| Background and objectiveSick sinus syndrome(SSS), characterized as brad arrhythmia, is a common life-threatening disease, which cannot be cured by medicines except artificial cardiac pacemakers. Although the implantation of artificial cardiac pacemakers relieves symptoms, that treatment is still obsessed by many problems such as the insufficient reactivity under stress, the limited battery life, the complications caused by implanting foreign bodies, the interference from the electromagnetic fields beyond the artificial cardiac pacemakers and so on. Therefore, to replace artificial cardiac pacemakers, the construction of biological pacemakers that could pace themselves would be the alternative method and becoming one of the hotpots in the area of heart electrophysiology.The hyperpolarization-activated cyclic nucleotide-gated channel(HCN) has several genotypes. Thereinto, HCN4 mainly mediates the formation of funny current(If) which is the pivotal step of the spontaneous diastolic depolarization in sinoatrial node cells. However, it is reported that the pacing function inevitably reduced or even faded away in a series of studies on HCN4 to reconstruct biological pacemakers. Thus, the reconstruction of the iron channel of If can hardly generate lasting effective biological pacemakers. Recently, it is found that short stature homobox 2(Shox2), which participates in the cardiac development in early embryo, can remarkably suppress Nkx2.5 and up-regulate the expression of HCN4 to control the differentiation of embryo heart cells into sinoatrial node cells rather than cardiomyocyte-like cells. Therefore, the optimal choice for biological pacemaker studies would be to employ Shox2 as a transcription regulator to induce the differentiation of pluripotent stem cells into sinoatrial-node-like cells with pacing function.Our study, funded by the Chinese National Natural Science Foundation(No. 81270246), aims to evaluating the canine bone marrow mesenchymal(cMSCs) with over-expressed Shox2 gene as the seed cells of biological pacemaker. In this study, we transfected cMSCs with the lentiviral vector pLentis-mShox2-RFP to induce the differentiation of cMSCs in vitro, and use western-blot, patch-clamp, etc. to test Shox2-MSCs in the aspects of physiology and biochemistry.Methods1.Bone marrow specimens were extracted from an adult healthy canine. cMSCs were isolated from the bone marrow specimens by gradient centrifugation and adherent method, and then identified by flow cytometry, while their morphological characteristics were observed by inverted microscope the morphological characteristics.2.The lentiviral vector pLentis-mShox2-ERFP and pLentis-ERFP, which contain the multidrug resistance gene against puromycin for cell purification, were constructed according to the sequence of mice Shox2 mRNA available at NCBI.3.cMSCs were transfected with the lentiviral vector pLentis-mShox2-ERFP and pLentis-ERFP respectively, and then co-cultured with neonatal rat ventricular myocytes(CMs). After that, puromycin was introduced to isolate and purify those cMSCs.4. Western-blot, RT-PCR and immunofluorescence assay were used to detect in the experimental groups the expression of Cx43/45, HCN4, Tbx3, Nkx2.5, etc. specific to myocytes.5.The ion channel characteristics of If were observed by whole cells patch clamp in the Shox2-cMSCs induced by CMs and control group respectively.Results1.When gradient centrifugation and adherent method were introduced to purify cMSCs, the cells of passage 3 appeared typically polygonal or long fusiform, well-stacked cell body and obviously refractive, and had great potential of proliferation and induced differentiation.Flow cytometry suggested the cMSCs were positive for CD29 and CD44(93.2±3.6%), negative for CD34 and CD45,2.At a MOI of 20, fluorescence microscope showed that the cells growth kept steady and the transfection efficiency reached at 85±4.5%(n=5), which indicates that the tranfecting capacity of the lentivirus is satisfactory.3.After the positive transfected cMSCs were induced to differentiate, the whole experiment was divided into four groups: group A(the cMSCs transfected with RFP, RFP-cMSCs group), group B(the cMSCs transfected with RFP and co-cultured with CMs, RFP-cMSCs +CMs group), group C(the cMSCs transfected with mShox2-RFP, Shox2-RFP-cMSCs group), and group D(the cMSCs transfected with mShox2-RFP and co-cultured with CMs, Shox2-RFP-cMSCs +CMs group).4.Compared with other groups, Shox2-RFP-cMSCs +CMs group expressed more HCN4, Cx45 and Tbx3(P<0.05)after being co-cultured with CMs, while RFP-cMSCs +CMs group expressed more Nkx2.5 and Cx43 than other groups did.5.The whole-cell patch clamp shows that the hyperpolarization-activated inward current sensitive to Cs+ was recorded in Shox2-RFP-cMSCs +CMs group, which displayed time and voltage dependent manner. Based on the start-up curve and the reversal potential of the inward current according to the tail current, the calculation shows that current’s half-maximal activation(V1/2), the slope and the reversal potential were-89.4±3.7mV,-16.3±1.5 and-28.9±6.7mV respectively, while no hyperpolarization-activated inward current was recorded in RFP-cMSCs group or RFP-cMSCs +CMs group.Conclusions1.Purified population of cMSCs was completed in the early passage by gradient centrifugation and adherent method.2.After transfected with pLentis- mShox2-RFP lentivirus successfully,the cMSCs expressed Shox2 stably and generated HCN4 channel protein,which is negative in RFP groups..3.After induced to differentiation by co-cultured with CMs,the Shox2-MSCs group expressed more Cx45、Tbx3 and HCN4,accord with the conduction system of heart;While the control group expressed of Cx43 and Nkx2.5 highly,consistent with the work cardiac myocytes4..After induced to differentiation,Shox2 genetic modification of MSCs possessed the electrophysiological characteristics of biopacemaker and recorded the funny current,which is not recorded in the RFP-MSCs and RFP MSCs + CMs groups. |
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