| Background Ventricular arrhythmias are often precipitated by physical oremotional stress. An increased release of the endogenous catecholamines changes thecharacteristics of ionic currents in the cardiomyocytes, which is an important reasonfor ventricular arrhythmia. There are many ionic currents in the cardiomyocytemembrane. The rapid component of the delayed rectifier potassium current, Ikr, is themajor outward current involved in ventricular repolarization and inhibiton of Ikr maylead to action potential duration prolongation. The human ether-a-go-go-related gene(hERG) encodes the voltage-gated potassium channel?-subunit underlying Ikr.Reduction of hERG currents due to mutations in hERG produces congenital long QTsyndrome (LQTS-2). On the other hand, hERG channels are a primary target for thepharmacological management of cardiac arrhythmias with class III antiarrhythmicagents. Excessive blockade of hERG channels by antiarrhythmic or nonantiarrhythmicdrugs may lead to acquired long QT syndrome (aLQTS). Stimulationof the sympathetic nervous system (SNS) in response to exercise or emotional stresscauses stimulation of adrenergic receptors. At least nine adrenoceptor subtypes havebeen identified:?1A,?1B,?1D,?2A,?2B,?2C,?1,?2 and?3. In human heart, the mainlypresence of adrenoceptor subtypes are?1 and?adrenoceptors. Recent investigationsrevealed that the rapid component of the delayed rectifier potassium current may beinhibited by either?1 or?adrenergic stimulation. However, the effects of adrenergicreceptor subtypes on the Ikr and the underlying mechanisms need further study.Objectives 1. Whole-cell patch clamp techniques were used to investigate the regulation of Ikr in guinea pig cardiomyocytes by?1 and?adrenergic receptor andtheir subtypes. 2. Their underlying signal pathway involved in Ikr were studied,which may help us to study the mechanism that stress induced ventriculararrhythmias.MaterialMaterials and Methods1. Myocyte isolation: Single ventricular myocytes were isolated from male guineapig(weighted 250-300g)hearts using a standard enzymatic technique withLangendorff perfusion.2. Patch clamp recording of Ikr tail currents in guinea pig ventricular myocytes:Isolated ventriular myocytes were placed in experimental chammber and wholecellpatch clamp recording technique was used to record Ikr. Ikr was elicited usingthe following test pulse protocol: after a holding potential of ?40 mV, test pulseswere applied at various voltages from ?40mV to +40mV (step width 20 mV, stepduration 200 ms) before returning to ?40mV for tail current recording.3. Regulation of Ikr currents by?1-adrenergic stimulation.3.1.?1-adrenergic effects on Ikr currents: Ikr currents were recorded before andafter application of specific?1-adrenergic receptor agonist phenylephrine(0.0001?mol/L?100?mol/L). The ventricular myocytes were applicatedprazosin (?1-adrenergic antagonist), 5-MU (?1A-adrenergic antagonist),chloroethylclonidine (?1B-adrenergic antagonist) or BMY7378 (?1Dadrenergicantagonist) respectively before phenylephrine application and Ikrcurrents were recorded before and after application of phenylephrine.3.2. Role of protein kinase A (PKA) and protein kinase C (PKC) in?1-adrenergic effects on Ikr currents: The ventricular myocytes werepretreated with specific PKA inhibitor (KT5720, 2.5?mol/L) or specific PKC inhibitor (chelerythrine, 1?mol/L) for 1 hour. Then Ikr currents wererecorded before and after application of phenylephrine.4. Regulation of Ikr currents by?-adrenergic stimulation.4.1.?-adrenergic effects on Ikr currents: Ikr currents were recorded before andafter application of unspecific?-adrenergic receptor agonist isoproterenol(0.001?mol/L?100?mol/L). The ventricular myocytes were applicatedpropranolol (?-adrenergic antagonist), CGP20712A (?1-adrenergicantagonist) or ICI118551 (?2-adrenergic antagonist) before isoproterenolapplication and Ikr currents were recorded before and after application ofisoproterenol.4.2.?1-adrenergic effects on Ikr currents: Ikr currents were recorded before andafter application of specific?1-adrenergic receptor agonist xamotorol (0.01?mol/L?100?mol/L).4.3. Role of protein kinase A (PKA), phospholipase C (PLC) protein kinase C(PKC) and calcium/calmodulin-dependent protein kinase II (CaMKII) in?1-adrenergic effects on Ikr currents: Cardiomyocytes were pretreated withspecific PKA inhibitor (KT5720, 2.5?mol/L), specific PLC inhibitior(U73122, 100nmol/L), specific PKC inhibitor (chelerythrine, 1?mol/L) for1 hour respectively, or specific CaMKII inbitor KN93 (10?mol/L) for 30minutes. Then, Ikr currents were recorded before and after application of?1-adrenergic receptor agonist xamotorol.5. Cross talk between?1 and?adrenergic effects on Ikr currents.5.1. Effects of isoproterenol on Ikr in the presence of phenylephrine: Theventricular myocytes were application of phenylephrine for 10 min beforeapplication of isoproterenol. Ikr currents were recorded before and afterapplication of phenylephrine and after application of isoproterenol. 