Anti-ventricular Remodeling Effect And Mechanism Of The Inward Rectifying Potassium Channel(I_K1)-Specific Agonist Tetramisole

Objective:By drug repurposing strategy and molecular docking,tetramisole,an anthelminthic agent,is to be identified as a potential specific I_K1agonist,and showed promising protection on ventricular arrhythmias and remodeling.The study will provide a key preclinical proof in translating the I_K1agonist from the benchtop to the bedside.Methods:1.The whole-cell patch clamp technique was applied to identify whether tetramisole is a selective I_K1agonist comparing its potential influences on other channels or transporters,such as voltage-gated Na⁺channel(I_Na),L-type Ca⁺channel(LTCC,I_Ca-L),transient outward K⁺channel(I_to),sustained outward K⁺current(I_Ksus),and Na⁺-Ca²⁺exchanger(I_NCX).2.Molecular docking technique was used to predict the binding of Tet to predominant subunits of ventricular ionic channels.The crystal structure of Kir2.1（PDB ID 1U4F）,pore-forming isoforms Kv4.3/4.2 underlying I_tofast component currents（PDB ID 2I2R and 7F0J,respectively）,and the voltage-gated sodium channel Nav1.5underlying I_Na（PDB ID 6UZ3）were downloaded from RCSB Protein Data Bank.There is not appropriate crystal structure of Cav1.2（voltage-dependent L-type calcium channel subunit alpha-1c）in PDB,we applied Alpha Fold protein structure database（Alphafold Q13936）.The 3D structure of tetramisole was downloaded from NCBI Pub Chem Compound.The molecular docking study was performed using Autodock 4.0 or Glide.3.Induction of cardiac hypertrophy and remodeling Cardiac hypertrophy was induced by daily injection of isoproterenol（Iso,3 mg/kg/d,Sigma,USA）for 10 days in rats in vivo.Pharmacological treatments were as follows:Iso,tetramisole（Tet,0.54mg/kg/d,i.p.）,Iso+Tet,Iso+Tet+CQ（7.5μg/kg/d,i.p.）.Control rats were administered with the same volume of saline.The dose of Tet and CQ were applied according to our previous study¹³and preliminary experiment.Ventricular remodeling were evaluated by echocardiography and histology.GE Vivid 7 Pro ultrasound system（10S probe,probe frequency 8.0 MHz,with 2D strain imaging software and Echo PAC workstation）in M mode for rodent heart.Approximate probing angles were 15-30°,depths of 2-3cm,frame rates>250/s and maximum frame rates up to 400/s.Localization criteria were LV long-axis views.Parameters measured included LV dimensions in end-diastole（LVIDd）and end-systole（LVIDs）,septal thickness in end-diastole（IVSd）and end-systole（IVSs）,LV posterior wall thickness in end-diastole（LVVPWd）and end-systole（LVVPWs）,and LV ejection fraction（EF）and LV short-axis fractional shortening（FS）.LV samples from all groups were fixed in10%phosphate-buffered formalin and subjected to routine histological processing.Transverse LV sections（5μm thick）were cut with a frozen sectioning machine.After hematoxylin and eosin（HE）staining,the cross-sectional area of myofibers（×400 magnification）was measured with a microscope（Olympus,Tokyo,Japan）under high magnification field of view（HPF）.Fibrosis was assessed by Masson’s trichrome staining,and the collagen content of the interstitial space was estimated by analyzing the images of each group.The total collagen area was calculated and expressed as a percentage of the total ventricular area under HPF.4.ARVMs were isolated to establish an in vitro model of cardiomyocyte remodeling induced by Iso（1μmol/L）.The cells were randomly divided into four groups,Control,Iso（1μmol/L）,Iso+Tet（30μmol/L）,and Iso+Tet+Ba Cl₂（1μmol/L）,and the cellular[Ca²⁺]i was detected by laser confocal microscopy after agent treatment for 24 h.In parallel,ARVMs post well-recalcification were randomly subdivided