| Background and objective:Nowadays there have two strategies to construct biological pacemaker:gene-based and cell-based.The former method employs single or combined gene transfer interventions which were designed based on characteristic ion channel of sinoatrial node?SAN?cells or critical transcriptional factor for embryonic SAN.One of the most favorable gene-based strategies is the overexpression of hyperpolarization-activated cyclic nucleotide-gated?HCN?channels,this method can generated the pacemaker current If which strongly respond to autonomic stimulation.This kind channels generate inward currents during diastole avoiding interferer of the action potential.The T-box gene?Tbx?family is the main regulators of myocardial proliferation and patterning.Transcriptional regulators,such as Tbx3,Tbx5,and Tbx18,play critical roles in embryonic sinoatrial node?SAN?development,upstream of these factors is Tbx18.One ingenious study induced ventricular cardiomyocytes into pacemaker-like cells using single Tbx18 showed a encouraging results for construction of biological pacemaker.The latter method adopts cell-based approaches which include two aspects:conversion of cells into SAN-like pacemaker?iSAN?cells directly and utilization cells as a gene delivery vehicle.As the ideal stem cells for biological pacemaker therapy,bone marrow-derived mesenchymal stromal stemcells?MSCs?have the advantage of immune privilege,are electrically quiescent.They can couple with adjecent cells through gap junction easily.MSCs have the potential to directly differentiate into cardiomyocytes,but this cardiac cellular phenotype differentiation potential is weak.c-kit+is a marker of cardiac progenitors,and expressing in MSCs.Canine bone mesenchymal stem cells?cMSCs?,as well as cardiomyocytes,are derived from early mesoderm,becoming committed to their fate under the influence of different differentiation factors.Therefore,we hypothesized that the overexpression of Tbxl8 could induce c-kit+cMSCs to iSAN cells.We examined whether the overexpression of Tbx18 can induce the differentiation of c-kit+ cMSCs into a phenotype similar to that of native pacemaker cells and whether these transfected cells can couple to adjacent atrial cells with functional consequences in co-culture condition.The electrical coupling of heart is mediated by gap junctions.Transmembrane channel clusters composed of connexin?Cx?form gap junctions.Based on different types and arrangement of connexins,there are four kinds:homometic homotypic,hetermetic homotypic,homometic heterotypic and hetermetic heterotypic.Gap junctions provide low-resistance pathways for electrical impulse propagation through allowing small molecules and ions to diffuse along their electrochemical gradient.In human,there have already discovered twenty-one connexins which have different biophysical properties including conductance,voltage,PH and selective permeation of ions and small molecules?fluorescent dyes?.The number of connexins and activity of channels composed of connexins determine cell-cell coupling which strictly governed by upstream transcription factors.So far,four connexins are found express in the heart:Cx43,Cx40,Cx45 and Cx37.Differential existence of these connexins is thought to confer distinctive passive electrical properties to different regions of the heart.In a wide range of mammalian species,Cx45 which is the most sensitive connexin to voltage changes of gap junctions is widely established as the principal connexin of SAN cells.When the cytoplasm is negative compared to the surrounding cell potential,the Cx45 channel will be closed.This function effectively blocks the depolarization current reentry to SAN from adjecent myocardium and avoids disturbing the conduction