I_K1 Agonist Alleviates Ventricular Remodeling Via Regulating Calcium Homeostasis And Autophagy In Rats

ObjectiveThe purpose of this study is to testify the hypothesis that enhancement of inward rectifier potassium channel(I_K1)currents is a compensation for I_K11 deficit and a novel modulation for cardiac Ca²⁺homeostasis and autophagy in pathological remodeling in a time course rat model.MethodsHealthy Male SD rats weighing 200-220 g were randomly divided into three batches.Isoproterenol（Iso）was administered by intraperitoneal injection（i.p.）once a day for 3,10and 30 days,respectively,to establish temporal cardiac remodeling,they were feed for 3days,10 days and 30 days,respectively.Each batch was set up with Control group,Iso(3mg·kg^-1)model,Iso+Zac(15μg·kg^-1)and Iso+Zac+Chlo(7.5μg·kg^-1).Age-matched control rats were administered with the same volume of saline.In addition,for the purpose of exclusion the potential effects of 5-HT receptors and agents per se,we set the groups of Zac alone,Chlo2alone,Iso+Chlo,Iso+Zac+RS23597(0.27mg·kg^-1·d^-1)2and Iso+Zac+m-CPBG(0.19mg·kg^-1·d^-1)except the groups mentioned above in 10 days.Echocardiography was used to detect the left ventricular diastolic diameter（IVIDd）,left ventricular systolic diameter（LVIDs）,interventricular septal thickness（IVS）,left ventricular posterior wall thickness（LVPW）and left ventricular ejection fraction（EF）,left ventricular short axis shortening rate（FS）.Standard western blotting were performed using respective antibodies to quantify the relative levels of inward rectifying potassium channel protein1subunits2Kir2.1（KCNJ2）,the ratio of phosphorylated ryanodine2receptor 2（p-RYR2）/RYR2,the2radio2of2 phosphorylated Ca²⁺/calmodulin-dependent protein kinase II（p-CaMKⅡ）/CaMKⅡ,SEARCA2 and cleaved caspase 3.Besides,changes of expression of autophagy related proteins LC3B and P62 were detected.Confocal microscopy was used to observe intracellular calcium stained by Fluo-4 and sarcoplasmic reticulum Ca²⁺(SR Ca²⁺)stained by Fluo-5/N in adult rat cardiomyocytes.Histomorphology were used to survey changes in cardiomyocytes,such as cell structure,cross-sectional area and collagen deposition.Result1.The mortality rate of Iso-modeled rats daily Iso infusion for 3 d,10 d and 30 d led to sudden death in some rats,the mortalities rates were 40%,41.7%（P<0.01 vs.control）and50%（P<0.05 vs.control）respectively.In Zacopride treated groups,the mortalities were16.7%,23.8%and 30.0%for 3 d,10 d and 30 d respectively,but did not reach a statistical significance compared with the Iso group.Co-application of cZacopride and Chloroquine(I_K11 channel blocker)increased the mortalities respectively to 30%（3d）,37.5%（10 d,P<0.01 vs.control）and 58.3%（30 d,P<0.05 vs.control）.All control rats,rats treated with Zacopride alone or Chloroquine alone survived in the entire period of the experiments.These results indicate that 15μg/kg Zacopride and 7.5μg/kg Chloroquine per se had no significant toxicity to the rats.2.Post 3 days of Iso infusion,IVSd and IVSs were increased（P<0.01）,LVIDd（P<0.05）and LVIDs（P<0.01）were reduced compared with age-matched control rats（Table 2and Figure 3 A-B）.Ten days after Iso treatment,IVSd（P<0.01）,IVSs（P<0.01）and LVPWd（P<0.05）were increased,while LVIDs and LVIDd had no significant differences compared with age-matched control.LVEF and LVFS were increased both in 3 days（P<0.05）and 10 days（P<0.01）Iso groups compared with age-matched controls.Zacopride treatment prevented the thickening of interventricular septum and the decrease of LV volume（P<0.01 or P<0.05）.Thirty days after Iso treatment,LVIDd（P<0.05）and LVIDs（P<0.01）were significantly increased,LVEF and LVFS（P<0.01）were decreased compared with age-matched control.The thickness of IVS was decreased