STVNa Attenuates Isoproterenol-Induced Cardiac Hypertrophy Response through the HDAC4 and Prdx2/ROS/Trx1 Pathways

Figures

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  Figure 1. Cellular effects of STVNa after Iso-induced cardiac hypertrophy in vivo. Rats were allocated into three groups (n = 8): the control group received 0.9% NaCl injections; the Iso group received subcutaneous injections of 5 mg/kg/d Iso for 7 consecutive days to create a model of cardiac hypertrophy; and the Iso + STVNa group received Iso (5 mg/kg) and isosteviol sodium (4 mg/kg). (A) The LW/TL ratio was calculated. (B–D) Real-time quantitative PCR results showing mRNA levels of ANP, β-MHC and BNP. (E) Hematoxylin -eosin (HE)-staining results of left ventricle cross-sections. Scale bar: 50 μm. (F) Determination of cardiomyocyte surface area by HE staining. The values shown in this graph represent means ± SD. of data from three independent experiments (n = 30). ** p < 0.01, *** p < 0.001 versus the control group; ## p < 0.01, ### p < 0.001 versus the Iso group. Iso, isoproterenol; BNP, brain natriuretic polypeptide; β-MHC, β-myosin heavy chain; ANP, atrial natriuretic peptide.

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  Figure 2. STVNa inhibited cardiomyocyte hypertrophy induced by Iso in vitro. H9c2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) which containing 1% fetal bovine serum (FBS) under the following conditions: control, DMEM only; Iso, 10 μmol/L Iso; Iso + STVNa, 10 μmol/L Iso and 5 μmol/L STVNa. (A) The surface area of H9c2 cells determined by rhodamine-phalloidin staining in all groups (B, n = 30). Scale bar: 50 μm. (C) ANP mRNA expression in H9c2 cells. (D) The cardiomyocytes were isolated from the rat heart (n = 5) and the surface area was visualized by a microscope (scale bar: 50 μm). The representative cell surface area in each group was calculated (E, n = 30). (F) The mRNA level of β-MHC in H9c2 cells. The values shown in this graph represent means ± S.D. from three independent experiments. * p < 0.05, *** p < 0.001 versus the control group; # p < 0.05, ## p < 0.01, ### p < 0.001 versus the Iso treatment group.

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  Figure 3. Effect of STVNa on mitochondrial integrity after Iso induction. Cells were seeded in 24-well plates and treated with JC-1 at 37 °C for 20 min to measure the Mitochondrial Membrane Potential (MMP). Confocal micrographs of JC-1 staining in H9c2 cells (A) and primary cardiomyocytes (B). Scale bar: 50 μm. (C) Mitochondrial morphology (scale bar: 10 μm) showing the length of mitochondria (E, n = 15). The results for relative mitochondrial membrane potential (MMP) were calculated using Zeiss 2012 imaging processing and 30 cells in each group were included. Data are shown as percentages of the control level. Images are representative of three independent experiments. 30 μg of total protein was used and the relative protein expression level were expressed as the ratio of target protein/ glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (D) Western blot analysis and quantitative results (H) of mitochondrial fission-related proteins Fis1 and Drp1 in cardiomyoctyes and H9c2 cells. (F,G). The values shown in this graph represent means ± S.D. of data from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the control group; # p < 0.05, ## p < 0.01, ### p < 0.001…

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  Figure 4. STVNa inhibited Iso-induced ROS generation, Trx1 and Prdx2 expression in H9c2 cells and rat cardiomyocytes. In total, 10 μM DCFH-DA was used to measure the intracellular ROS. Confocal images showing ROS levels in H9c2 cells (A) and cardiomyocytes (C). The micrographs are representative of three independent experiments. Scale bar: 50 μm. (B,D) Graphs of ROS levels in H9c2 cells (B, n = 30) and cardiomyocytes (D, n = 30). In total, 30 μg of total protein was extracted and the western blot was performed. (E) Immunoblotting showing the expression of Prdx2 and Trx1 in H9c2 cells and cardiomyocytes and the relative fold change in protein levels of Trx1 and Prdx2 compared to β-actin (F). The values shown in this graph represent means ± S.D. of data from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the control group; # p < 0.05, ## p < 0.05 versus the Iso treatment group.

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  Figure 5. STVNa inhibited the translocation of HDAC4 from the nucleus to the cytoplasm in H9C2 cells. (A) IF staining showing HDAC4 shuttling between the cytoplasm and nucleus. Green: rhodamine-phalloidin; red: HDAC4; blue: 4′, 6-diamidino-2-phenylindole (DAPI). Representative images from four independent experiments. Scale bar: 20 μm. (B) The ratio of nuclear HDAC4 to total HDAC4. The results were calculated using Zeiss 2012 imaging processing and 30 cells in each group were included. The values shown in this graph represent means ± S.D. of data from three independent experiments. *** p < 0.001 versus the control group; ## p < 0.01 versus the Iso treatment group.

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  Figure 6. Mechanistic illustration of the effects of STVNa on Iso-induced cardiac hypertrophy. STVNa elevates Trx1 and Prdx2 expression, inhibits cellular ROS overload and ROS-induced cell mitochondrial fission, thus restoring MMP and mitochondrial morphology. This in turn decreases ROS expression. In addition, Trx1 reciprocally inhibits the translocation of HDAC4 from the nucleus to the cytoplasm, thus inhibiting cardiac hypertrophy induced by Iso. STVNa: isosteviol sodium; βAR: β-adrenergic receptor; Iso: Isoprenaline; Prdx2: Peroxiredoxin 2; Trx1: Thioredoxin 1; Fis1: mitochondrial fission 1; Drp1: Dynamin-related protein 1; HDAC4: histone deacetylase 4.