Research Of The Effect Of Electrical Pulse Current Stimulation On Differentiation Status Of MSCs And Cardiac Myocytes Coculture Monolayer

Background and aimsCardiovascular diseases are types of illness which have severe harmful effect onhuman health.The acute infarct, for example,the block of coronary artery and the followingIschemia lead to irreversible damage to cardiomyocytes in15to20minutes,the remodelingprocess after infarct and replacement of the cardiomyocytes to fibroblasts lead to thedeterioration in function of the remaining cardiomyocytes and the Left ventricular dilatationhappens.MSC is a kind cell type as suitable candidate for transplantation. Afterinfarct,MSC transplation can minimize necrosis,secrete factors which can supportangiogenesis and improve the heart function,there are also reports regard that mesenchymalstem cells act as a kind of supportive cell of cardiac stem cells, they can recruit endogenousstem cells which repair the site of injury, not only the MSCs themselves. In the research ofSick sinus syndrome,the HCN gene modified MSCs are used to reconstruct pacemaker.Howto improve the function of MSCs tranplatation is a direction for further research,molecularapproaches are effective methods,but since the application of Lentiviral infection is stilllimited and in cardiology therapy, physical treatment is widely used clinically,we wondercan biophysical methods be used in the process of MSC differentiation or to affect thedifferentiation status of MSC and cardiac myocytes coculture monolayer?In normalphysiological state, endogenous current and electrical field play an important role in thedifferentiation of the embryo.The aim of the study is to find the possibility and feasibility ofusing exogenous electrical signal to promote the cardiogenesis process of MSCs or theMSC and cardiac myocyte coculture monolayer, to find relative suitable parameter and toestablish the electrical pulse current stimulation (EPCS) system,find the degree of MSCdifferentiation which act as a basic for finding the targets of EPCS and primarily determinthe relative suitable time for EPCS. Methods:1. With the feedback of the EPCS effect for several times, we establish an EPCSculture system.2. Using the pathch clamp test the effect of direct current on the membrane potentialchange of MSC in the msc and cardiomyocytes coculture monolayer.3. The MSCs was divided into two groups–the EPCS group and controlgroup.CCK-8and PCNA was detecdted to see if the EPCS will affect the proliferation ofMSCs; biomarker was detected to see if EPCS will affect the molecular markers on MSCs;the gap junction protein was detected to see if EPCS-treated MSCs can still generate gapjunctions after coculture with cardiac myocytes. Detect the effect of EPCS on the beatingbehavior of cardiomyocytes, and to detecte troponin t, troponin I, myosin heavy chain6andmyosin heavy chain7at mRNA level.4. The5-aza treated (3w)mouse MSCs was divided into two groups–the the EPCSgroup and control group to test the if EPCS have effect on the induction of cardiacdifferentiation.5.The CX43，Nav1.5，CaMKⅡ expression in the MSC and cardiac myocytescoculture monolayer was visualized with immunofluorescence,the MSCs and cardiacmyocytes was cocultured at a rate of4:1and the CaMKⅡ level was tested at48h,72h and96h using western-blotting, to check the dynamic changes of CaMKⅡ.6. After cocuturing for5days and the expression of cardiac transcriptional factorsGATA4, MEF2c,TBX5,and it was visualized with immunofluorescence. Detect thekaryopherin expression differention of MSC and cardiac myocytes in the coculturemonlayer.7. The monolayer was divided into two groups: the Ca2+channel block group and thecontrol group,after coculture for5days,the VSCC(voltage sensitive ca channel) and Catransient was detected with immunofluorescence and fluo-3staining， detect thecalcineurin(CaN)activity and Troponin T， NAV1.5， CACNA1G/H and CX43proteinlevel in the two groups.8. To test the effect of mild electrical pulse current stimulation on MSC and cardiacmyocyte coculture monolayer. The MSC and cardiac myocytes were seeded at a primaryratio of3:1, and the monolayer was treated with EPCS for1h/d，3h/d and6h/d, EPCS was given after24h of primary culture and last for4days,and repeat for3times,thecontemporary monolayer were used as controls. TroponinT and CX43level was detected inthe4groups.9.Immunofluorescence was used to visualize the S100A4，MEF2C, troponin T, CX43and GATA4expression in the EPCS(constant5days) group and control group.Results:1. A stable and realiable EPCS given and recipient system was established, the signalcan be monitored when EPCS is given, the system is easy to use and can give cell accuratestimulation.2. The membrane potential has some Minor fluctuations of MSCs in the MSC andcardiac myocytes coculture monolayer when direct current is given.3. EPCS can keep the contractional behavior and structure of few regions of cardiacmyocytes monolayer