into four groups,control,Iso（1μmol/L）,Iso+Tet（30μmol/L）,Iso+Tet+Ba Cl₂（1μmol/L）,and a 1-h pharmacological treatment was performed before calcium imaging.5.H9c2（2-1）cardiomyocytes were selected and an in vitro model of cardiac hypertrophy was established by applying isoproterenol（Iso）.H9c2 were randomly divided into 10 groups:Control group,Tet 1,10,30,100μmol/L,Iso（1μmol/L）,Iso+Tet（10μmol/L）,Iso+Tet（30μmol/L）),Iso+Tet10+Ba Cl₂（1μmol/L）,Iso+Tet30+Ba Cl₂.After 48 h of culture,cells were collected for Western Blot to detect the expression of Kir2.1 and SAP97,PKA,p-PKA,AKAP5,Kir6.1 and other proteins.In addition,cells were inoculated in 24-well plates or 35-mm confocal culture dishes and treated with the above drugs for 48 h.Cellular[Ca²⁺]_iwas detected by laser confocal microscopy.H9c2（2-1）cardiomyocytes were randomly divided into 10 groups:normal control,Tet 1,10,30,100μmol/L,Iso infusion（1μmol/L）,Iso+Tet（10μmol/L）,Iso+Tet（30μmol/L）,Iso+Tet10+Ba Cl₂（1μmol/L）,Iso+Tet30+Ba Cl₂.After 48-h incubation,cells were collected for Western blotting.Besides,cells were seeded in a 24-well plate or35mm plates,and treated with above mentioned agents for 48 h.The resting[Ca²⁺]_iof cardiomyocytes was detected by laser confocal microscopy.6.The intracellular Ca²⁺fluorescence in native ARVMs were indicated by dual-wavelength Ca²⁺indicator Fura-2 AM.The intracellular Ca²⁺fluorescence in cultured H9c2（2-1）cells were indicated either by single-wavelength Ca²⁺indicator Fluo-4 AM or by Fura-2 AM.Cells were incubated with 5μM Fluo-4 AM or 3μM Fura-2 AM in the dark at 37°C for 30 min.The loaded cells were then washed three times with Hank’s Balanced Salt Solution（HBSS）and kept in HBSS for another 30 min to allow de-esterification of calcium indicator in cell.The resting[Ca²⁺]_ifluorescence indicated by Fluo-4 was measured by an FV1000 laser scanning confocal microscope.The data was collected and analyzed with Fluo View 1.7a software.The Ca²⁺fluorescence indicated by Fura-2 was measured as fluorescence ratios（excitation at 340and 380 nm;emission at 510 nm）from single cells using an Olympus IX71 inverted fluorescence microscope and a Luca EMCCD camera,and collected at 2s intervals.The data were recorded and analyzed by Meta Fluor?Fluorescence Ratio Imaging System.7.Recombinant lentivirus The Open Reading Frame（ORF）of Kir2.1 was inserted to the MCS sites of p HBLV-CMV-MCS-3FLAG-EF1-Zs Green-T2A-PURO to generate lentiviral vector overexpression of Kir2.1（Hanbio Biotechnology,Shanghai,China）.The lentiviral vectors mediated Kir2.1 knockdown were generated by inserting three sh RNA sequences target for Kir2.1 respectively to p HBLV-U6-MCS-CMV-Zs Green-PGK-PURO（Hanbio Biotechnology,China）.To generate lentiviruses,the lentiviral vectors and packaging vectors were co-transfected into 293T cells.Cells were lysed after well-transfection,and extracts were subjected to Western blot analysis.8.Western blotting Proteins from samples of LV tissue or H9c2（2-1）cells were loaded on 12%acrylamide gels.After electrophoretic transfer,the nitrocellulose membranes were incubated overnight at 4°C with primary antibodies against Kir2.1,Synapse-associated protein-97（SAP97,DLG1）,phosphorylated protein kinase A（p-PKA）,PKA,Kir6.1,AKAP5,GAPDH.Quantification was performed using Image J.Results:1.Combined with the results of the group’s previous experiments,it was concluded that Tetramisole specifically increased I_K1currents in ARVMs without affecting other ion channels and sodium/calcium exchangers.2.Molecular docking was performed to predict the possible interaction between tetramisole and the most important ion channel protein contributing to ventricular action potentials.Tetramisole was