Experimental Studies On The Effects Of Combining Meglumine Cycle Adenylate Phosphate And Transplantation Of Mesenchymal Stem Cells In Rat Model Of Heart Failure And Its Mechanism

Dilated Cardiomyopathy (DCM) with end-stage heart failure is no effective treatment method at present. Therefore, search for a new and better treatment has become a top priority. A large number of animal experiments and clinical trials confirm that Mesenchymal Stem Cells(MSCs) can differentiate into cardiomyocyte. The poor differentiation rate and survival rate of the donor cells in the host myocardium hampers the efficacy of MSCs transolantation. Studies have shown that cAMP has influential on cell growth, differentiation. cAMP regulates cell differentiation mainly through the cAMP/PKA signaling pathway to play a role. Study found that cAMP/PKA signal transduction pathway have correlation of the transcription factor GATA-4 and Connexin 43 (Connexin43, Cx43). Meglumine Cycle Adenylate Phosphate (MCA) is a positive inotropic drug for heart failure treatment. MCA, as a cAMP analogue, has the same effect as cAMP. We designed in vitro and in vivo experiments to characterize role of MCA on the proliferation of MSCs, MCA differentiate of MSCs into to cardiomyocyte-like cells, the effect of MSCs combining MCA on heart failure and mechanism of MCA. Whether the differentiation of MCA through the cAMP/PKA signaling pathway to play? The research aim is finding a drug who can enhance the survival of transplanted MSCs rate and differentiation rate, effectively improve cardiac function and no adversely affect, for clinical treatment of DCM heart failure with MSCs provide a theoretical basis.Objective:The study was to assess the effect of MCA on growth, differentiation of MSCs, the effects of MSCs combining MCA treatedt heart failure on improved cardiac function, and its possible mechanisms. Given PKA inhibitor H89 for observation of cardiac-specific gene and protein expression of any changes to explore the MCA mainly through cAMP/PKA signaling pathway play a role in inducing differentiation mechanism.Methods:This study from the five aspects to inquiry: PartⅠ:Mesenchymal Stem Cells:Isolation, Cultivation, Identification, Labelling and Differentiation in VitroUsing the whole bone marrow adherent culture method were isolated, cultured and amplified to observe its growth characteristics. Using MTT method determinated MSCs growth curve. Flow-Cytometryanalysis MSCs surface marker. Labelling MSCs with BrdU and DAPI. The 3th to 6th of MSCs with 10μM of 5-azacytidine(5-Aza) incubation for 24h, and culture 2weeks,3weeks,and 4 weeks, extracted total mRNA. RT-PCR techniques observed MSCs express of cardiac-specific geneβ-MHCmRNA.PartⅡ:Meglumine Cycle Adenylate Phosphate induced differentiation of Mesenchymal Stem Cells into cardiomyocytes in vitro(1)Take 3rd generation cells, using MTT method determinated impaction of MCA on the growth capacity of MSCs. (2) 5-Aza as a positive control, the optimal concentration of MCA induced differentiation of MSCs into cardiomyocyte was tested. (10-2.10-3.10-4.10-5M) concentration of MCA induced differentiation of MSCs. Expression of cardiac specific gene GATA-4,β-MHC, Cx43 mRNA were detected fluorescent quantitative RT-PCR analysis.. (3) On the base of MC A-induced concentration of the best, given different induction time and culture time, comparative analysis of the optimum induced program. (4)By fluorescent quantitative RT-PCR and flow cytometry, comparated the effect of MCA, MCA combined 5-Aza with 5-Aza on differentiation. (5) Determined the expression of cardiac-specific protein cTNI by Western Blot analysis and cardiac-specific protein a-actin, desmin, cTNI, Cx43 of MSCs after MCA-induced differentiation by immunocytochemistry. Electron microscope examination was to identify ultrastural change of MSCs by MCA-induced differentiation.PartⅢ:Transplantation Mesenchymal Stem Cell and combined with Meglumine Cycle Adenylate Phosphate Treatment Heart Failure of Adriamycin-induced Dilated Cardiomyopathy in RatsPrepare the modle of heart failure caused by adriamycin-induced dilated cardiomyopathy male wistar rats. The model rats were randomly divided into 6 groups:normal group, MSC group, MCA group, MSC+MCA group, induced-MSC group and HF group. At 4 weeks after transplantation, brain natriuretic peptide (BNP) level in serum before and after all the therapy was mearsured by ELISA. Echocardiography measured left ventricular end-systolic diameter (LVSD), left ventricular