compared with that in 3 day and 10 days Iso groups（P<0.01）.Collectively,these results indicated that longer（30 days）Iso exposure led to progression of cardiac pathological remodeling,dysfunctions and even failure.Zacopride treatment prevented LV chamber dilatation（P<0.05）and preserved the systolic function（P<0.01）.These effects were largely reversed by I_K11 channel antagonist Chloroquine（P<0.01 or P<0.05）.3.The results of Western blot showed that the expression of p-RYR2/RYR2 and p-CaMKⅡ/CaMKⅡincreased in the Isoproterenol group,SERCA2 declined compared with the control group in 10 and 30 days（P<0.01）,but no obviously changes in 3 days.The expression of Kir2.1 was significantly reduced（P<0.01）.In addition,the cleaved caspase3 enhanced（P<0.05 or P<0.01）.These changes could be effectively reversed by Zacopride（P<0.05 or P<0.01）.Chloroquine could block the consequence of Zacopride（P<0.05 or P<0.01）.Compared with control,Zacopride treatment（15μg/kg/d）alone for 10 days upregulated Kir2.1 channel protein by 48.6%（P<0.01）,while Chloroquine（7.5μg/kg/d）treatment alone for 10 day decreased Kir2.1 channel protein level by 44.4%（P<0.01）.Co-application of RS23597-190（5-HT₄R antagonist）or m-CPBG（5-HT₃R agonist）could not counteract the effect of Zacopride on Kir2.1 expression.These data show that Zac can adjust Ca²⁺-related protein by agitated I_K1which recover calcium homeostasis.In the time course models,compared with age-matched control LC3B expression was increased in the model group（P<0.01）,but was downregulated after treatment with Zacopride.The expression of P62 declined during myocardial hypertrophy（P<0.05 or P<0.01）and upregulated after administration of 1Zac.Chloroquine could inhibit the effect of Zacopride.4.The results of intracellular calcium stained by Fluo-4 and sarcoplasmic reticulum calcium stained by Fluo-5/N in the 3,10,and 30 days groups showed the same trend.After Isoproterenol treatment,intracellular calcium increased（P<0.01）compared to sarcoplasmic reticulum calcium（P<0.05）,the circumstances improved after Zac-treated,overloading calcium in intracellular declined（P<0.01 or P<0.05）.The intensifyed of reticulum calcium concentration relieves the intracellular calcium overload.The I_K11 blocker Chloroquine can inhibit the effect of Zac.5.Histomorphological examination depicted that compared with controls,the cardiac myofibers in Iso treated rats are disorganized and hypertrophic,with certain degree of cell necrosis and relatively light staining of the cytoplasm.In Zacopride-treated rats,cardiac myofibers are better arranged and with normal size.I_K11 channel blocker Chloroquine abolished these protective effects from Zacopride as shown by worsened manifestation,indicating that the anti-remodeling effect of Zacopride is mediated by the activation of I_K1channels.After 10-days Iso infusion,rat hearts exhibited significant fibrosis,validated by increased collagen deposition（P<0.01 vs.control）.Zacopride treatment dramatically attenuated the fibrosis（P<0.01）,and this effect was largely abolished by Chloroquine（P<0.01）.Zacopride（15μg/kg）or chloroquine（7.5μg/kg）per se had no significant effects on cardiac structure or function.5-HT₄ receptor antagonist RS23597-190 and 5-HT₃receptor agonist m-CPBG could not counteract the effects of Zacopride on cardiac remodeling including changes of hypertrophy and fibrosis,suggesting that the protective effects of Zacopride on LV remodeling is mediated by I_K11 channel but not by 5-HT receptors.Conclusion1.By enhancing cardiac I_K1,Zacopride prevents Iso-induced electrical remodeling around intracellular Ca²⁺overload,thereby attenuates cardiac structural disorder and dysfunction.2.Autophagy is involved in the cardioprotection of Zacopride on cardiac remodeling.