5.2. EffectEffects of phenylephrine on Ikr in the presence of isoproterenol: Theventricular myocytes were application of isoproterenol for 10 min beforeapplication of phenylephrine. Ikr currents were recorded before and afterapplication of isoproterenol and after application of phenylephrine.Results1. Patch clamp recording of Ikr tail currents in guinea pig ventricular myocytes: Ikrtail current of guinea pig ventricular myocyte was recorded using whole cell patchclamp and it can be completely blocked by 1?mol/L dofetilide. Ikr tail currents aremarkedly sensitive to temperature changes. The Ikr tail current density at +40 mVincreased from 0.28±0.07 pA/pC at 22°C to 0.62±0.07 pA/pC at 37°C.2. Regulation of Ikr currents by?1-adrenergic stimulation.2.1.?1-adrenergic effects on Ikr currents: The dose-dependent effect ofphenylephrine, an?1-adrenoreceptor agonist, on Ikr was examined infreshly isolated guinea pig ventricular myocytes. The amplitude of Ikr wasreduced to 0.67±0.03 in response to 0.1?mol/L phenylephrine. This effect wasblocked by the?-adrenoreceptor antagonist prazosin and the selective?1Aadrenoreceptorantagonist 5-MU, but not by the?1B-adrenoreceptor antagonistchlorethylclonidine or the?1D-adrenoreceptor antagonist BMY7378.2.2. Role of protein kinase A (PKA) and protein kinase C (PKC) in?1-adrenergic effects on Ikr currents: When cardiomyocytes were pretreatedwith chelerythrine or KT5720 for 1 hour, the amplitude of Ikr was reduced to0.98±0.02 and 0.77±0.03 respectively in response to 0.1?mol/L phenylephrine.By comparison, phenylephrine elicited a reduction to 0.67±0.03 on Ikr in theabsence of chelerythrine and KT5720.3. Regulation of Ikr currents by?-adrenergic stimulation. 3.1.?-adrenergi-adrenergic effects on Ikr currents: The dose-dependent effect ofisoproterenol, a nonspecific?-adrenoreceptor agonist, on Ikr was alsoexamined in freshly isolated guinea pig ventricular myocytes. The amplitudeof Ikr was reduced to 0.62±0.03 in response to 10?mol/L isoproterenol. Thiseffect was blocked by the?-adrenoreceptor antagonist propranolol and theselective?1-adrenoreceptor antagonist CGP20712A, but not by the?2-adrenoreceptor antagonist ICI118551.3.2.?1-adrenergic effects on Ikr currents: Stimulation of?1-adrenergic receptorsusing xamotorol also reduced Ikr current in a dose-dependent manner. 10?mol/L xamotorol decreased Ikr current to 0.56±0.04.3.3. Role of protein kinase A (PKA), phospholipase C (PLC) protein kinase C(PKC) and calcium/calmodulin-dependent protein kinase II (CaMKII) in?1-adrenergic effects on Ikr currents: When xamotorol was combined withKT5720 (2.5?mol/L), a specific inhibitor of PKA, the inhibitory effect wasdrastically reduced. The amplitude of Ikr relative current was reduced to0.87±0.03, it was significantly different from xamotorol elicited a reductionto 0.56±0.04 on Ikr in absence of KT5720. When cardiomyocytes were pretreatedwith PLC inhibitor, U73122 (100 nmol/L) or a general PKC inhibitor,chelerythrine (1?mol/L) for 1 hour prior to the recording the xamotoroleffect on Ikr. Xamotorol inhibited Ikr to 0.91±0.07 or 0.71±0.01, respectivelyin the presence of U73122 or chelerythrine. They were both significantlydifferent from xamotorol elicited a reduction to 0.56±0.04 on Ikr in theabsence of chelerythrine and U73122. When cardiomyocytes were pretreatedwith KN93 (10?mol/L for 30 minutes), a synthetic CaMKIIinhibitor, the inhibitory effect of xamotorol on Ikr was unaffected by KN93,xamotorol inhibited Ikr to 0.68±0.07 compared to 0.56±0.04 without KN93. 4. Cross talk between?1 and?adrenergic effects on Ikr currents.4.1. Effects of isoproterenol on Ikr in the presence of phenylephrine: Whencardiomyocytes were firstly acute stimulated by phenylephrine, an?1-adrenoreceptor agonist, the Ikr tail currents were not decreased byisoproterenol, a?-adrenoreceptor agonist. In an other word, isoproterenol cannot reduce Ikr tail currents in cardiomyocytes pretreated with phenylephrinebefore. It was significantly different from the cardiomyocytes not firstly acutestimulated by phenylephrine group.4.2. Effects of phenylephrine on Ikr in the presence of isoproterenol: Whencardiomyocytes were firstly acute stimulated by isoproterenol, a?-adrenoreceptor agonist, the Ikr tail currents were only deceased to 0.80±0.02by phenylephrine, an?1-adrenoreceptor agonist, which was significantlydifferent from the cardiomyocytes not firstly acute stimulated by isoproterenolgroup.Conclusions1. Phenylephrine, an?1-adrenoreceptor agonist, can reduce Ikr current in a dosedependentmanner. This effect is mediated by the?1A-adrenoreceptor subtyperather than other?1-adrenoreceptor subtypes.2. When cardiomyocytes were pre-treated with chelerythrine or KT5720 for 1 hour,both chelerythrine and KT5720 drastically reduced the phenylephrine-inducedeffects, indicating possible involvement of PKC and PKA in the?1-adrenergicinhibition of Ikr.3. Isoproterenol reduced Ikr current in a dose-dependent manner. This effect ismediated by?1-adrenoreceptor subtype rather than other?2-adrenoreceptorsubtype. 4. When xamotorol was combined with KT5720, a specific inhibitor of PKA, theinhibitory effect was drastically reduced. Both Chelerythrine, a specific inhibitorof PKC, and U73122, a inhibitor of phospholipase C (PLC) inhibited thecurrent decreasing induced by xamotorol. However, KN93, an inhibitorCaMKII, may not attenuate the inhibtory effects of xamotorol on Ikr. Our datasuggest a link between Ikr and the?1-adrenergic receptor, involving activationPKA , PLC and PKC.5. Signaling“cross talk”between?1- and?-adrenergic cascades might be involvedin regulation of Ikr tail current in guinea pig ventricular myocyte. |
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