Suberoylanilide Hydroxamic Acid Promotes Cardiomyocyte Differentiation Of Mesenchymal Stem Cells And The Mechanisams

PART 1 SAHA promotes cardiomyocyte differentiation of mesenchymal stem cells in vitroObjective: This study was to investigate the effect of SAHA on cardiomyocyte differentiation of MSCs in vitro and the cooperative effect between SAHA and 5-aza.Methods: Rat MSCs were isolated and induced to differentiate into cardiomyocyte with SAHA or 5- aza or a combination of SAHA and 5-aza. The expression of cardiogenesis-related gene and cardiomyocyte-specific protein were detected by immunofluorescence staining, qPCR and Wstern-blot during the differentiation.Results:1.The mRNA levels of the transcriptional expression of GATA4, NKx2.5, and MEF2c after the 7 days induction on MSCs. (1)The induction with SAHA alone: the expressions of GATA4, NKx2.5, and MEF2c increased significantly (p<0.05), and were dose dependent. The expressions were increased highest when induction with 1uM SAHA, about 10-15 times higher than the group which with no induction, nevertheless, the expressions of which induction with 2uM SAHA were relative reduced. (2) The induction with 5-aza alone: the mRNA levels of GATA4, NKx2.5 and MEF2c increased only 2-4 times when induction with 10uM 5-aza(p<0.05). (3)The induction with SAHA + 5-aza:When MSCs were induced with both SAHA and 5-aza, the mRNA levels of GATA4, NKx2.5, and MEF2c were similar with those induced by SAHA alone(p>0.05).2.The expressin of cTnT after 4 weeks induction on MSCs:(1) The induction with SAHA alone: The immunofluorescence staining detect that green plaguliform fluorescences were dense in the cells. The Western blot detect that cTnT when induction with 1uM SAHA was increased about 8 times than the group with not induction(p<0.05),whereas the expression of the cTnT protein was decreased when induction with 2uM SAHA.(2) The induction with 5-aza alone: The immunofluorescence staining detect that green plaguliform fluorescences were sparseness in the cells.The Western blot detect that cTnT only increased about 1.3 times than the group with not induction ( p>0.05 ) . (3) The induction with SAHA + 5-aza: The immunofluorescence staining results and the Western blot results were similar with the group which induced by SAHA(p>0.05).Conclusion: 1.SAHA promoted cardiomyocyte differentiation of rat MSCs with a significant increase in the expression of cardiogenesis-related gene GATA4, NKx2.5, and MEF2c, and cardiomyocyte-specific protein cTnT in vitro. SAHA was much more effective on cardiomyocyte differentiation of MSCs than 5-aza.2.The cooperative effect between SAHA and 5- aza was not found during the differentiation of MSCs into cardiomyocyte.PART 2 The acetylation mechanism of cardiomyocyte differentiation of MSCs induced by SAHA in vitroObjective: This study was to investigate whether the increase of cardiogenesis-related genes transcription in MSCs was related to hyper-acetylation on their promoters DNA after induction by SAHA. Whether the repression of HDAC4 lead to the hyper-acetylation.Methods: Rat MSCs were collected after 7 days induced with 1μM SAHA. Then the DNA of these cells were precipitated by acetylated H3 antibody or HDAC4 antibody with ChIP . The primers were designed in the 1000bp region before the cardiogenesis-related genes first exon. The quantity of these genes promoter region DNA were evaluated using qPCR by the primers. Results: The DNA level of GATA4, MEF2c and NKx2.5 promotor region in MSCs after induction with SAHA were 3 times, 1.4times and 2 times higher than that in MSCs with not induction when acetylated H3 antibody was used with ChIP(p<0.05). The DNA level of GATA4, MEF2c and NKx2.5 in MSCs after induction with SAHA were decreased about 0.41 times, 0.31 times and 0.43 times than that in MSCs with not induction when HDAC4 antibody was used with ChIP(p<0.05).Conclusion:1.The increase of GATA4,MEF2c and NKx2.5 genes transcription may be related to the hyper-acetylation on their promoters DNA after induction with SAHA.2. The hyper-acetylation in GATA4,MEF2c and NKx2.5 promotor region probably related with the supression of HDAC4.And this is possible one of the reasons that differentiation of MSCs into cardiomyocyte induced by SAHA.PART 3 The effect of SAHA on cardiac function after MSCs transplantion into rat heart of doxorubicin heart failure model Objective: This study was to investigate the effect of SAHA on cardiac function after MSCs transplantion into rat heart of doxorubicin heart failure model.Methods: Doxorubicin heart failure model were constructed in rats. MSCs and MSCs which induced with 1μM SAHA by 7 days were transplanted into the myocardial tissue of the models. Cardiac function was evaluated with UCG 3 weeks after the transplantion, a nd the cardiac function on rats were assessed by EF and FS.Results: The EF was increased from 65.92±2.98 to 73.25±3.58 and the FS was increased from 32.25±1.66 to 37.46±3.12 in the MSCs transplantation rats. And in the rats which transplanted with MSCs induced by SAHA, the EF was increased from 66.04±2.02 to 75.13±2.05 and the FS was increased from32.29±0.96 to 38.87±1.82. In the two groups EF and FS were improved markedly after the transplantation than before(p<0.05).But there was no significant difference between the two groups after the transplantation(p>0.05).Conclusion: The cardiac function of doxorubicin heart failure rats was improved three weeks after the transplantion of MSCs which induced with SAHA or not. But there was no significant difference between the two groups.