| Background and objectionElectronic pacemakers are currently the primary treatment to sustain the heart rate forsick sinus syndrome or complete atrioventricular block. Although electronic pacemakers areeffective to treat these diseases and have already saved millions of lives, they are notoptimal because such devices have many shortcomings, such as lack of biologicalresponsiveness to autonomic neurotransmitter changes, unstable electrode position, limitedbattery life, severe infection, electronic and magnetic interference. For these reasons,biological pacemaker based on cell and gene therapy is becoming an important candidate totreat bradyarrhythmias.As one type of adult pluripotent stem cell, mesenchymal stem cells (MSCs) may be themost promising candidate. They can easily be acquired from bone marrow. The injectedMSCs can differentiate into cardiac myocytes after being implanted directly into themyocardium. In our recent experiments, transplanted canine MSCs carryinghyperpolarization-activated cyclic nucleotide-gated4(HCN4) genes could inducespontaneous ventricular rhythms in models of complete atrioventricular block byallografting into the host heart. However, the beating rate ranged from40to50bpm, whichwas markedly lower than that of MSCs transfected with HCN4co-cultured withcardiomyocytes in vitro. A high level of donor cells death may be one of the adversefactors.Some in vivo studies have demonstrated that the majority of cells implanted into theheart die within the initial2days following implantation and that acute inflammation mayplay an important role in donor cell death. Other studies have also shown that the number ofsurviving engrafted cells was limited to20–30%, possibly due to ischemia, apoptosis,inflammation, or immunological rejection. The therapeutic effects of stem celltransplantation into the heart are hindered by the poor survival of the implanted cells. So it is very important and necessary to find ways to promote the therapeutic effects byimproving the survival rate of grafted cells within this arduous microenvironment.Triptolide is a small molecule extracted from the traditional Chinese herbal plantTripterygium wilfordii Hook F (TWHF). Currently, triptolide has been shown to possessboth anti-inflammatory and immunosuppressive properties. Maybe triptolide can prolongthe survival of donor cells by its anti-inflammatory and immunosuppressive roles in vivo.This study was designed to investigate the role of the inflammatory response in acute celldeath after implantation into the myocardium and the effects of triptolide.Methods1. Under sterile conditions, male rat mesenchymal stem cells were isolated from bonemarrow of the bilateral femurs and tibias by gradient centrifugation and the character ofadherence to culture plates, and further purified and amplified by changing the mediumfrequently in vitro.2. The mHCN4lentiviral vector pLenti6.3-mHCN4-IRES2-EGFP was used totransfect rat MSCs. The infected rat MSCs were selected by using blasticidin and theexpression of mHCN4and EGFP protein were detected by using immunohistochemicalmethod and fluorescence microscope. Cell proliferation was assessed by the Cell-CountingKit-8(CCK-8) assay.3. Sex-mismatched cell transplantation models were used in our experiments. MSCswere isolated from male Sprague-Dawley (SD) rats and transduced with mHCN4(HCN4-MSCs) and then injected into syngeneic female rats. The total number of maleHCN4-MSCs grafted into the female heart was analyzed by quantitative reversetranscription polymerase chain reaction (RT-PCR) for the male-specific Y chromosome(Sry). Intact female hearts were collected and injected ex vivo with known cell numbers ofHCN4-MSCs (2.0×10~2，2.0×10~3，2.0×104，2.0×10~5and2.0×10~6). The samples wereanalyzed for the amount of Sry gene to calculate the numbers of HCN4-MSCs and to obtaina standard curve.4. According to the standard curve, different cell numbers of HCN4-MSCs wereinjected directly into the myocardium at one site to explore the optimal transplanted numberby quantitative RT-PCR for the male-specific Y chromosome (Sry). 5. All of the female rats were given intramyocardial injections containing the optimaltransplanted number of HCN4-MSCs, and the rats were randomly divided into three groups(n=30in each group):i) The triptolide group (Tri), in which rats were injected intraperitoneally (i.p.) with200μg/kg/d triptolide for10d (from day-3to7);ii) The dexamethasone group (Dex), in which the rats were injected i.p. with400μg/kg/d dexamethasone for10d (from day-3to7);iii) The control group (Con), in which rats were injected i.p. with sterile normal salinefor10d (from day-3to7).6. The rats in each group were divided into five subgroups according to different timepoints (24h,72h,7d,14d and28d) after cell transplantation (n=6in each subgroup). Theanimals were sacrificed at the indicated time points, the blood and the hearts werecollected.(1) The surviving number of male donor HCN4-MSCs in the female host heart wasestimated by the amount of Sry gene in the implanted area of each sample by referring tothe standard curve.