Protocatechuic acid attenuates isoproterenol-induced cardiac hypertrophy via downregulation of ROCK1–Sp1–PKCγ axis

Figures

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  Figure 1. Protocatechuic acid reduces isoproterenol-induced cardiomyocyte hypertrophy in vitro. (A) H9c2 cells were treated with protocatechuic acid (0, 1, 10, 100 μM) for 24 h as indicated and cell viability was determined using the MTT assay (n = 8). NS indicates not significant. (B,C) H9c2 cells were serum-starved overnight and then treated with vehicle or protocatechuic acid in the presence or absence of isoproterenol (10 μM) for 24 h. Cell size was quantified by measuring the cell surface area of Alexa Fluor 488 phalloidin-stained cells (n = 40 cells). Scale bar = 50 μm. ***P < 0.001; ###P < 0.001. (D‒H) H9c2 cells were treated with isoproterenol (10 μM) in the presence or absence of protocatechuic acid (10 μM) for 6 h. The mRNA expression levels of Nppa (D), Nppb (E), and Myh7 (F) were determined by RT-PCR and normalized to Gapdh (n = 10). **P < 0.01 and ***P < 0.001; #P < 0.05 and ###P < 0.001. (G) Representative western blot images are shown; β-actin was used as a loading control. (H) Quantification of BNP protein levels (n = 6). ***P < 0.001; ###P < 0.001. ISO, isoproterenol; PCA, protocatechuic acid. Data are presented as the mean ± S.E. Statistical analysis: one-way ANOVA…

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  Figure 2. Protocatechuic acid attenuates isoproterenol-induced cardiac hypertrophy in vivo. (A) Representative images of gross hearts from sham, isoproterenol, and isoproterenol + protocatechuic acid treated mice. (B,C) Heart weight to body weight ratio (HW/BW) and heart weight to tibia length ratio (HW/TL) of the mice described in (A) (n = 8). ***P < 0.001; ###P < 0.001. (D) Representative B-mode and M-mode echocardiograms are shown. Cardiac hypertrophy was determined by echocardiography after 5 days of isoproterenol administration. (E,F) Left ventricular posterior wall thickness (LVPWd, mm) and interventricular septum thickness (IVSd, mm) in mice (n = 8). ***P < 0.001; ##P < 0.01 and ###P < 0.001. ISO, isoproterenol; PCA, protocatechuic acid. (G) Representative images of H&E (upper panel) and wheat germ agglutinin (WGA, lower panel) staining of left ventricle papillary muscle from sham, isoproterenol, and isoproterenol + protocatechuic acid-treated mice. Scale bar = 50 μm. (H) Cross-sectional area quantification of H&E-stained hearts described in (G), n = 161 cells. ***P < 0.001; ###P < 0.001. (I,J) Total RNA was isolated from the hearts of mice (n = 8). mRNA levels of Nppb (I) and Myh7…

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  Figure 3. ROCK1 knockdown reduces isoproterenol-induced cardiomyocyte hypertrophy. (A) Heart tissues from sham, isoproterenol, and isoproterenol + protocatechuic acid treated mice were analyzed by western blotting using anti-ROCK1 and GAPDH antibodies. Representative images. (B) Quantification of ROCK1 protein levels in heart tissues (n = 6); the results were normalized to GAPDH. ***P < 0.001; ##P < 0.01. (C − E) H9c2 cells were serum starved overnight and then treated with isoproterenol (10 μM) in the presence or absence of protocatechuic acid (10 μM) for 6 h. (C) mRNA levels of Rock1 (n = 6). **P < 0.01; ##P < 0.01. (D) Western blot analysis. Representative images of ROCK1. (E) Quantification of western blots presented in (D, n = 6). **P < 0.01; #P < 0.05. (F) H9c2 cells were transfected with either control or ROCK1 siRNA for 2 days. The mRNA levels of Rock1 were analyzed using RT-PCR. ***P < 0.001, Student’s t-test. (G) RT-PCR analysis of Rock1, Nppa, and Nppb expression in H9c2 cells transfected with either control or ROCK1 siRNA in the presence or absence of isoproterenol (10 μM, n = 6). ***P < 0.001; ###P < 0.001. (H) Western blot analysis of cells described in (G). Representative…

