Modulation Of Cardiac Iksby Norepinephrine And Angiotensin Ⅱ And The Signaling Mechanism

Cardiomyopathies such as cardiac hypertrophy and heart failure are oftenaccompanied with arrhythmia such as atrial fibrillation and ventricularfibrillation and sometimes torsade de pointes (Tdp). An often seencharacteristic of cardiac electrophysiological changes accompanying thesearrhythmias is the prolongation of action potential duration (APD). Theseabnormal electrophysiological activities during the cardiomyopathies are theresults of pathological remodeling of cardiac ion channels. Norepinephrine(NE) and angiotensinⅡ (Ang II), two endogenous important modulators ofcardiac electrophysiology and function, play an major role both in normalphysiological condition as well as in pathological remodeling. Norepinephrinemediates the effect of activation of sympathetic nerves which activatesβ1-adrenoceptor, which in turn activates adenylate cyclase (AC)-cAMP-PKApathway, and increases the activity of L-type calcium channel and contractilityof cardiac myocytes through excitation-contraction coupling. Under thepathological condition, NE level is increased due to the overexcitation ofsympathetic nerve which will promote the remodeling of ion channel, aprocess with characteristic of changes of expression or malfunction of manycardiac ion channels. It has been reported that overly induced NE duringoverexcitation of sympathetic nerve would increase the automaticity throughcAMP-mediated increase of Ifwhich accelerates the diastolic depolarization;Itodensity is reduced by NE through activation of α1-adrenoceptor which leadto prolongation of action potential duration; NE-mediated activation of PKCin pathological condition reduces IK1which results in prolongation of actionpotential duration, and increases the probability of spontaneous depolarizationand delayed after depolarization. These remodeling of cardiacelectrophysiology all can result in arrhythmia. In physiological condition, NE can increase the slowly activating delayed-rectifier K+current (IKs) throughβ1-adrenoceptor-PKA path way. In contrast, NE reduces IKsin heart failure dueto the long exposure to NE stimulation. These effects of NE will reduce thecardiac repolarization reserve accompanied with prolongation of QT intervaland action potential duration. As such, modulation of ion channel functions byNE plays an important role in cardiac function under both physiological andpathological conditions.Ang II is the main biologically active peptide in the renin-angiotensinsystem (RAS), which is important in maintaining the cardiovascularhomeostasis. In addition, Ang II is involved in diseased conditions such asmyocardial infarction, heart failure, cardiac hypertrophy, where Ang II ispossibly one of the central mechanisms. Furthermore, Ang II-inducedinterstitial fibrous tissue accumulation is noted to impair electrical conduction.Ang II also affects the function of cardiac ion channels. Ang II, throughactivation of AT1receptor, prolongs the time course of activation andinactivation of L-type calcium channel. It has been shown that Ang IIdown-regulates Itodensity in isolated rat ventricular myocytes. The reduced Itodensity has also been reported on mice with cardiac-specific overexpression ofhuman Ang II type1(AT1) receptors. It has been demonstrated that specificoverexpression of Ang II genes in mice hearts tissues can prolong actionpotential duration and induce ventricular arrhythmia.Slowly activating delayed-rectifier potassium current (IKs) is one of majorrepolarzing currents in the hearts. The IKschannel is formed by thepore-forming α-subunits encoded by KCNQ1(Kv7.1) and β-subunits encodedby KCNE1. It is one of the most important potassium currents in cardiacrepolarization reserve. Congenital defects, pharmacological inhibition, andacquired loss of IKscan all result in prolongation of QT-interval and actionpotential duration.It has been demonstrated IKschanged significantly in the animal modelsof hypertrophy and heart failure induced NE and Ang II. However the signalpathways for modulation of IKsby NE and Ang II are far from being well understood. This study is planned to investigate the effect of NE and Ang II onIKsin the atrial and vetricular myocytes of guinea pig and to compare theirsignaling mechanisms of actions.Part1The effect of norepinephrine (NE) on IKsin guinea pig cardiacmyocytesObjective: To assess the effect of NE on IKsin guinea pig ventricular andatrial myocytes.Methods: Single ventricular and atrial myocytes were enzymaticallydissociated from the hearts of adult guinea pigs. The IKswas recorded by usingthe whole-cell patch-clamp technique. The external solution contained (in mM)NaCl132，KCl4，MgCl21.2，CaCl21.8，glucose5，Hepes10(pH adjustedto7.4with NaOH). L-type Ca2+current was blocked by1μM Nimodipine(Nim). IKrwas blocked by5μM E4031. The pipette solution contained (inmM) potassium aspartate125，KCl20，MgCl21，Mg-ATP5，EGTA10，Hepes5(pH adjusted to7.2with KOH). Cardiomyocytes