(2) At different time points (24h and72h) after cell injection, enzyme linkedimmunosorbent assay (ELISA) and RT-PCR were used to measure the protein and mRNAlevels, respectively, of nuclear factor (NF)-κB and pro-inflammatory cytokines (IL-1β, IL-6and TNF-α).(3) The apoptosis of grafted HCN4-MSCs was tested by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) assay and apoptosis-regulatingproteins (Bax and Bcl-2) were analyzed by western blotting. The pacemaker current (If) ofthe donor cells was detected by using patch-clamp technique.(4) The rats were sacrificed28days after the transplantation of HCN4-MSCs. Theheart tissue was fixed in4%paraformaldehyde, and embedded in paraffin. Serial sectionswere cut at4μm. Immunohistochemical staining was done for cardiac troponin T (cTnT)and connexin43(CX43).Results1. The second passage of MSCs were attached to the culture flasks tightly and spread in the shape of spindles or polygons. Purified MSCs were positive for CD29and CD90, butnegative for CD45with the use of flow cytometry analysis.2. Rat MSCs were transfected with pLenti6.3-mHCN4-IRES2-EGFP vectorsuccessfully and the transfection efficiency were about67%at multiplicities of infection(MOIs) of10and over90%after antibiotic selection. The cells stably expressed both EGFPand HCN4protein by immunofluorescent staining. The cell proliferation assay by CCK-8showed that HCN4-MSCs exhibited significantly increased cell viability beforetransplantation.3. Intact female hearts were collected and injected ex vivo with known cell numbers(2.0×10~2,2.0×10~3,2.0×10~4,2.0×10~5and2.0×10~6) of the mHCN4-infected male MSCs,respectively. The samples were analyzed for the amount of Sry gene by quantitativeRT-PCR and obtained a standard curve.4. Sex-mismatched cell transplantation models were used in our experiments. Differentcell numbers of HCN4-MSCs were injected directly into the myocardium of syngeneicfemale rats. Grafted cell survival was analyzed by RT-PCR for the Sry gene from heartsharvested after72hours of cell transplantation by referring to the standard curve. We foundthat1×10~6may be the optimal transplanted number of engrafted HCN4-MSCs at one site.5. All of the rats were given intramyocardial injections containing1×10~6HCN4-MSCs.The numbers of surviving HCN4-MSCs injected were29.27±0.93%,17.55±1.24%,7.37±0.34%,4.15±0.38%and3.40±0.14%at24h,72h,7d,14d and28d in the Tri group,respectively. Which were much higher than those found in the controls by RT-PCR(14.74±0.94%,6.90±0.19%,3.17±0.16%,1.41±0.40%and0.76±0.36%) at each time point(P＜0.01). The numbers of surviving cells injected in Dex group were28.17±1.76%,15.63±0.36%,6.08±0.26%,3.38±0.32%and2.55±0.12%at the same time point,respectively (n=4in each point). Which were also much higher than those found in thecontrols (P＜0.01).6. Direct intramyocardial injection caused mechanical injury and acute inflammation,which caused the engrafted cells to die. Triptolide exhibited anti-inflammatory activity andsignificantly decreased the expression levels of IL-1β, IL-6and TNF-α, as well as NF-κB.The short-term administration of triptolide can promote the early graft survival of HCN4-MSCs in myocardium after transplantation.7. Pretreatment with triptolide significantly reduced the apoptosis of donor cells. Inaddition, triptolide downregulated the Bax level but upregulated the Bcl-2level in theinjected region.8. The results of patch-clamp experiments suggested that the allografted HCN4-MSCssurvived for over4weeks and expressed funny current (If) in the host heart. The activity ofHCN4-MSCs was unaffected by triptolide in vivo.9. Immunohistochemical results showed that some donor HCN4-MSCs were positivefor both EGFP and troponin T (cTnT) or connexin43(CX43) in the host heart at4weeksafter transplantation, which indicated that the implanted HCN4-MSCs differentiated intonew cardiomyocytes.Conclusions1. Sex-mismatched cell transplantation models were used in our experiments. Differentcell numbers of male HCN4-MSCs were injected directly into the myocardium of syngeneicfemale rats. The number of engrafted HCN4-MSCs was estimated by using the amount ofSry gene of each sample by quantitative RT-PCR. We found that1×10~6may be the optimalnumber of engrafted HCN4-MSCs at one site by referring to the standard curve.2. The short-term administration of triptolide can promote the survival ofHCN4-MSCs in myocardium after transplantation. Direct intramyocardial injection causedmechanical injury and acute inflammation, which caused the engrafted cells to die.Triptolide exhibited anti-inflammatory activity and significantly decreased the expressionlevels of IL-1β, IL-6and TNF-α, as well as NF-κB.3. The results of patch-clamp experiments suggested that HCN4-MSCs expressedfunny current (If) in vivo and the activity of donor cells was unaffected by triptolide. SomeHCN4-MSCs were positive for both EGFP and troponin T (cTnT) or connexin43(CX43)in the host heart at4weeks after transplantation, which indicated that the implantedHCN4-MSCs differentiated into new cardiomyocytes. |
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