| Impaired ventricular repolarization can lead to long QT syndrome (LQT),a disease with high risk of developing lethal ventricular tachyarrhythmia.Human ether-a-go-go-related gene (hERG) encodes the pore forming subunitof the channel underlying IKr, which is crucial for the cardiac repolarization. Areduction in hERG currents due to either genetic defects, or adverse drugeffects leads to hereditary or acquired LQT. Therefore, one potentiallyinteresting approach to treat LQT would directly target the deficiency in netrepolarizing current through activating hERG channels. In the last few years,more than10compounds have been identified to enhance IKrcurrent throughdifferent mechanisms and are classified into two groups, type1and2. Themechanism of type1compounds is to slow the rate of channel deactivationand the type2compounds primarily slow or remove inactivation. Morerecently, Zhang et.al have reported that a type2IKractivator dose-dependentlyshortens APD in induced pluripotent stem cells derived from LQT1patient,and in contrast, a type1compound is not effective. Earlier research alsoshows that the representative type1compound RPR260243shortens APDonly at the highest concentration tested (30μM). These results suggest thatreduction of voltage dependence for inactivation is more effective inmodulating APD and thus type2activators may have more potentantiarrhythmic activity.However, up to now, only few of type2compounds have been tested fortheir effectiveness of antiarrhythmia under pathological conditions in intacthearts, although many type2compounds have been demonstrated to enhancesIKrcurrent and shorten APD. NS1643is the best examined hERG activator andexperimental studies with NS1643have shown its antiarrhythmic properties inrabbit models of acquired LQT and in the isolated hypokalaemic murine heart. ICA-105574is by far the most potent compound to remove the channelinactivation, and it remarkably increases the hERG current amplitude inheterologous expression cells and shortens APD in guinea pig ventricularmyocytes. Recently, it has been revealed that ICA-105574significantlyshortens monophasic action potential duration, QT intervals in guinea-pighearts as well as in anesthetized dogs. However, effectiveness of ICA-105574in normalizing the prolongation of APD and antiarrhythmia in pathologicalLQT conditions has not been assessed.The prolongation of APD and thus QT interval in ECG are the mostimportant features of electrical remodeling in myocardial hypertrophy or heartfailure. The enhanced inward (including INaLand ICaL) and/or reduced outwardcurrents (including Ito, IKsand IKr) contribute to the delay of ventricularrepolarisation although the mechanism underlying the dysfunction ofcardiac ion channels in cardiac hypertrophy and heart failure is stillunknown. The increasing evidence has indicated that the stimulation ofmyocardial α, β, AT1, endothelin receptor directly modulate the function ofcardiac ion channels. It is well known that activation of those receptors and itssignal transduction pathway play an important role in the myocardialhypertrophic structural remodeling. Thus, the activation of those signalingpathways may also result in electrophysiological remodeling in pathologicalsituations. Up to now, the question about the causation relationship betweenmyocardial hypertrophic and electrophysiological remodelling has not beenyet resolved. It is traditionally accepted that cardiac electrical remodeling issecondary to structural hypertrophic remodelling. However, it has beenrevealed that the dysfunction of ion channels and arrhythmias precedes thehypertrophic remodeling in some experimental models. For example,electrical remodeling precedes heart failure in a cardiac-specificoverexpression of endothelin-1-induced model of cardiomyopathy. Mice withcardiac-specific overexpression of human AT1receptor undergo cardiacremodelling and die prematurely of sudden death. The increased incidence ofarrhythmia and the repolarization defects also occurrs in much younger AT1R mice that do not present