) mV,(-83.3±7.6) mV and (-84.7±5.1) mV (all n=6, P＞0.05);transformed APA from (112.5±6.3) mV to (109.8±8.5) mV,(114.2±4.9) mV and(115.9±6.2) mV (all n=6, P＞0.05); augmented APD90from (164.6±21.4) ms to(188.3±11.5) ms,(221.6±25.7) ms and (278.7±38.2) ms, as well as APD50,from (71.8±11.8) ms to (86.9±10.7) ms,(100.5±14.1) ms and (123.6±25.4)ms, respectively (all n=6, P<0.05). While applied with0.1μM,1μM,10μM,100μM,1mM and10mM concentration of puerarin, the stable current densityof KvLQT1was supressed by (2.35±0.50)%,(5.69±2.88)%,(17.20±2.74)%,(57.71±3.45)%,(72.94±3.77)%and (75.78±2.04)%, respectively(all n=6, P＜0.05); as well as KvLQT1tail, by (5.21±0.39)%,(8.12±2.37)%,(15.32±4.75)%,(63.23±2.82)%,(75.97±1.99)%and (80.42±3.90)%, respectively(all n=6, P＜0.05); the same as IKs, by (3.19±4.11)%,(8.87±3.66)%,(17.81±2.52)%,(40.44±1.90)%,(60.99±3.21)%and (63.18±2.18)%, respectively(all n=6, P＜0.05); so did IKs tail, by (4.98±0.45)%,(7.78±1.63)%,(14.32±1.75)%,(45.23±2.82)%,(63.25±2.73)%and (65.42±3.90)%, respectively(all n=6, P＜0.05). Once given10mM,50mM,100mM and200mM concentration ofpuerarin, the stable current density of Kir2.1was decreased by (28.20±2.80)%,(52.36±1.76)%,(52.42±3.07)%and (53.11±3.81)%, respectively(all n=6, P＜0.05); as well as Kir2.3, by (11.04±2.89)%,(25.89±2.21)%,(38.56±3.39)%and (57.18±4.15)%, respectively(all n=6, P＜0.05).100mM puerarin inhibitedthe open rate of normalized single channel of Kir2.3by (32.91±4.60)%(all n=6,P＜0.05),while it had no significant effect on channel conductance of(14.42±0.14) pS;100μM purarin supressed the open rate of normalized singlechannel of KvLQT1and IKsby (49.78±7.65)%and (54.25±8.22)%,respectively (all n=6, P＜0.05), however, it also had no significant effect on channel conductance of (4.43±1.47) pS and (15.89±3.83) pS. Additionally,KvLQT1channel was inhibited by the pretreatment of1mM puerarin, thecurrent was also decreased by (38.81±6.42)%,(46.89±3.80)%,(56.37±7.97)%,(63.53±4.98)%and (52.34±5.73)%while applied with β receptor activatorIsoproterenol(100nM), protein kinase A activator8-Br-cAMP(100uM),adenylate cyclase activator Forskolin(10μM), PI3K inhibitor LY294002(10μM)and PLC inhibitor U73122(10μM), respectively(all n=6, P＜0.05). Conclusions:Puerarin remarkably prolonged action potential duration (APD) in aconcentration dependent manner, the antiarrhythmic performance of puerarinwas based on its suppressive effect of KvLQT1, IKs, Kir2.1and Kir2.3channelcurrent. Not only acted on the inside surface of membrane, but purarin played arole in inhibitting KvLQT1channel current by β receptor/cAMP signalingpathway. Additionly, LY294002and U73122could increase KvLQT1channelcurrent by enhancing intracellular PIP2level, while puerarin acted as acompetitive antagonist in these progress. |
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