Myocardin and Stat3 act synergistically to inhibit cardiomyocyte apoptosis

Figures

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  Figure 1. Myocardin and Stat3 inhibit staurosporine-induced cardiomyocyte apoptosis (A) Afterstimulating postnatal rat cardiomyocytes with 1μM staurosporine for 30 min to induce cardiomyocyte apoptosis, cells were transfected with the plasmids pcDNA3.1, Myocardin, Stat3, and Myocardin/Stat3 for 24 hours. Then we used TUNEL Assay to observe the change of apoptotic rate from treatment with staurosporine or transfection with the plasmids pcDNA3.1, Myocardin, Stat3, and Myocardin/Stat3 in the presence of staurosporine. (B) Statistical software was used to analyze the change in apoptotic rate by the TUNEL Assay. **, p<0.01, #, p>0.05. n=3. (C) Afterstimulating postnatal rat cardiomyocytes with 1μM staurosporine for 30min induce cardiomyocyte apoptosis, cells were transfected with the plasmids pcDNA3.1, Myocardin, Stat3, and Myocardin/Stat3 for 24 hours. Annexin V-FITC apoptosis detection assay was used to observe the change of apoptotic rate by treatment with staurosporine or transfection with the plasmids pcDNA3.1, Myocardin, Stat3, and Myocardin/Stat3 in the presence of staurosporine.

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  Figure 2. Myocardin and Stat3 enhance the expression of anti-apoptotic genes (A) Afterstimulating postnatal rat cardiomyocytes with 1μM staurosporine for 30min induce cardiomyocyte apoptosis, cells were transfected with the plasmids pcDNA3.1, Myocardin, Stat3, and Myocardin/Stat3 for 24 hours. Total RNAs were isolated, and the expression of Bcl-2 and Mcl-1 was examined by RT-PCR (including quantifications performed with Quantity One software). **, p<0.01. n=3. (B) Afterstimulating postnatal rat cardiomyocytes with 1μM staurosporine for 30min induce cardiomyocyte apoptosis, cells were transfected with the plasmids pcDNA3.1, Myocardin, Stat3, and Myocardin/Stat3 for 24 hours. Total proteins were extracted, and the protein levels of Bcl-2 and Mcl-1 were detected by western blot analysis (including quantifications performed with Quantity One software). β-actin was used as a loading control. **, p<0.01. n=3. (C and D) Representative images of immunostained cardiomyocytes. The left panels (green) respectively show anti- Bcl-2 and Mcl-1 antibody reactivity to demonstrate gross morphology. The middle panels (blue) show the DAPI staining for nuclei. The right panels respectively show double…

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  Figure 3. Myocardin and Stat3 mediated anti-apoptosis by the intrinsic or mitochondrial apoptotic pathway (A and B) The expression of Bcl-2 and Bax was examined by RT-PCR and western blot analysis. GAPDH and β-actin were used as loading controls. (C and D) The ratio of Bcl-2 to Bax was determined by statistical software. **, p<0.01. n=3. (E and F) After postnatal rat cardiomyocytes were transfected with pcDNA3.1, Myocardin, Stat3, and Myocardin/Stat3 for 24 hours, the expression of Apaf-1, caspase-9 and caspase-3 were examined by RT-PCR and western blot analysis (including quantifications performed with Quantity One software). **, p<0.01, *, p<0.05. n=3.

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  Figure 4. Myocardin and Stat3 interact in vivo Coimmunoprecipitation assays were used to identify the interaction of Myocardin and Stat3 after cotransfection of cardiomyocytes with myc-Myocardin and flag-Stat3 for 24 hours.

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  Figure 5. The molecular mechanism of Myocardin and Stat3 ranti-apoptosis marker gene regulation (A) Schematic of the -878 Bcl-2 promoter, containing the CArG box element, which was linked to a luciferase reporter and the -878 Bcl-2 promoter with a mutation in CArG box. (B) The postnatal rat cardiomyocytes were transfected with the wild-type -878 Bcl-2 promoter, or the -878 Bcl-2 promoter with a mutation in CArG box, and transfected with pcDNA3.1, Myocardin, Stat3, and Myocardin/Stat3 for 24 hours. Luciferase reporter assay was used to test the transactivity of Bcl-2. (C) Schematic of the -479 Mcl-1 promoter, containing CArG box elements and the -479 Mcl-1promoter with mutation CArG box. *, p<0.05. n=6. (D) Postnatal rat cardiomyocytes were transfected with the wild-type -479 Mcl-1 promoter, or the -479 Mcl-1 promoter with a mutation in CArG box, and transfected with pcDNA3.1, Myocardin, Stat3, and Myocardin/Stat3 for 24 hours. Then the luciferase reporter assay was used to test the transactivity of Mcl-1. **, p<0.01, *, p<0.05. n=6. (E and F) The postnatal rat cardiomyocytes were transiently transfected with pcDNA3.1, Myocardin, Stat3, and Myocardin/Stat3 for 24 hours. ChIP assays were…