order of normal electrical impulses.Studies in vivo had only transfected MSCs with HCN4 vector shown that this kind biological pacemaker generated the spontaneous rate significantly lower than that in normal SAN.This research employed connexin ?7?GJA7?gene encoding Cx45 and HCN4 gene to investigate whether co-transfected HCN4 and GJA7 genes could promote the HCN4 transfected cMSCs differentiation toward native pacemaker cells.Injection of HCN2 overexpressed MSCs into left ventricular free wall of atrioventricular block dogs can gain the pacing frequency satisfying physiological needs and responsiveness to catecholamine.However,the pacing function decayed after eight weeks of injection.This phenomenon was due to migration of the transplanted cells because of no rejection and apoptosis.Therefore,using biocompatible materials to enwrap and fix the cells is one of the current research focuses.Chitosan/?-glyceral phosphate disodium thermosensitive hydrogel can transform solution to gel at 37? and has less chemical crosslink toxicity.Aim to facilitate the future research in vivo,this research chose chitosan/?-glyceral phosphate disodium thermosensitive hydrogel which is an injectable biopolymer to enwrap the transfected cMSCs for evaluating whether this biopolymer can be used as cell delivery vehicles to provide a suitable matrix environment for cells survival and retention.Methods:1.Culturing cMSCs and sorting of c-kit+cMSCs.Canine mesenchymal stem cells?cMSCs?were first isolated and cultured.Part of the third passage cells were used for flow cytometric live cell sorting,the others were normal cultured and passaged.After the c-kit+cMSCs were sorted,CD45/CD29/CD44 were tested using flow cytometric analysis.2.Construction of nTbx18 lentiviral vector and transfected c-kit+cMSCs;construction of hHCN4 and hGJA7 lentiviral vectors simply transfected or co-transfected cMSCs.2.1 The lentiviral vector of pLV[Exp]-Puro-EF1A>hTBX18[NM00108050 8.2]:T 2A:{EYFP} was constructed by Gateway recombination cloning using Gateway-adapted vector as an experimental group.The lentiviral vector of pLV[Exp]-Puro-EF1A>?EYF P} was constructed as a control group simultaneously.The c-kit+ cMSCs were transfec ted with pLV-hTbx18-EYFP or pLV-EYFP respectively to gain Tbx18-EYFP-c-kit+cM SCs or EYFP-c-kit+ cMSCs.Pre-experiments determined the optimal multiplicity of inf ection and optimal concentration of polybrene.After 4 days of infection,RT-PCR veri fied the hTbx18 overexpression in Tbx18-EYFP-c-kit+cMSCs.2.2 The lentiviral vector of pLV[Exp]-EGFP:T2A:Puro-EF1A>hHCN4[NM005477.2]and pLV[Exp]-mCherry:T2A:Puro-EF1A>hGJC1[NM005497.3]were respectively constructed by Gateway recombination cloning using Gateway-adapted vectors as experimental groups.The lentiviral vectors of pLV[Exp]-EGFP:T2A:Puro-Null was also constructed simultaneously as a control group.Pre-experiments determined the optimal multiplicity of infection and optimal concentration of polybrene.In co-transfected condition,The HCN4-cMSCs were first purified five days using 2.0ug/mL puromycin,then added with pLV[Exp]-mCherry:T2A:Puro-EF1A>hGJC1.Through above mentioned procedure,we gained Tbx18-EYFP-c-kit+cMSCs and his control group EYFP-c-kit+cMSCs;HCN4-GFP-cMSCs,GFP-cMSCs,GJA7-RFP-cMSCs and HCN4+GJA7-cMSCs groups.3.Preparation of canine SAN cells and atrial cells and co-culture conditions in vivo.The canine SAN cells were isolated and cultured as suspending cells,these fresh cells were used as positive control group and tested within 6 hours after isolation.The atrial cells were isolated from right atriums of neonatal dogs,digested and cultured with differential attachment and 5-BrdU treatment.In co-culture condition,above mentioned transfected cells were respectively mixed