after long term of culture.4. In the aspect of safety: EPCS do not affect the beating behavior of cardiac myocytes;EPCS do not affect the proliferation process of MSCs(cck-8assay and PCNA detection)andthe gap junction genetation potential; EPCS slightly reduce the CD44expression onMSCs,and the amplitude of reduction do not increase with treating time/day;EPCS do notaffect the troponin and myosin heavy chain at mRNA level after9days(from the third daythe EPCS was given,12days.)EPCS do not affect the process of5-aza induction of MSCdifferentiation.5. The potential of canine MSC differentiation in cardiogenesis aspect.After coculture with the cardiomyocytes,MSCS express gap junctions,Na+channel(Nav1.5)，CaMKⅡ and transcriptional factor GATA4，MEF2C and TBX5.Gata4has ahigher level (4.49fold)in the nuclei of cardiac myocytes than it in the nuclei of MSCs.In aMSC and cardiac myocyte coculture monolayer(4:1),the CaMKⅡ level strongly increasewith coculture time,especially after coculture for72-96h. The concentrate of Karyopherinα3is higher in the cytoplasm of cardiomyocytes than it in the MSCs, the Karyopherinα4and Karyopherinα5is higher in some cadiac myocytes than that in the MSCs,andKaryopherin β1，Karyopherin α1/6，and Karyopherin α2makes no differntion in levelin between cardiac myocytes and MSCs. Among these Karyopherins,the percentage ofcardiac myocytes have high level of Karyopherinα3is coincidence with the percentage of cardiac myocytes show high level nuclei GATA4concentration(90%).The Ca2+channelblock culture lead to a high rate proliferation of fibroblasts in the primary cardiac myocytesmonolayer and the S100A4level increases.The generation of Ca2+channel on the MSCsdepends on the Ca2+current in the MSC and cardiac myocytes coculture monolayer.Blockof Ca2+chanel lead to a Ca2+transient amolitude and duration downregulation in MSCs.Block of Ca2+chanel lead to a up-ragulation in cardiac transcriptional factors, but thestructure protein do not elvated.6. Gap junction protein and troponin T show an increase after EPCS with treatingtime/d. We found that the gap junction protein Cx43increased with treating time—in theEPCSgroup, it exhibited1.5and1.7fold increase in the3h/day group and6h/day group(P<0.01), and troponin T exhibited3.6and4.4fold increase in the3h/day group (P<0.01)and6h/day group (P<0.05).7. After5days exposure, EPCS can enhance the expression of S100A4, which is2.33fold in cardiacmyocytes (P<0.01) and1.99fold inMSCs (P<0.01) in gray value.Asignificant increasing expression of the myocyte enhancer factor (MEF.1.63fold) andGATA4(1.57fold) is detected in neonatal rat cardiac myocytes (P<0.01) compared withcardiac myocytes in the cotemporary coculture monolayer in the control group. Also, EPCScan trigger the assembly of MEF2c in the nuclei. In addition, more cardiac myocytes werefound to have two nuclei. But MSCs fail to active MEF2C transcriptional factor like that incardiac myocytes after EPCS exposure. The elevation of MEF2in both cytoplasm andnuclei of cardiac myocytes can always make a clear distinction of the cardiac myocytes andMSCs in coculture. Some factors show strong upregulation tendency with EPCS in bothcardiac myocytes and MSCs—these include the troponin T (2fold,P<0.01) and Cx43(1.4fold,P<0.05) in cardiac myocytes, and troponin T (1.88fold,P<0.01) and Cx43(1.94fold,P<0.01) in MSCs. S100A4and troponin t nuclei concentrate phenomenon is alsofound in the EPCS treated cardiac myocytes in the control coculture monolayer, very fewMSCs also has this phenomenon.Conclusion1. Suitable EPCS intervention is a very effective and multi-targets physicalintervention to stimulate differntion in the MSC and cardiac myocytes coculture monolayer.2. The nuclei concentration of GATA4of the MSCs in the MSC cardiac myocytes coculture monolayer is far below that in the cardiac myocytes nuclei. Among theKaryopherins, the concentrate of Karyopherin α3is higher in the cytoplasm ofcardiomyocytes than it in the MSCs, the Karyopherinα4and Karyopherinα5is higher insome cadiac myocytes than that in the MSCs.3. The generation of Ca2+channel on the MSCs depends on the Ca2+current in theMSC and cardiac myocytes coculture monolayer.4.MSCs are not so comprehensive and sensitive recipients of EPCS like cardiacmyocytes.After EPCS treatment, though S100A4and TroponinT upregulated, thephenomenon of these proteins concentration in the nuclei is always detected atcardiacmyocytes but only on very few MSCs,It implies that the barriers of differentiation inMSCs may correlate with the failer of importion of important proteins to the nuclei.In thecoculture monolayer, cardiac myocytes is sensitive to EPCS in MEF2c, but the MSCs donot respond to EPCS in ME2c，which implies limit receptivity of MSCs to EPCS.5EPCS can upregulate the S100A4level in the MSCs, in reports，parietal endodermsecreted S100A4and promote cardiogenesis，it implies that EPCS may enhance the abliy ofsupport role of MSCs in the coculture monolayer.