end-diastolic diameter (LVDD), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). Haemodynamic were measured including left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVDP), the maximum rate of LV systolic pressure rise(+dp/dtmax) and the maximum rate of LV systolic pressure descend(-dp/dtmax). Determination of whole heart weight index (HW/BW) and left ventricular weight index (LW/BW). Myocardial tissue samples were identified by immunohistochemical staining, HE and Masson pathology.PartⅣ:The mechanism of Transplantation Mesenchymal Stem Cell combining with Meglumine Adenosine Cyclophosphate Treatment Heart Failure of Adriamycin-induced Dilated Cardiomyopathy in RatsPrepare the modle of heart failure caused by adriamycin-induced dilated cardiomyopathy male wistar rats. The model rats were randomly divided into 6 groups:normal group, MSC group, MCA group, MSC+MCA group, induced-MSC group and HF group. At 4 weeks after transplantation:Myocardial tissue samples were identified by immunohistochemistry, HE and Masson staining. Expression of cardiac tissue-specific protein of GATA-4, Cx43 and cTNI were detected by Westren Blot analysis. cAMP contents of myocardial was measured by ELISA.Part V:Mechanism of Meglumine Cycle Adenylate Phosphate induces Differentiation of Mesenchymal Stem Cells into CardiomyocyteMSCs were randomly divided into 5 group:MCA group, H89+MCA group, MCA+5-Aza group, H89+MCA+5-Aza group, and blank group. Application of MTT method determinated the impact on the growth capacity of MSCs with H89. cAMP contents of MSCs was measured by ELISA. SYBR-Real Time RT-PCR determinated gene expression of GATA-4, Cx43 and P-MHC mRNA. Western Blot determinated proteins expression.of cTNI, Cx43 and GATA-4.Results:1. The cultured MSCs showed adherent growth, cell morphology as a spiral arrangement of long spindle cells similar to fibroblasts. The growth curve of MSCs was S-type. Cellular proliferative growth curve shape is similar to the proliferation on the 2th,4th,6th generation of MSCs. BrdU labelling rate is 94%, DAPI labeling rate is 100%. Flow-cytometric analysis of MSCs expended to three passeges. Most of the MSCs ecpressed CD44 and CD71, and did not express for CD45.β-MHC relative expression levels of 5-Aza induced MSCs was significant increase.2. (1) Inhibited MSCs proliferation by MCA was time-and dose-dependent.10-2M of MCA was particularly remarkable on the inhibition of cell proliferation (2) Compared with blank group,GATA-4,β-MHC, Cx43mRNA expressed in MCA group were significantly higher (P<0.05).The amount of GATA-4,β-MHC mRNA expression in the MCA10-3M group is significantly higher(P<0.05). Cx43 mRNA expression in the MCA10-5M group was significantly higher than the concentration of the other three groups (P＜0.05). (3) Give 10-3M of MCA induced MSCs for 1d,3d,5d,7d,9d time, GATA-4,β-MHC, Cx43 mRNA was significant increased. With culture time extended GATA-4,β-MHC, Cx43 mRNA expression was gradually increased.10-3M MCA-induced 3 days of MSCs, GATA-4,β-MHC, Cx43 mRNA expression is highest. (4) GATA-4, P-MHC, Cx43 mRNA expression in the MCA group was higher than 5-Aza group(P＜0.01); MCA+5-Aza group of GATA-4,β-MHC, Cx43 mRNA gene expression was significantly higher than MCA group and 5-Aza group(P＜0.01). Differentiation rate by flow cytometry, MCA group was higher than 5-Aza group. Differentiation rate of MCA+5-Aza group significantiy improved, compared with the MCA group and 5-Aza group (5) WB determination have clear specific band of the expression of cardiac-specific proteins cTNI. Compared with 5-Aza group and MCA group, MCA+5-Aza group was significant increased. MCA group is significant increased compared with 5-Aza group. MSCs were positive for cardiac markers of a-actin, desmin, cTNI and Cx43 on MCA group,5-Aza group and MCA+5-Aza group by Immunocytochemical staining tested. Cell ultrastructure show those cells within the newborn cells, micro-filaments, mitochondria, ribosomes and well-developed Golgi apparatus, organelles in the nuclear accumulation around consistent with myocardial ultrastructure.3.