| Background and objection:Life threatening rhythm disorders such as sick-sinus syndrome,completeatrioventricular nodal block often require the implantation of artificial electronicpacemakers.Although electronic pacemakers are certainly effective in their function, suchdevices are not optimal because they lack the biologic responsiveness of native tissues andpresent other shortcomings, such as lack of autonomic modulation, limited battery life, theneed for permanent catheter implantation into the heart, unstable electrode position, andelectronic and magnetic interference. For these reasons, several approaches have beenexplored as potential alternatives.Gene-based methods include overexpression ofβ2-adrenergic receptors, overexpression of hyperpolarization-activated cyclic nucleotide(HCN)-gated channels, and down-regulation of K+currents. These gene-based methodsare typically accompanied by inherent problems, with both viral and non-viral approachesof gene delivery. Cell-based methods involve the transfer of spontaneously beatingcardiomyocytes derived from various types of stem cells which pose problems such as theimmunogenicity of the transplanted cells, the potential for neoplasia, identifyingappropriate pure cardiac lineages, spatial non-uniformity of implants, and the possibility ofdifferentiation into lines other than pacemaker cells. However, among the different stemcell types, marrow mesenchymal steml cells (MSCs) may be the most promising candidate.Bone marrow-derived MSC can be transfected by lentiviral vectors (LV). In addition, LVcan be integrated into the host genome for long-term transgene expression, which rendersthese vectors as ideal candidates for managing a chronic condition, such as sick sinussyndrome.Because funny channels (If) have a specific role in pace-making and do notdirectly affect phases of the action potential other than the diastolic depolarization,HCN-based biologic pacemakers appear to be the basis for a potentially successful approach. Implantation of MSCs carrying the HCN2gene into the left ventricular anteriorwall can create catecholamine-responsive biologic pacemakers that function stably for6weeks. The HCN-gated channel4(HCN4) gene is the predominant and most cyclicadvenosine monophosphato (cAMP)-responsive isoform in the sinoatrial node of the heart.HCN4gene-transduced myocytes result in an increase in spontaneous beating rate andresponsiveness to autonomic modulation. Thus the HCN4gene is an attractive candidate forthe development of a biologic pacemaker. We hypothesize that adult canine MSCs (cMSCs)transduced with the HCN4gene and transplanted into the heart may be capable of inducingin vivo heart pacing under proper conditions.With this study, we demonstrate that cMSC are effectively transfected by a lentiviralvector encoding a mouse HCN4(mHCN4) gene and an enhanced green fluorescent protein(GFP) gene and are capable of expressing functional mHCN4channels in vitro and in vivo.mHCN4gene expression in cMSC provides an If-based current sufficient to drive thecanine ventricle in vivo under proper conditions.Methods1. cMSCs were isolated from bone marrow using a novel method in which the mediumis changed frequently during the initial phase of culturing and the duration of trypsinizationis diminished over time. The cell dynamic morphologic changes were observed byphase-contrast microscopy.2. A cell proliferation potentia was performed using MTT cell proliferation assay, andfreshly isolated cells would be induced to differentiate into osteoblastic, adipocytic andchondrocytic lineages in appropriate medium.3. Immunophenotypes of these cells were analyzed by fluorescence-activated cellsorting (FACS) and immunofluorescence staining.4. cMSCs were transduced with a lentiviral vector encoding an GFP gene, the longterm transgene expression and the cell dynamic morphologic changes of cMSCs transfectedwith GFP were observed.5. cMSCs were transfected by pLenti6.3-HCN4-IRES2-EGFP vector and the controlvector pLenti6.3-IRES2-EGFP respectively, and the expression of GFP was observed byfluorescence microscope, and the cells stably expressed the gene of mHCN4after selected by FACS. The expression of mHCN4was detected by immunocytochemistry and wersternblotting. The cell dynamic morphologic changes were observed by phase-contrastmicroscopy.6. The kinetics characters of