found to formed a special S...O bond at residue Glu224 and an alkyl-πinteraction at residue Arg260 from Chain B of Kir2.1.Further docking study showed that a Pi-Pi interaction was observed between benzene ring of tetramisole and PHE 98 residue of Kv4.3,but there was no binding activity between tetramisole with Kv4.2,Nav1.5,or Cav1.2.3.Echocardiographic observations showed the typical features of concentric hypertrophy and enhanced pump function.After 10 days of Iso infusion,IVSd,IVSs,EF and FS were increased（P<0.01）;LVIDs（P<0.05）were reduced compared with control rats.Tetramisole treatment prevented septal thickening,increased LV volume（P<0.05）,and normalized cardiac pumping function（P<0.01）.the I_K1antagonist chloroquine largely reversed this effect（P<0.05）.h&e and Masson’s staining showed myocardial fiber disorder and hypertrophy in Iso perfused rats compared to controls,with some degree of cell necrosis and relatively lighter staining of the cytoplasm.In tetraimidazole-treated rats,myocardial fibers were better aligned and normal in size.The I_K1channel blocker chloroquine counteracted this protective effect,suggesting that the anti-remodeling effect of tetraimidazole was mediated by I_K1activation.After 10 days of Iso infusion,rat hearts showed significant fibrosis,as verified by increased collagen deposition.Tetramisole significantly attenuated fibrosis（P<0.01）,and this effect was largely abolished by chloroquine（P<0.01）.4.In H9c2（2-1）cardiomyocytes,the Ca²⁺imaging data showed that 1μmol/L Iso induced electrical remodeling as evidenced by higher[Ca²⁺]_icompared to controls（P<0.01）.Tetramisole at 10 or 30μmol/L alleviated Iso-induced[Ca²⁺]_ioverload（P<0.01）,and the effect could be reversed by Ba Cl₂（P<0.01）,respectively.Notably,without Iso stimulus,tetramisole at 1-100μmol/L had no effect on intracellular[Ca²⁺]_i,suggesting that the protection of tetramisole against Ca²⁺overload is a compensation for Ca²⁺dyshomeostasis in remodeled cardiomyocytes.In isolated ARVMs,30μmol/L tetramisole showed significant cardioprotection on Iso-induced Ca²⁺overload whether by acute or chronic infusion（P<0.05 or P<0.01）,and the effect could be reversed by Ba Cl₂（P<0.01）.Pease see the detail in S1 Figure.5.Western blot results showed that Tet significantly upregulated SAP97 expression in normal H9c2（2-1）cardiomyocytes（P<0.01）,consistent with the upregulation of Kir2.1 expression.Iso downregulated Kir2.1（P<0.05）,and Tet at 10 or 30μmol/L normalized Kir2.1 expression（P<0.05 or P<0.01）.I_K1blocker barium chloride reversed this effect.Iso had no significant effect on SAP97,and b Ba Cl₂reversed the effect of Tet on SAP97（P<0.05）.Tet at 30μmol/L inhibited the activation of the PKA-AKAP signaling pathway,as shown by the decreasein PKA phosphorylation（p-PKA/PKA）,AKAP5expression compared with Iso-group（P<0.01）.These effects could be reversed by Ba Cl₂.Notably,in the absence of Iso-stress,1-100μmol/L tetramisole had no significant effects on the expression of AKAP5 and the phosphorylation of PKA.6.The interaction between SAP97 and Kir2.1 was further observed in H9c2（2-1）cells by knocking down or overexpressing Kir2.1 using lentiviral transfection.The results showed that compared with negative control,neither Kir2.1 knockdown nor Kir2.1overexpression had effects on the expression of SAP97.Conclusions:1.Tetramisole is a specific agonist of I_K1and binds specifically to Kir2.1 subunit.2.Tetramisole inhibits Iso-induced calcium overload and ventricular remodeling in rats.3.The cardioprotection of tetramisole was associated with the facilitation of I_K1channel forward trafficking,deactivation of PKA signaling,and inhibition of intracellular calcium overload.