signs of hypertrophy, confirming that thesearrhythmogenic changes are not secondary to cardiac remodelling. Alteredintracellular Ca2+handling appears to play a central role during theprogression of cardiac hypertrophy and heart failure. APD prolongationincreases the open time of L-Ca2+channel and thus increases intracellularCa2+concentration. Therefore, the electrophysiological abnormality maycontribute to structural remodeling and thus reversing of APD prolongation isexpected to inhibit hypertrophic remodeling under pathological situations. Astudy has shown that Itoreductions and APD prolongation is the mainmechanism of α receptor-mediated hypertrophy in neonatal rat ventricularmyocytes (NRVMs). APD prolongation results in larger Ca2+influx whichactivates the hypertrophic factor calcineurin. Overexpression of Kv4.2prevents hypertrophy induced by the stimulation of α-receptor. Thisexperiment suggests that the acceleration of action potential repolarizationmay reverse cardiac hypertrophic remodeling. IKris the crucial current for thecardiac2and3phase repolarization and play an important role on the lengthof theaction potentials. The effects of accelerating repolarization by increasingHERG current on cardiac hypertrophy have not been tested.Therefore, the present study was designed to examine the effects ofcompound ICA-105574on prolonged APD in guinea pig ventricular myocytesand arrhythmias caused by delayed repolarization in isolated guinea pig hearts.It is known that NS1643has moderate effect on channel inactivationcompared to ICA-105574. For comparison, the effect of NS1643was parallelobserved to address the issue of whether the antiarrhythmic activity isproportional to the potency for removing channel inactivation. In addition, weevaluated the effects of ICA105574on cellular hypertrophy induced byphorbolester (PMA) in cultured neonatal rat ventricular myocytes. It aimed toprovide experimental and application basis for the development of HERGchannel activator. Part1The reverse effects of ICA-105574on blocked IKrand prolongationof APDAim: To observe the effects of ICA-105574and NS1643on blockedHERG current and action potentials in guinea-pig ventricular myocytes.Methods: Whole-cell HERG current was recorded on stably transfectedHEK-293cell using the conventional patch-clamp technique. We examinedthe effects of ICA-105574and NS1643on the inhibition of hERG currents bydofetilide or moxifloxacin. A regular pulse protocol was used to record hERGcurrents, which was elicited from a holding potential of-80mV to depolarizeto20mV for a4-s duration and was followed by a test pulse to-60mV toevoke large, slowly decaying outward tail currents. A ventricular actionpotential (VAP) waveform was used to evoke hERG currents. This protocol,illustrated, was applied every1s. Experiments were conducted at roomtemperature（20℃～25℃）. The pipette solution contained (in mM) KCl,140;MgCl2,1; EGTA,5; magnesium-ATP,4and HEPES,10(pH7.2with KOH).The external solution contained (in mM) NaCl,140; KCl,5.4; MgCl2,1;CaCl2,2; glucose,10; and HEPES,10(pH7.4with NaOH).Action potential recordings were performed in isolated guinea pigventricular myocytes using the perforated patch technique. We examined theeffects of two hERG activators on APD and further examined the effects ofICA-105574on APD90under different stimulus frequencies and theeffectiveness of ICA-105574for converting the pathological prolongation ofAPD. Action potentials were evoked at different rate with suprathresholdcurrent pulse of46ms duration applied via patch electrodes in thecurrent-clamp mode. Experiments were performed at36±1°C using a WarnerInstrument TC-324B Automatic Temperature Controller. Cells werecontinuously perfused with extracellular buffer containing (in mM) NaCl,138;KCl,4; MgCl2,1; CaCl2,2; NaH2PO4,0.33; glucose,10and HEPES,10(pH7.4with NaOH). Borosilicate glass electrodes were tip-dipped in amphotericinB-free internal solution for several seconds and back-filled with internalsolution added amphotericin B (240g/ml, Sigma-Aldrich). The