were depolarizedfrom a holding potential of-30mV to various prepulse potentials of-30to+70mV for2s, and repolarized to-30mV to evoke outward tail currents. Allexperiments were performed at room temperature (22-23℃).Results: NE markedly potentiated the IKscurrents in guinea pigventricular myocytes. The tail current densities at+70mV were1.8±0.1pA/pF and2.6±0.2pA/pF (n=6) in the absence and presence of NE (10M).The potentiation of IKstail current reached saturation about2-3min afteraddition of NE (10M) and was abolished by a IKsspecific inhibitor,293B(30M). NE increased the amplitude of IKsin a concentration-dependentmanner with an EC50of0.66±0.09M. The V1/2of IKsin the absence andpresence of NE were36.9±1.8mV and35.7±2.4mV (n=6, P>0.05),respectively. The voltage dependence of IKsactivation was found to be notaffected by NE.NE also potentiated IKsin guinea pig atrial myocytes. The tail currentdensities at+50mV increased from0.65±0.10pA/pF to0.95±0.10pA/pF (n=5)and were abolished by a IKsspecific inhibitor,293B (30M). The V1/2of IKsin the absence and presence of NE were34.45±4.24mV and33.05±5.86mV (n=5,P>0.05), respectively. The voltage dependence of IKsactivation was found to benot affected by NE.Conclusion: NE activates IKscurrents in both ventricular and atrialmyocytes, which were not achieved through affecting the voltage-dependentactivation.Part2The effect of angiotensin II (AngII) on IKsin guinea pig cardiacmyocytesObjective: To assess the effect of AngII on IKsin guinea pig ventricularand atrial myocytes.Methods: Single ventricular and atrial myocytes were enzymaticallydissociated from the hearts of adult guinea pigs. The IKswas recorded by usingthe whole-cell patch-clamp technique. Cardiomyocytes were depolarized froma holding potential of-30mV to various prepulse potentials of-30mV to+70mV for2s, and repolarized to-30mV to evoke outward tail currents.Results: AngII markedly inhibited the IKscurrents in guinea pigventricular myocytes. The tail current densities at+70mV were1.9±0.1pA/pF and1.4±0.01pA/pF (n=6) in the absence and presence of AngII (100nM). The inhibition of IKstail current reached saturation about5-10min afteraddition of AngII (100nM). AngII decreased the amplitude of IKsin aconcentration-dependent manner with an IC50of30.42±5.97nM. The V1/2of IKsin the absence and presence of AngII were33.3±0.8mV and31.6±1.9mV(n=6, P>0.05), respectively. The voltage dependence of IKsactivation wasfound to be not affected by AngII.AngII also inhibited IKsin guinea pig atrial myocytes. The tail currentdensities at+70mV were decreased from1.76±0.12pA/pF to1.29±0.08pA/pF(n=5) by AngII. The V1/2of IKsin the absence and presence of AngII were37.94±1.19mV and38.90±2.47mV (n=5, P>0.05), respectively. The voltagedependence of IKsactivation was found to be not affected by AngII.Conclusion: AngII inhibits IKscurrents in both ventricular and atrialmyocytes, which were not achieved through affecting the voltage-dependent activation.Part3Signaling mechanism for norepinephrine-andangiotensinII-induced modulation of guinea pig cardiac IKsObjective: To examine the signal transduction pathways mediating themodulation of IKsby NE and AngII.Methods: The IKswas recorded in guinea pig ventricular myocytes byusing whole-cell patch-clamp technique to examine the effects of PKAinhibitor, PKC inhibitor and PLC inhibitor on IKsinduced by NE or AngII.Results: Prazosin (1M) and doxazosin (1M) as α1-adrenoceptorblockers only partially reduced NE-induced increase of IKs. The increase wasreduced from42.3±1.9%to21.9±0.6%or26.1±0.1%by prazosine ordoxazosin, respectively. PLC inhibitors U73122(1M) or edelfosine (1M)reduced NE-induced potentiation of IKsfrom40.9±2.3%to21.0±0.4%or12.7±1.2%by U73122or edelfosine, respectively. PKC inhibitorbisindolylmaleimide (Bis-1)(100nM) reduced NE-induced potentiation of IKsfrom40.9±2.3%to22.6±0.6%.Phenylephrine (PE), a selective agonist of α1-adrenoceptor did not affectIKsat a concentration of10M. However, IKswas increased by24.4±0.8%by PE at a higher concentration of60M. In the presence of propranolol (1M), a specific inhibitor of β-adrenoceptor, PE induced an inhibition ratherthan potentiation of IKs. Propranolol (1M) almost totally abolishedNE-induced increase of IKs(potentiation was reduced from40.9±2.3%to5.78±0.2%). NE-induced potentiation of IKswas reduced from40.9±2.3%(n=6) to5.4±1.1%by H89(30M), a PKA blocker.Specific AT1receptor blocker, losartan (1μM), antagonized the inhibitoryaction of Ang II on IKs. PLC inhibitors U73122(1M) or edelfosine (1M)reduced Ang II-induced inhibition of IKsfrom29.8±0.9%to15.2±2.7%or12.9±0.3%by U73122or edelfosine, respectively. PKC inhibitorbisindolylmaleimide (Bis-1)(100nM) reduced Ang II-induced inhibition ofIKsfrom29.8±0.9%to8.9±1.0%.Conclusion: NE activates IKsvia α1-PLC-PKC and β-PKA pathways. And a crosstalk between α1-and β-adrenoceptor pathways occurred for theeffect of NE which contributes to the NE-induced enhancement of IKs. Ang IIinhibits IKsvia AT1-PLC-PKC pathway.