The Cardiac I_k1 Agonist Inhibits Isoproterenol-induced Ventricular Remodeling In Rats

Background:Ventricular remodeling is characterized by myocyte hypertrophy and interstitial fibrosis in responsed to exercise or damage as an adaptive mechanism to improve pumping function by increasing the amount of contractile units and reducing wall stress.It eventually causes ventricular maladaptive structural and functional changes and heart failure?HF?.In the process of ventricular remodeling,changes occure in ion channel expression and function,for instance,altered ICa-L,reduced Ito,outward I_K1,IKs,IKr,enhanced NCX and downregulated pump are named electrical remodeling which usually accompanied with structural remodeling.A recent study indicates that electrical remodeling might be a primary abnormality in electrophysiology and precedes ventricular dysfunction.Early ‘electrical' interventions may prevent the progression to,or possibly even reverse,the manifestations of ventricular dysfunction.Researchers need to reconcieve of the relationship between electrical remodeling and structure remodeling,and develop ion channels as effective targets to reverse the electrical dysfunction and thus prevent the development of HF.Convinced data show that the ATP sensitive potassium channel?KATP channel?plays an important role in ventricular remodeling,and KATPchannel agonists significantly repress remodeling.I_K1 and KATPare consist of Kir2.x channel and kir6.x channel respectively,and both of them belong to the inward rectifier potassium channel family.They have similar molecular structure of channel subunits.Experiments also confirmed that I_K1 decline is a hallmark of electrical remodeling in heart failure.Therefore,we hypothesize that I_K1 channel is involved in the development of ventricular remodeling and activation of myocardial I_K1 may prevent or attenuate ventricular remodeling.We first reported a selective I_K1 /Kir2.1 channel agonist,zacopride,and showed its anti-arrhythmic effect in aconitine-toxic and acute myocardial infarction rat model.By this pharmacological tool,via enhancing I_K1 /Kir2.1,zacopride significantly hyperpolarized the resting membrane potential?RMP?and shorten the action potential duration?APD?,thus effectively suppressed the delayed afterdepolarization?DAD?.Ca²⁺plays important roles in the excitation-contraction coupling,metabolism,cell cycle and communication,gene expression.The content of intracellular Ca²⁺is strictly regulated by rapidly cycling between the cytosol?where it activates the myofilaments?and the sarcoplasmic reticulum?SR?to maintain the Ca²⁺homeostasis.A large number of clinical and experimental study showed that calcium overload is associated with the progress of hypertrophy and heart failure,in parallel with the prolongation of APD and effective refractory period,which made the cardiomyocyte prone to early afterdepolarization?EAD?,DAD and triggered activity.Severe calcium overload is also a predisposing factor for cell apoptosis.Objective:To investigate the effect of inward rectifier potassium channels(I_K1)agonist zacopride on isoproterenol?Iso?-induced ventricular remodeling and involved mechanisms.Methods:Part 1 In vivo adult rat experiment SD rats were randomly divided into six groups: control,zacopride,Iso model,Iso+zacopride,Iso+zacopride+chloroquine and Iso+captopril groups.Cardiac hypertrophy was developed by administration of Iso?ip.3 mg/kg/d,10 d?and determined by the increase of heart-to-body weight ratio and left ventrical-to body weight ratio.The morphology of myocardium and interstitial collagen deposition are monitored by HE and Masson's staining.The changes of voltage-gated calcium current?ICa-L?,the RMP and the morphology of action potential?AP?of rat ventricular myocytes were detected by whole cell patch clamp technique.The expression of Kir2.1,cleaved caspase-3,p-Ca MKII and total Ca MKII,SERCA2 protein were detected by western blotting.Part 2 In vitro neonatal rat cardiomyocyte experiment Neonatal rat ventricular myocytes?NRVMs?were isolated from 1-3-day-old Sprague-Dawley rats by 0.08% trypsin and 0.04% type II collagenase,and purified using differential adherence method and 5-bromine deoxyuridine?5-Brdu?.Cultured neonatal cardiomyocytes were randomly separated into six groups: control,zacopride,Iso,Iso+zacopride,Iso+zacopride +Ba Cl2 and Iso+zacopride +chloroquine groups.The surface area and the [Ca2 +]i of cardiomyocytes were recorded using a laser confocal scanning microscope,and apoptosis was measured by FCM?flow cytometry?.Results:Part 11.In Iso group,compared with control group,the whole heart hypertrophic index and left ventricular hypertrophic index were increased significantly?P< 0.01?.Patch clamp data suggested that the RMP of ventricular myocytes is reduced,action potentials duration?APD?was obviously prolonged.Zacopride treatment obviously retarded myocardial hypertrophy which compared favorably with captopril.HE and Masson's staining showed that myocardial fibers were disordered and thickening with collagen deposition on the extracellular matrix and cardiac fibrosis.Patch clamp data showed that zacopride increased the RMP and shorten APD in Iso stressed cardiomyocytes?P< 0.01?.Application of low-dose I_K1 atagonist chloroquine can reverse the effect of zacopride?P< 0.01?.2.In cultured neonatal cardiomyocytes,Iso increased the cell area and [Ca2 +]i.Zacopride treatment restored the hypertrophic morphology of cells to normal or near normal levels,and significantly attenuated calcium overload.I_K1 blocker Ba Cl2 or chloroquine reversed the effect of zacopride.3.The long-term use of zacopride had no obvious effect on ICa-L in normal rats.4.Western blot analysis demonstrated that the expression of Kir2.1,SERCA 2 were downregulated,while cleaved caspase-3 and phospho-Ca MKII were upregulated.Zacopride treatment significantly reverses the maladaptive expresson of p-Ca MKII and activated caspase 3,restores Kir2.1 and SERCA 2.Application of low-dose I_K1 atagonist chloroquine can reverse the effect of zacopride.Part 21.In cultured neonatal cardiomyocytes,Iso increased the cell area and [Ca2 +]i?P<0.01?.Zacopride treatment restored the hypertrophic morphology to normal or near normal level,and significantly attenuated calcium overload?P< 0.01?,2.I_K1 blocker-Ba Cl2 or chloroquine can reverse the effect of zacopride?P< 0.05?.3.Reduced apoptosis in zacopride-treated cardiomyocytes was demonstrated by the FCM?flow cytometry?.Conclusions:As an I_K1 agonist,zacopride significantly inhibits left ventricular remodeling induced by isoproterenol.Via enhancing I_K1,thus increasing RMP and shortening APD,zacopride might decrease Ca²⁺influx and inhibit intracellular calcium overload and resultant Ca²⁺-dependent remodeling signaling or apoptosis.