internalsolution contained (in mM) potassium aspartate,120; KCl,25; MgCl2,1;CaCl2,2and HEPES,10(pH7.3with KOH). Results:(1) Dofetilide (10n M) and moxifloxacin (100μM) evidentlyinhibited step and tail current as they were previously demonstrated. Thecompound ICA-105574at1μM reversed the inhibition induced by dofetilideand moxifloxacin, and enhanced the step current by4-5times withoutevidently affecting peak tail currents. NS1643(10μM) attenuated thereduction of dofetilide and moxifloxacin on step and tail current ampiltudes,but did not reverse the inhibition to the control levels. In addition, by actionpotential clamp, we found ICA-105574reversed the inhibition of peakrepolarizing currents by dofetilide and moxifloxacin to the control levels.Meanwhile, it remarkably increased the current during the initial phases andthe current thus displayed an AP-like configuration. NS1643at10μM did notchange the “hump” shape of hERG current, but it only partly reversed thedecrease of peak repolarizing currents produced by dofetilide andmoxifloxacin.(2) Both ICA-105574and NS1643induced concentration-dependentshortening of APD. However, effects of ICA-105574on APD were evidentlystronger than NS1643and action potential was not able to evoke in three ofsix cells when its concentration was10μM. The concentration-responsecurves for percentage shortening in APD50and APD90were constructed anddata were fit to Hill equation for comparison. The maximum effects (efficacy)and IC50values in shortening of APD50were100.6±0.3%and2.6±0.1μM forICA-105574, and52.1±1.0%and4.7±0.1μM for NS1643, respectively. Theefficacy and IC50values in shortening of APD90were95.5±0.1%and2.1±0.1μM for ICA-105574, and48.5±1.0%and4.9±0.1μM for NS1643,respectively. The results revealed a more potent effect of ICA-105574thanthat of NS1643on APD.(3) ICA-105574shortened APD90at all of test stimulus frequencies, butthe rate-dependent change of APD90was almost lost when its concentrationwas more than3μM. In contrast, NS1643had no visible influence on therate-dependence of APD90although it also shortened APD90at all of teststimulus frequencies. (4) Upon application of IKrinhibitor, dofetilide (100n) or moxifloxacin(100μM), or IKsinhibitor Chromanol293B (20μM) or hypokalaemia (2.1mM),action potential durations were evidently prolonged in differently extent. TheAPD prolongation was completely reversed by co-application of ICA-1055743μM or NS164310μM. Both ICA-105574and NS1643did not affect thechanges in RP and APA.Conclusion: ICA-105574and NS1643may antagonize currents reducedby HERG inhibition; both of them could shorten APD of isolated guinea-pigventricular myocytes and QT and QTc intervals of Langendorff-perfusedguinea-pig hearts in a concentration dependent manner; and can antagonizethe prolonged APD by dofetilide, moxifloxacin, Chromanol293B orhypokalaemia. ICA-105574is stronger than NS1643on these roles.Part2ICA-105574prevents arrythmias induced by cardiac delayedrepolarizationAim: The present study was designed to examine the effects ofICA-105574on arrythmias caused by either IKror IKsinhibitors in isolatedguniea-pig hearts. For comparison, the effect of NS1643was parallel observedto address the issue of whether the antiarrythmic activity is proportional to thepotency for removing channel inactivation. We also determined theproarrythmic activities of ICA-105574and NS1643Methods: ECG was recorded via two silver electrodes attached to theventricular apex and the aortic root of Langendorff-perfused hearts. All datawere acquired using the BIOPAC/MP100system. Empirical procedures wereas follows:(1) To determine the potential proarrhythmic activities of hERGactivators，the experiment was initiated with a20min vehicle control (0.1%DMSO) after stabilization. The perfusate was switched to the reservoircontaining a test compound (NS1643or ICA-105574) at the firstconcentration. The heart was repeatedly exposed to another ascendingconcentration (1,3,5,10μM) of test compound after20min. In this study,ventricular tachyarrhythmias included ventricular fibrillation (VF) andventricular tachycardia (VT), the later was defined as three or more ventricular extrasystoles in succession.