(1) After 4 weeks treatment, compared with HF group, BNP content in MSC+MCA group, MSC group, induced-MSC group and MCA group were significant decline. (2) Echocardiography results:compared to pre-treatment, LVSD, LVDD in MSC group, MSC+MCA group, induced-MSC group and MCA group were significant decrease after 4 weeks treatment, and compared with HF group, also were significant decrease. Compared with MSC+MCA group, MSC group and induced-MSC group, the change were not signification, but the decline extent of MSC+MCA group were decreased by a big range. LVEF, LVFS of after treatment in MSC group, MSC+MCA group, induced-MSC group were signification increase, compared with pre-treatment. LVEF, LVFS in MSC group, and MSC+MCA group were significant improve, compared with MCA group and induced-MSC group. (3)Hemodynamic:Compared with HF group, LVSP,±dp/dtmax were significantly higher and LVDP decreased significantly in MSC group, MSC+MCA group, MCA group and induced-MSC group. MSC+MCA group was statistically significant. compared with other treatment groups (4) HW/BW in MSC group, MSC+MCA group, MCA group, induced-MSC group were significant difference, compared with HF group. LW/BW in MSC group, MSC+MCA group, induced-MSC were statistically significant, compared with HF group. HE staining showed HF group showed myocardial cell degeneration, and the changes can be seen to reduce in the treatment groups. Masson staining showed that myocardial fibrosis of the treatment groups were still present, but some relief compared with HF group.4.(1)BrdU-positive cells by Immunohistochemical staining can be seen in the three MSCs transplantation groups. The number of the positive cells in the MSC+MCA group more than the MSC group and inducd-MSC group.Immunohistochemical double-labeled in the three MSCs transplantion groups were seen scattered BrdU positive cells, yellow-brown cytoplasm of fiber structure can be seen in those BrdU positive cells. (2)WB results:Compared with HF group, GATA-4, Cx43, cTNI protein expression was significant change in MSC group, MSC+MCA group and induced-MSC group. Compared with MSC group and induced-MSC group, MSC+MCA group express GATA-4, Cx43, cTNI protein was significantly increased (P<0.05). (3)ELISA measured the level of myocardial cAMP content:MSC+MCA group, MCA group was significantly higher than HF group. Given MCA could increase the myocardial cAMP levels. 5. (1) 25Mm of H89 treatment for 30min and then 10-3M of MCA deal MSCs12h and 24h, OD values respectively were significant increase, compared with MCA group. H89 can reduce the effect of MCA inhibition on cell growth. (2) cAMP levels:cAMP content of groups was significantly higher than blank group at timepoints. With increasing of MCA concentration, cAMP content also increased. cAMP concentration reached its peak after 6h, then decreased gradually. (3) Compared with blank group, GATA-4, MHC, and Cx43 mRNA expression was significantly increased in MCA group and MCA+5-Aza group induce differentiation of MSCs. Compared with MCA group, GATA-4, MHC, Cx43 mRNA expression of H89+MCA group was significantly reduced.Compared with MCA+5-Aza group, GATA-4, MHC, Cx43 mRNA expression levels of H89+MCA+5-Aza group was also significantly reduced.(4) Compared with blank group, MCA group was increased significantly of GATA-4, Cx43, cTNI protein expression. Compared with MCA group GATA-4, Cx43, cTNI significantly reduced protein expression in H89+MCA group (P<0.05).Conclusion:1. The whole bone marrow adherent culture method can get pure MSCs, express CD44 and CD71, but not express CD45. The 2th,4th and 6th generation MSCs have the same growth capabilities. BrdU and DAPI labeling positive cells have the high rate. MSCs have the ability of differentiation into cardiomyocyte-like cells after 5-Aza-induced MSCs.2. MCA inhibited the proliferation of MSCs. MCA can induce differentiation of MSCs into cardiomyocytes. The optimal induced concentration and time of MCA is 10-3M, and three days. Induce effection of MCA is stronger than 5-Aza, whose combined with 5-Aza can play a collaborative effect.3. Simply transplanted MSCs, transplanted MSCs induced by MCA and transplantation of MSCs combined MCA, can improve cardiac structure and function. Transplanted directly MSCs can play the same effects and treatment effects as MCA induced-MSCs.4. Given MCA into the MSCs increased intracellular cAMP levels, the promotion of the expression of GATA-4 and Cx43, so that MSCs expressed cardiac-specific protein expression was increased Transplanted MSCs combined MCA can improve the survival rate and differentiation rate of MSCs into cardiomyocyte-like cells.5. Given MCA into the MSCs increased intracellular cAMP levels, the promotion of the expression of GATA-4 and Cx43, so that MSCs expressed cardiac-specific protein expression was increased. H89 can inhibit the expression of these genes and proteins. MCA play a role through the cAMP/PKA signaling pathway.