with atrial cells and plated at a ratio of 1:4?20%c-kit+cMSCs?after atrial cells cultured 24 hours.4.Functional testing of transfected cells in each group.4.1 After 4 days of transfection,Tbx18-EYFP-c-kit+cMSCs and EYFP-c-kit+cMSCs were respectively collected to test the specific characteristics of SAN cells which compared with that of neonatal dogs' SAN cells;Tbx18-EYFP-c-kit+cMSCs and EYFP-c-kit+cMSCs were respectively co-cultured with neonatal dogs' atrial cells for three days.Using whole-cell patch-clamp technique and Lucifer fluorescent yellow dye test,the If currents of each group were recorded and cell-cell coupling of each group were evaluated,both compared with that of neonatal dogs' SAN cells.4.1.1 Intracellular cyclic adenosine monophosphate?cAMP?assay:used to determine cAMP levels in canine SAN cells,Tbx18-EYFP-c-kit+cMSCs and EYFP-c-kit+cMSCs.4.1.2 RT-qPCR:evaluated the relative mRNA levels of Cx45,Kir2.1,PLB and a-actinin in SAN cells,Tbx18-EYFP-c-kit+cMSCs and EYFP-c-kit+cMSCs.4.1.3 Immunoblotting?WB?:used to test the HCN4,Cx45,PLB,p-PLB,and a-actinin protein expression in SAN cells,Tbx18-EYFP-c-kit+cMSCs and EYFP-c-kit+cMSCs.4.1.4 Immunofluorescence:used to test the Cx45 immunofluorescence image of SAN cells,Tbx18-EYFP-c-kit+cMSCs and EYFP-c-kit+cMSCs.4.1.5 Electrophysiological effects of transfected cells under co-culture conditions:using whole-cell patch-clamp technique and perforated whole-cell patch-clamp technique to record the functional If currents of each group according to activation and inactivation voltage protocol in order to test the activation and inactivation features of these voltage-gated ion channels and test the isoproterenol stimulation response.Dye transfer experiment to evaluate the gap junction condition.4.2 HCN4-GFP-cMSCs,GFP-cMSCs,GJA7-RFP-cMSCs and HCN4+GJA7-cMSCs were respectively co-cultured with neonatal dogs' atrial cells for three days.Adding 2.0 ug/mL puromycin 5 days to clear atrial cells,cells of each group were respectively collected to test the specific characteristics of SAN cells.Using whole-cell patch-clamp technique,the characteristic If currents of each group were recorded.4.2.1 RT-qPCR:evaluated the relative mRNA levels of Cx45,Cx43,Tbx3,Tbx18 and gene coding homeobox protein Nkx-2.5?Nkx2.5?in HCN4-GFP-cMSCs,GFP-cMSCs,GJA7-RFP-cMSCs and HCN4+GJA7-cMSCs.4.2.2 Whole-cell patch-clamp technique:used to compare the recorded characteristic If currents of each group according to activation and inactivation voltage protocol.5.Transfected cells and thermosensitive hydrogel co-cultured and function evaluation.5.1 Extraction experiment and Cell Counting Kit-8?CCK-8?test:used to evaluate the cytotoxicity of thermosensitive hydrogel.The pure medium containing no cells was used as blank control group,the medium containing 0.64%naphthol was used as a positive control group,the pure medium without extract was used as a negative group.Each group set 6 multiple pores besides the experiment groups.5.2 CCK-8 test:used to evaluate the proliferation of transfected cells in thermosensitive hydrogel.After 72 hours of transfection,Tbx18-EYFP-c-kit+cMSCs,HCN4-GFP-cMSCs,GJA7-RFP-cMSCs were co-cultured with thermosensitive hydrogel respectively.cMSCs,Tbx18-EYFP-c-kit+cMSCs,HCN4-GFP-cMSCs,GJA7-RFP-cMSCs used as control groups,each group set 6 multiple pores besides the experiment groups.CCK-8 reagent were tested on 1,4 and 7day respectively to observe the cell proliferation in gel.5.3 Laser confocal microscope image:used to observe the shape of Tbx18-EYFP-c-kit+cMSCs,HCN4-GFP-cMSCs,GJA7-RFP-cMSCs in gel after co-cultured with thermosensitive hydrogel 4 days.5.4 iCelligence system real-time test:used to evaluate the real-time growth of Tbx18-EYFP-c-kit+cMSCs,HCN4-GFP-cMSCs,GJA7-RFP-cMSCs in gel.cMSCs,Tbx18-EYFP-c-kit+cMSCs,HCN4-GFP-cMSCs,GJA7-RFP-cMSCs used as control groups.Result:1.Flow cytometric live cell sorting was used to separate c-kit+from c-kit-cells.Post-sort analysis indicated a purity of c-kit+cells>90%with minimal cell death?