| IntroductionIt has been documented that angiotensin-Ⅱis effective in enhancing myocardial contractility, promoting cardiac hypertrophy and inducing cardiac arrhythmia.All of these actions are based on cardiomyocyte electrophysiological changes.The biological activities of angiotensin-Ⅱare primarily mediated by angiotensinⅡAT1 receptors.In recent years,autoantibodies against angiotensin-ⅡAT1 receptors have been found in the sera of patients with preeclampsia and malignant hypertension.These autoantibodies were demonstrated to display a stimulatory "agonist-like" activity on their corresponding receptors,suggesting that they might be implicated in the pathogenesis of these diseases.In view of the fact that AT1 receptors play an important role in the regulation of cardiac pathological changes,it is of significance to explore the relations of autoantibody against cardiac AT1 receptors to cardiac diseases.In order to clarify the role of anti-AT1 receptor autoantibodies in the pathogenesis of cardiac diseases,firstly we should be aware of the direct effects of these autoantibodies on the heart.Therefore,in this study we explored the effects of anti-AT1-receptor antibodies on cardiomyocyte action potential and the related ionic currents as well as cardiac inotropism.Objective1.To explore the effects of anti-AT1-receptor autoantibodies on rat ventricular action potential and L-type calcium current(ICa-L);2.To clarify the effects of anti-AT1-receptor autoantibodies on the in vivo cardiac inotropism.Methods1.Peptide synthesis,active immunization of rat model,detection of anti-AT1-receptor autoantibodies by SA-ELISA assay,Affinity purification of IgG..2.Whole cell patch clamp techniques were used to study the effects of anti-AT1-receptor autoantibodies on rat ventricular myocyte action potential and L-type calcium current(ICa -L),which were compared with those of Ang-Ⅱ.The effects of losartan,a specific antagonist of AT1-receptor,on the effects of anti-AT1-receptor autoantibodies and of Ang-Ⅱwere also analyzed.3.Healthy Wistar rats were divided randomly into six groups:(1)control group,n=6;(2) negative IgG group,n=6;(3)anti-AT1-receptor autoantibodies(AT1-R AA)group,n=6; (4)AT1-R AA +losartan group,n=6;(5)angiotensin-Ⅱ(Ang-Ⅱ)group,n=6;(6) angiotensin-Ⅱ(Ang-Ⅱ)+losartan group,n=6.4.For measurement of cardiac inotropism,Wistar rats were randomly divided into 5 groups: (1)control group(n=5),(2)AT1-R AA group(n=5),(3)AT1-RAA+losartangroup(n=5), (4)angiotensin-Ⅱgroup(n=5),(5)angiotensin-Ⅱ+ losartan group(n=5).Cardiac function in rats in vivo,including the left ventricular systolic pressure(LVSP),maximal positive and negative values of the instantaneous first derivative of LVP(+dP/dtmax and -dP/dtmax),was digitally processed via a ms2000 analyzing system.Results1.Titres of AT1-R AA in the sera of immunized rat began to increase from second week after active immunization(Fig 1,Tab 1).At 8th week,the antibody titres in the sera of rats increased from the pre-immunization value of 1:25.56±9.22 to 1:1490.00±847.58 (p＜0.01).No changes were observed for control group.2.Purified immunoglobulin fractions(IgG)from the positive sera were prepared using a MabTrap kit(Amersham).The concentration of IgG after purification is 2.38 mg/ml.3.Effects on action potential:AT1-R AA significantly shortened action potential duration at 50%(APD50)and 90%(APD90)repolarization(Fig3,Fig5,Tab2).The effects of Ang-Ⅱwere in the same way as the effects of AT1-R AA showing that the antibody against AT1-receptor displayed an "agonist-like" activities on the cardiac AT1 receptors (Fig,Tab).Both the effects of AT1-R AA and Ang-Ⅱcould be blocked by losartan indicating that the effects were mediated by AT1-receptors(Tab2).4.Effects on ICa-L:AT1-R AA remarkably and dose-dependently increased the ICa-L(Fig7, Fig 9,Tab4).For example,AT1-R AA at 50nmol/L increased the ICa-Lfrom the control value of 4.11±0.58(pA/pF)to 8.55±0.13(pA/pF)(p＜0.01).The effects of Ang-Ⅱare in the same way as the effect of AT1-R AA(Fig,Tab).The effects of AT1-R AA could be blocked by losartan indicating that the effects were mediated by AT1-receptors(Tab6). 5.Effects on the action potential in which INa/Cais inhibited by lithium:AT1-R AA could no longer change the APD50and APD90of action potential(Fig 11,Tab 6).The effects of Ang-Ⅱare in the same way as the effect of AT1-RAA(Fig 12,Tab 7).6.Effects on cardiac function in rats in vivo:AT1-R AA could significantly increase LVSP, +dp/dtmax and -dp/dtmax(Fig 13,Fig 14,Fig 15).The effects of Ang-Ⅱare in the same way as those of AT1-R AA.Conclusion1.In this study,we have successively produced enough anti-AT1-receptor autoantibodies (AT1-R AA)in the sera from rats immunized with antigenic peptide corresponding to the second extracellular loop of human angiotensinⅡAT1 receptors.2.Our results demonstrated that AT1-R AA significantly shortened the APD50and APD90of action potential.Its effects were the same as that of Ang-Ⅱindicating that AT1-R AA has an "agonist-like" activity on cardiac AT1-receptors.The effects of AT1-R AA could be blocked by losartan indicating that the effects were mediated by AT1-receptors.The mechanism responsible for the shortening effects of AT1-R AA might be due to the increased outflow of potassium current by enhanced AT1-R AA-induced influx of ICa-L.3.AT1-R AA remarkably and dose-dependently increased the ICa-Lwhich are in the same way as the effect of Ang-Ⅱ.This effect can account for its shortening APD effect on action potential.4.AT1-R AA can increase the cardiac contractility via stimulation of AT1 receptor.The increased ICa-Lby AT1-R AA can also account for its positive inotropism.5.AT1-R AA could enhance cardiac function,which verified its positive inotropic effects.
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