(2) The antiarrhythmic activities of hERGactivators were determined using pharmacologically modeled LQT1andLQT2by IKsinhibitor chromanol293B and IKrinhibitor moxifloxacinrespectively. Tachyarrhythmias were induced by IKror IKsinhibitor combinedwith hypokalemia (2.1mM, the reduced amount of KCl was substituted withan equivalent molar quantity of NaCl) according to the literature. Langendorffhearts were perfused in normal Tyrode solution containing vehicle (0.1%DMSO) for10min and a test compound was added. Ten minutes later theperfusate was then switched to the reservoir containing a test compound andmoxifloxacin or chromanol293B in hypokalemic medium for up to60min.To determine whether test compounds could stop tachyarrhythmias, theperfusate was randomly switched to different reservoir containing a testcompound at different concentrations after moxifloxacin or chromanol293Bin hypokalemic medium induced visible ventricular tachyarrhythmias.Results:(1) ICA-105574at3,5, and10μM significantly shortened QTcfrom182.2±5.1ms (control) to140.5±10.2ms (n=6, p<0.01),123.8±11.0ms(n=6, p<0.01), and113.2±6.4ms (n=6, p<0.01) respectively. Anotherprominent shape change upon application of ICA-105574on ECG was theinversion of T-wave and the amplitude of inverted T-wave displayed aconcentration-dependent manner. ICA-105574at10μM evoked VT in2out of6hearts. NS1643at5,10μM also shortened QTc from180.5±4.0ms to160.7±10.2ms (n=6, p<0.01) and146.4±11.2ms (n=6, p<0.01) respectively.NS1643did not induce obvious every wave change on ECG.(2) In combined with the perfusion of hypokalaemic solutionsmoxifloxacin (100μM) and Chromanol293B (10μM) caused VT or VF in4out of8and5out of10hearts respectively. In the presence of ICA-105574(3μM), both moxifloxacin and Chromanol293B induced no arrhythmias in all oftest hearts. However, in the presence of NS1643(10μM), moxifloxacincaused arrhythmias in two out of eight hearts and Chromanol293B evokedarrhythmias in eight out of ten hearts.(3) Different concentrations of ICA-105574(1,3,5μM) and NS1643(3, 5,10μM) did not stop VT or VF once they were induced by moxifloxacin orChromanol293B.Conclusion: The present study demonstrates that the compoundICA-105574, but not NS1643, is able to prevent ventricular arrhythmias inpharmacologically induced LQT1and LQT2models in intact guinea-pighearts, but it may have the potential risk of proarrhythmia under higherconcentrations. Thus, anti-arrhythmic benefits and potential pro-arrhythmicrisks should be seriously taken consideration for further developing this typeof hERG activators.Part3The effects of ICA-105574prevent cellular hypertrophyAim: To evaluate the effects of HERG activator ICA-105574on thecellular hypertrophy in NRVMs.Methods: The NRVMs were isolated from the hearts of1–2-day-oldSprague-Dawley rat pups via trypsin digestion. Dissociated cells werepreplated for1h in serum-free DMEM-F12to selectively remove nonmusclecells. Myocytes were then plated in DMEM-F12with10%fetal bovine serumand0.1mM BrDU at a density of1×105cells/ml onto plastic dishes orchamber slides and left undisturbed in a5%CO2-95%room air incubator for24h. NRVMs were assigned into control, ICA-105574（0.3、1、3μM）, PMA(1μM) and PMA plus ICA-105574（0.3、1、3μM）groups. The followingstudies were performed48h after the treatment.（1） NRVMs was marked by3,3’-dioctadecyloxacarbocyanineperchlorate(DiO). The surface area and the membrane capacitance changewere observed by confocal microscope.（2）RT-PCR was performed to detect the BNP, β-MHC mRNAlevels.