<10%?.The c-kit+cMSCs expression of CD29,CD44 and absence of CD45.2.The c-kit+cMSCs were respectively transfected with pLV-hTbx18-EYFP or pLV-EYFP 24 hours in the presence of 5 ?g/mL of polybrene at a multiplicity of infection?MOI?of 100.At 48 hours after transfection,the infected c-kit+cMSCs exhibited yellow fluorescence.The transfection rates of pLV-hTbx18-EYFP-c-kit+cMSCs and pLV-EYFP-c-kit+cMSCs were 94± 3.5%and 96±2.5%respectively,which were analyzed by laser confocal microscopy in six different random fields.RT-PCR analysis confirmed the designed TBX18 primer based on detection of the TBX18 gene in Tbx18-c-kit+cMSCs.The cMSCs were respectively transfected with pLV-hHCN4-GFP,pLV-GFP,pLV-hGJA7-RFP 24 hours in the presence of 6?g/mL of polybrene at a multiplicity of infection?MOI?of 50.At 72 hours after transfection,the infected cMSCs exhibited green,green and red fluorescence repectively.The transfection rates of above mentioned three transfected cells were all>90%,which were analyzed by fluorescence microscopy in six different random fields.In co-transfected condition,The HCN4-GFP-cMSCs added with pLV-hGJA7-RFP in the presence of 6?g/mL polybrene at a MOI of 50.Using 2.Oug/mL puromycin purified five days,the co-transfected cells exhibited yellow fluorescence.The transfection rate was>95%,which were analyzed by fluorescence microscopy microscopy in six different random fields.3.The fresh isolated primary SAN cells mostly showed slender hook,irregular rh ythmic beating.The fresh isolated primary atrial cells were totally adherent growth wit hin 24 hours,showed long rod-shape.Mixture of atrial cells and Tbx18-EYFP-c-kit+c MSCs or EYFP-c-kit+cMSCs or HCN4-GFP-cMSCs or GFP-cMSCs or GJA7-RFP-cM SCs or HCN4+GJA7-cMSCs at a ratio of 4:1?80%atrial cells?.The cell mixtures we re co-cultured for three days as isotropic monolayers with the medium changed every two days.4.Tbx18-EYFP-c-kit+cMSCs recapitulate the critical features of genuine SAN pac emakers.Intracellular cAMP level of Tbx18-EYFP-c-kit+cMSCs was significantly higher compared to EYFP-c-kit+cMSCs?n=3?,reproducing the higher intracellular cAMP level observed in SAN cells?n=3?.Relative mRNA levels of Cx45,Kir2.1,PLB,a-actinin comparing Tbx18-EYFP-c-kit+cMSCs showed similar pattern of normalized transcript levels of SAN cells.HCN4,Cx45,PLB,p-PLB,a-actinin protein expression levels of Tbx18-EYFP-c-kit+cMSCs alike SAN cells were significantly different with EYFP-c-kit+cMSCs.Among them,HCN4 and Cx45 protein expression in Tbx18-c-kit+cMSCs were 12-fold and 5.6-fold higher,respectively,than that in EYFP-c-kit+cMSCs.Cx45 immunofluorescence image showed the Tbx18-c-kit+cMSCs resembled native SAN cells,which had strong positive Cx45 expression compared to EYFP-c-kit+cMSCs.After co-cultured with canine atrial cells for three days,the electrophysiological characteristics were tested.The trend of typical If currents in Tbx18-EYFP-c-kit+cMSCs and SAN was highly similar,which reached statistical significance?I=-89.368+82.306 V-19.975 V2+0.868 V3,F=693.056,Sig.<0.01,R2=0.983 vs.I=-110.892+102.927 V-26.637 V2+1.204 V3,F=649.977,Sig.<0.01,R2=0.982?.The differences of V1/2 between Tbx18-EYFP-c-kit+cMSCs and SAN cells reached statistical significance?-82.5±4.6,n=22 vs.-77.216.3 mV,n=18;P<0.05?,but the k did not?-7.7±0.4,n=22 vs.-8.1±0.2 mV,n=18;P=0.573?.Tbx18-c-kit+cMSCs have a more positive reverse potential compared with SAN cells?-18.41±1.2 mV,n=12 vs.