Effects of the Activin A–Follistatin System on Myocardial Cell Apoptosis through the Endoplasmic Reticulum Stress Pathway in Heart Failure

Figures

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  Figure 1. General observations.

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  Figure 2. Expression levels of activin A–follistatin system, brain natriuretic peptide (BNP) and angiotensin II (Ang II) in the serum of rats. (A) ELISA method was used to determine the expression of activin A–follistatin system, BNP and Ang II. The data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the SO group. # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the one-week MI group. ΔΔ p < 0.01, ΔΔΔ p < 0.001, compared with the four-week MI group; (B) Activin A/Follistatin represents the ratio of activin A and follistatin.

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  Figure 3. The expression of activin A, follistatin, caspase-3, and C/EBP homologous protein (CHOP) in rats detected by immunohistochemical staining. The border zones were used to take photos in the eight-week MI group. The expression of activin A, follistatin, caspase-3, and CHOP was detected using the anti-activin A antibody, anti-follistatin antibody, anti-caspase-3 antibody, and anti-CHOP antibody; IgG from a normal rabbit served as a negative control (200× magnification and 400× magnification).

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  Figure 4. Expression levels of activin A, follistatin, BNP, caspase-3 and ERS-related molecules in the left ventricular noninfarction area. (A) The expression of activin A, follistatin, BNP, caspase-3, glucose-regulated protein-78 (GRP-78), caspase-12, and CHOP was detected by Western blot assay; (B) The graph shows the result of densitometry quantification of proteins relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. (a) GRP-78, (b) Caspase-12, (c) CHOP, (d) Follistatin, (e) BNP, (f) activin A, (g) Caspase-3; (C) Activin A/Follistatin represents the ratio of activin A and follistatin. The data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the SO group. ## p < 0.01, ### p < 0.001, compared with the one-week MI group. Δ p < 0.05, ΔΔΔ p < 0.001, compared with the four-week MI group.

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  Figure 5. Apoptosis in activin A-stimulated H9c2 cells. (A) H9c2 cells were stained with propidium iodide (PI) and Annexin V–fluorescein isothiocyanate (FITC) and sorted by fluorescence-activated flow cytometry. (a) control; (b) activin A, 6.25 ng/mL; (c) activin A, 12.5 ng/mL; (d) activin A, 25 ng/mL; (e) activin A, 50 ng/mL; and (f) activin A, 100 ng/mL; (B) Graph presented the percentage of H9c2 cell apoptosis. The data are presented as mean ± standard deviation. * p < 0.05, ** p < 0.01 versus the 100 ng/mL activin A group; (C) Caspase-3 activity assay was used to test the relative caspase-3 activity of the control. The data are presented as the mean ± standard deviation. *** p < 0.001 versus the 100 ng/mL activin A group.

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  Figure 6. Expression levels of follistatin and endoplasmic reticulum stress (ERS)-related molecules in activin A-stimulated H9c2 cells. (A) Western blot was performed to test the expression of follistatin and ERS-related molecules. Proteins were extracted from H9c2 cells, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and immunoblotted sequentially with antibodies. (1) Control, (2) activin A (100 ng/mL), (3) activin A (100 ng/mL) + p38MAPK inhibitor, (4) activin A (100 ng/mL) + ERK inhibitor, (5) activin A (100 ng/mL) + Smad3 inhibitor, (6) activin A (100 ng/mL) + JNK inhibitor; (B) The graph showed the result of densitometric quantification of proteins relative to GAPDH as an internal control. (a) CHOP, (b) caspase-12, (c) GRP-78, (d) Follistatin; (C) H9c2 cells were stained with PI and Annexin V-FITC and sorted by fluorescence-activated flow cytometry. (a) Control, (b) activin A (100 ng/mL), (c) activin A (100 ng/mL) + p38MAPK inhibitor, (d) activin A (100 ng/mL) + ERK inhibitor, (e) activin A (100 ng/mL) + Smad3 inhibitor, (f) activin A (100 ng/mL) + JNK inhibitor; (D) Graph presented the apoptotic rate of H9c2 cells. The data are presented as the mean ±…

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  Figure 7. Concentration and acting time of Ang II-stimulated H9c2 cell apoptosis. (A) Cell viability was assessed using the MTT assay. H9c2 cells were seeded at a density of 5 × 103 cells per well into 96-well plates. (a) Effect of Ang II on the viability of H9c2 cells cultured with different concentrations (0.01, 0.1, 1 μM·mol/L) for the same time; (b) Effect of Ang II on the viability of H9c2 cells cultured with the same concentration for different times (24, 48 or 72 h); (B) Caspase-3 activity assay was used to test the relative caspase-3 activity of the control. (a) Effect of Ang II on the caspase-3 activity of H9c2 cells cultured with different concentrations (0.01, 0.1, 1 μM·mol/L) for the same time; (b) Effect of Ang II on the caspase-3 activity of H9c2 cells cultured with the same concentration for different times (24, 48 or 72 h). The data are presented as the mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the control group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus the 0.01 μM Ang II group; Δ p < 0.05, ΔΔ p < 0.01, ΔΔΔ p < 0.001 versus the 0.1 μM Ang II group, $ p < 0.05, $$ p < 0.01.

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  Figure 8. Expression levels of follistatin and ERS-related molecules in Ang II-stimulated H9c2 cells cultured with a low concentration of activin A. (A) Western blot was performed to determine the expression of follistatin and ERS-related molecules. Proteins were extracted from H9c2 cells, separated by sodium dodecyl sulfate−polyacrylamide gel electrophoresis, and immunoblotted sequentially with antibodies. (1) Control, (2) Ang II (0.1 μM), (3) activin A (5 ng/mL) + Ang II (0.1 μM), (4) activin A (10 ng/mL) + Ang II (0.1 μM), and (5) activin A (50 ng/mL) + Ang II (0.1 μM); (B) The graph showed the result of densitometric quantification of proteins relative to GAPDH as an internal control. (a) CHOP, (b) caspase-12, (c) GRP-78, (d) Follistatin; (C) H9c2 cells were stained with PI and Annexin V–FITC and sorted by fluorescence-activated flow cytometry. (a) Control, (b) Ang II (0.1 μM), (c) activin A (5 ng/mL) + Ang II (0.1 μM), (d) activin A (10 ng/mL) + Ang II (0.1 μM), (e) activin A (50 ng/mL) + Ang II (0.1 μM); (D) Graph presented the apoptotic rate of H9c2 cells. The data are presented as the mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001.