（3）Patch-clamp recordings: The effects of ICA-105574on actionpotentials in NRVMs were investigated by using the conventional patch-clamptechnique. Action potentials were evoked at a rate of1Hz with suprathresholdcurrent pulse of1ms duration applied via patch electrodes in thecurrent-clamp mode. The pipette solution contained (in mM) KCl,135; EGTA,10; MgCl2,1; magnesium-ATP,5and HEPES,10(pH7.2with KOH). The external solution contained (in mM) KCl,5; NaCl,130; MgCl2,1; CaCl2,2;glucose,10; and HEPES,10(pH7.4with NaOH). Whole-cell Cs-IKrwasrecorded in NRVMs using the conventional patch-clamp technique, the effectsof ICA-105574on Cs-IKrcurrent was investigated. A regular pulse protocolwas used to record Cs-IKrcurrents, which was elicited from a holding potentialof-80mV to various prepulse potentials of-70to70mV for1s andrepolarized to-70mV to evoke outward Cs-IKrtail currents. The pipettesolution contained (in mM) CsCl,135; EGTA,10; magnesium-ATP,5andHEPES,10(pH7.2with CsOH). The external solution contained (in mM)CsCl,135; MgCl2,1; glucose,10; and HEPES,10, nimodipine10μM,(pH7.4with CsOH).Results:（1）The effect of ICA-105574on cellular hypertrophy: Afterincubating PMA for48hours, the cell surface area increased to179±38%(P <0.05, n=24cells). After co-incubation of ICA-105574at0.3,1,3μM cellsurface areas were reduced to135±23%(P <0.05, n=22cells),113±28%(P<0.05, n=39cells) and105±24%(P <0.05, n=28cells) respectively. PMA(1μM) increased membrane capacitance from31±4pF to72±13pF (P <0.05, n=20cells), but after co-application of ICA-105574at3μM, membranecapacitance was reduced to35±11pF (P<0.05, n=20cells).（2）The effect of ICA-105574on BNP, β-MHC mRNA expression:PMA (1μM) significantly elevated the BNP and β-MHC mRNAexpression(P<0.05, n=6), suggesting that intracellular signaling pathwaysrelated to cardiac hypertrophy was activated. In the case of pretreatment withICA-105574（1,3μM）, BNP mRNA expression was decreased in aconcentration-dependent manner(P<0.05, n=6). In the case of pretreatmentwith ICA-105574（0.3,1,3μM）, β-MHC mRNA expression was alsodecreased in a concentration-dependent manner(P<0.05, n=6). Pretreatmentwith different concentrations of ICA-105574（0.3,1,3μM）alone, BNP,β-MHC mRNA expression levels had no significant changes compared withthe control group. The result indicated that ICA-105574specificallyantagonized the activation of intracellular signaling pathways related to cardiac hypertrophy by PMA.（3）PMA(1μM) prolongedAPD50and APD90from160.2±16.5ms and250.2±16.6ms to225.7±23.3ms (n=8，P<0.01) and310.8±23.5ms (n=8,P<0.01), respectively. Long-term incubation of ICA-105574prevented theprolongation of action potential induced by PMA. ICA-105574（3μM）shortened APD50and APD90to187.3±17.6ms (n=8，P<0.01) and275.2±17.3ms (n=8， P<0.01). This suggested that the APD shortening maycontribute to its prevention from myocardial hypertrophy be related toICA-105574.（4）In the voltage range from-30to70mV, ICA-105574at3μMsignificantly increased Cs-IKrcurrents in control NRVMs and the enhancementof the current displayed a voltage dependence manner, i.e. More increase atmore positive voltage. The result suggested that enhancement on Cs-IKrcurrent contribute to its APD shortening of ICA-105574.Conclusion: The current results indicated that ICA-105574could preventcellular hypertrophy of NRVMs induced by PMA. The anti-hypertrophiceffect may be derived from its increasing IKrcurrent and shortening APD. Themechanism may be related to altereation of intracellular Ca2+handlingsecondary to the shortening of APD. Specific mechanisms need to be furtherinvestigated.Sunnary:1Both ICA105574and NS1643antagonized the inhibition ofHERG currents produced by the channel inhibitors and reversed theprolongation of APD in pathological conditions. Above effects of ICA-105574was more potent than NS1643.2ICA-105574, but not NS1643, completely prevented ventriculararrhythmias in pharmacologically induced LQT1and LQT2models in intactguinea-pig hearts. Both of them did not stop on-going ventricular arrhythmia.3ICA-105574may have the potential risk of proarrhythmia under higherconcentrations. Thus, potential pro-arrhythmic risks should be seriously takenconsideration for further developing this type of hERG activators.4ICA-105574prevented cellular hypertrophy of NRVMs induced by PMA. The mechanism may be related to the shortening of APD. Specificmechanisms need to be further researched. |
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