-23.19± 1.6 mV,n=10;P<0.05?.The activation time constant of Tbx18-EYFP-c-kit+cMSCs ranged from 660±27 ms at-120 mV to 1880± 103 ms at-80 mV,and the deactivation time constant of Tbx18-EYFP-c-kit+cMSCs was 1000±99 ms at-70 mV to 210± 19 ms at-40 mV.Given 3?mol/L isoproterenol,the average activation time of If?upon steps to-120 mV?decreased by 26±2%?n=8?.Additionally,the V1/2 was shifted approximately 6.1 mV?from-84.9±5.6 to-78.8±4.8 mV,n=8;P<0.05?toward the direction of depolarization.After 5 minutes of dye diffusion,two neighboring atrial cells of a Tbx28-EYFP-c-kit+cMSCs were also fluorescent whereas no neighboring atrial cells of EYFP-c-kit+cMSCs were fluorescent.And the other cell of SAN cells pair was fluorescent.HCN4+GJA7-cMSCs promotes HCN4-GFP-cMSCs or GJA7-RFP-cMSCs to differe ntiate into iSAN cells.In co-cultured condition,RT-qPCR results showed GJA7-RFP-cMSCs increased the expression of Tbx3?n=8;p<0.05?,reduced the expression of Cx43,Nkx2.5 compared to GFP-cMSCs?n=8;p<0.05?.The relative mRNA level of HCN4 and Tbx18 had no significant differences between GJA7-RFP-cMSCs and GFP-cMSCs?n=8,p>0.05?.HCN4+GJA7-cMSCs increased the expression of HCN4,Cx45,Tbx3?n=8,p<0.05?,reduced the expression of Cx43,Nkx2.5?n=8,p<0.05?compared to HCN4-GFP-cMSCs or GJA7-RFP-cMSCs.The relative mRNA level of Tbx18 had no significant difference between HCN4+GJA7-cMSCs and HCN4-GFP-cMSCs or between HCN4+GJA7-cMSCs and GJA7-RFP-cMSCs?n=8,p>0.05?After co-cultured with canine atrial cells for three days,the whole-cell patch-clamp technique was used to record the characteristic of If currents.There was no If currents elicited from GFP-cMSCs and GJA7-RFP-cMSCs.Compared with HCN4-GFP-cMSCs,the If current amplitude of HCN4+GJA7-cMSCs increased?-1173.8± 18.4 vs.-1057.9± 14.6 mV,n=3;p<0.05?,the current activation curve shifted to the right,the half-maximal activating voltage changed to-92.6±3.6 from-101.9±4.0 mV?n=3;P<0.05?and the absolute values of If channel reversal potential increased?-29.6±3.9 vs.-26.8±3.2 mV,n=3;p>0.05?5.On the seventh day of extraction experiment,the value of optical density?OD?in 12.5%,25%,50%,100%extraction solution group were 4.6247±0.099,4.5911 ±0.101,4.0057±0.087,4.5144±0.088 respectively.The relative growth rate?RGR%?of all tested groups were above 90%showing 0?I grade cytotoxicity.CCK-8 showed the value of OD in Tbx18-EYFP-c-kit+ cMSCs and gel co-cultured group on Dayl,Day4,Day7 were 3.6754±0.10501,4.3294±0.20432,3.7682±0.41215 respectively;the value of OD in HCN4-GFP-cMSCs and gel co-cultured group on,Day4,Day7 were 3.2820±0.00686,4.4947±0.23174,4.3849±0.43016 respectively;the value of OD in GJA7-RFP-cMSCs and gel co-cultured group on Day 1,Day4,Day7 were 3.0181±0.31104,4.1634±0.26420,4.1348±0.36887 respectively,all transfected cDaylells grew in gel for four days to reach the peak of proliferation,and about seven days,the trend of growth tend to be stable.Tbx18-EYFP-c-kit+ cMSCs,HCN4-GFP-cMSCs,GJA7-RFP-cMSCs were co-cultured respectively with thermosensitive hydrogel four days,laser confocal microscope image displayed yellow or green or red fluorescence cells grew in different layers of gel.iCelligence system tested showed the mean cell indexes of all transfected cells co-cultured with thermosensitive hydrogel were no significant change at different time point.Conclusion:1.The overexpression of Tbx18 in c-kit+ cMSCs induces their differentiation to SAN-like pacemaker cells.2.HCN4+GJA7 co-transfected cMSCs promoted the differentiation of HCN4-GFP-cMSCs into pacemaker-like cells through enhancing the amplitude of If current and facilitating the propagation of If current3.Thermosensitive hydrogel provide a beneficial environment for biological pacemaker survival and growth. |
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