Iflike current channels were detected by whole-cell patchclamp, in order to determine expression of the mHCN4channels in cMSCs transfected withmHCN4gene.7. cMSCs transfected with either control plasmid or the mHCN4gene construct wereinjected subepicardially into the canine right ventricular wall in situ. Animals recovered for14days, during which their cardiac rhythms were monitored. Sinus arrest was induced bygraded right and left vagal stimulations performed via standard techniques.and their cardiacrhythms were monitored by multi channel electrophysiological signal recording system.The region of the injection was paced via an epicardial electrode and electrocardiograph(ECG) was recorded.8. The right ventricular myocardium containing the injection sites was stained withhematoxylin and eosin to determine the distribution and survival of cMSC in relation to theneedle tract and whether cMSC were associated with inflammation. The expressions ofCD3, caspase3and IgG antigen were detected by immunohistochemical methods. Theexpressions of mHCN4and GFP protein were detected by immunofluorescent technique.Result1. Purified population of fibroblast-like cells was achieved in the third passage usingthis method. These cells maintained both proliferation and mesenchymal differentiationpotentials.2. These cells were strongly positive for CD29and CD44, but negative for CD34andCD45, and they were easily transfected with a lentiviral vector, and stably expressed thetransgene over the long term.3. cMSCs were transfected with pLenti6.3-HCN4-IRES2-EGFP vector successfully,and the cells stably expressing the protein of mHCN4were selected by FACS.4. A hyperpolarization-activated inward current was recorded by whole-cell patchclamp in cMSCs transduced with mHCN4gene. The current had obvious time and voltagedependences, and it also was high sensitive to extrocelluar Cs+. Meanwhile, this current could not be observed in control groups under the same conditions. The mHCN4channelcurrent’s half-maximal activation was-91.5±9.2mV, A slope factor was about19.4±3.8mV, the reversal potential was-29.7±2.5mV。The mHCN4channel current density at–140mV was26.4±1.8pA/pF.5. Both two groups had not ventricular arrhythmias and significant differences in theheart rates after cMSCs were injected subepicardially into the canine right ventricular wallin situ.During sinus arrest, all control hearts had spontaneous atrioventricular node rhythms[rate21±5beats per minute (b.p.m.)]. In the mHCN4group, six animals developedspontaneous ventricular rhythms of right-sided origin (rate45±9b.p.m.). Pace mappingclose to the cMSC injection site in the right ventricle showed QRS features identical tospontaneous ventricular rhythms.Normal sinus rhythm was restored in all animals afterright and left vagal stimulations stopped.6. cMSC survived in the right ventricular myocardium containing the injection HCN4gene sites without inflammation. mHCN4and GFP protein were expressed in c transfectedwith mHCN4and GFP gene in the canine heart in vivo. Only GFP protein was expressed inc transfected with GFP gene in vivo. CD3, caspase3and IgG antigen were not expressed inthe injection sites.Conclusions1. Purified population of cMSCs was achieved in the early passage using this method.2. cMSCs can stably express the mHCN4protein and If-like current after transfectedwith mHCN4and selected by FACS.It shows they have the electrophysiological capabilityof cardiac pacemaker cells. The mHCN4protein and If-like current were not expressed inthe cMSCs transfected with GFP and control cMSCs.3. During sinus arrest, all control animals had spontaneous atrioventricular noderhythms, in the mHCN4group, and six of eight animals developed spontaneous ventricularrhythms of right-sided origin. The mHCN4group show significant increase in heart rate.Pace mapping indicated that the spontaneous ventricular rhythms were originated from theregion injected with cMSC transfected with mHCN4.Normal sinusrhythm was restored in all animals after right and left vagal stimulations stopped.4. The cMSCs transplanted in situ survived in the canine heart in vivo. the mHCN4 protein was expressed in only transfected with mHCN4.5. The injected regions of cMSCs transplanted demonstrated neither apoptosis norimmunologic rejection rejection at2weeks. |
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