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  Figure 4. Sp1 knockdown reduces isoproterenol-induced cardiac hypertrophy in H9c2 cells. (A) Sp1 mRNA levels in H9c2 cells treated with isoproterenol (10 μM) in the presence or absence of protocatechuic acid (10 μM, n = 6). ***P < 0.001; ###P < 0.001. (B) Sp1 mRNA levels in heart tissues treated with protocatechuic acid (100 mg/kg/day) in the presence or absence of isoproterenol (25 mg/kg, n = 8). ***P < 0.001; ###P < 0.001. (C) Representative western blot images showing SP1 protein levels in mouse hearts. GAPDH was used as a loading control. (D) Quantification of SP1 protein levels (n = 6). ***P < 0.001; ###P < 0.001. (E‒G) H9c2 cells were transfected with control or Sp1 siRNA, serum starved overnight, and then treated with isoproterenol (10 μM). *P < 0.05 and ***P < 0.001; ###P < 0.001; @@@P < 0.001. mRNA levels of Sp1, Nppa, and Nppb (n = 9). (H) Merged images of H9c2 cells transfected with Sp1 siRNA, incubated with or without isoproterenol stimulation (10 μM) for 24 h, and then stained with Alexa Fluor 488 phalloidin (green) and DAPI (blue). (I) Cell size was quantified by measuring the cell surface area of the Alexa Fluor 488 phalloidin-stained cells (n = 20 cells). Scale bar = 50 μm.…

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  Figure 5. Protocatechuic acid ameliorates Sp1 overexpression-induced cardiac hypertrophy. H9c2 cells were transfected with empty vector or pCMV-Sp1 for 24 h. (A) Transcript levels of mouse Sp1 were quantified (n = 8). **P < 0.01, Student’s t-test. (B) The expression of hypertrophic marker genes (Nppa (n = 6), Nppb (n = 8), and Myh7 (n = 6)) was determined by RT-PCR. **P < 0.01 and ***P < 0.001, Student’s t-test. (C,D) Representative western blot images and the quantification of Sp1 and BNP in pCMV-Sp1 and vector-transfected cells (n = 6). **P < 0.01 and ***P < 0.001, Student’s t-test. (E,F) Quantification of Sp1 and Nppb mRNA levels in cells transfected with vector or pCMV-Sp1 and treated with protocatechuic acid (10 μM, n = 6). ***P < 0.001; ###P < 0.001. (G) Sp1 and BNP protein expression in cells described in (E,F), representative Western blot images. (H) Representative images of the cells transfected with vector or pCMV-Sp1, incubated with or without protocatechuic acid (10 μM), and then stained with anti-Sp1, Alexa Fluor 488 phalloidin, and DAPI. Sp1-positive cells (red); nuclei (blue); actin filaments (green). Scale bar = 50 μm. (I) Quantification of cell surface area (n = 30 cells).…

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  Figure 6. Protocatechuic acid attenuates isoproterenol- or Sp1 overexpression-induced PKCγ expression. (A) The quantification of PKC isoforms in H9c2 cells treated with isoproterenol (10 μM) in the presence or absence of protocatechuic acid (10 μM) for 6 h (n = 6). ***P < 0.001; ##P < 0.01 and ###P < 0.001; NS not significant. Data are presented as the mean ± S.E. Statistical analysis: one-way ANOVA followed by Bonferroni post hoc tests. (B) The quantification of PKC isoforms in H9c2 cells transfected with pCMV-Sp1 and treated with protocatechuic acid (10 μM, n = 6). ***P < 0.001; ##P < 0.01; NS not significant. Data are presented as the mean ± S.E. Statistical analysis: one-way ANOVA followed by Bonferroni post hoc tests. (C) Representative western blots (the same blots as presented in Fig. 5C) of pCMV-Sp1-transfected cells; β-actin was used as a loading control. (D) Quantification of western blots presented in (C, n = 6). ***P < 0.001, Student’s t-test. (E − G) H9c2 cells were transfected with control, ROCK, or Sp1 siRNA for 24 h, serum starved, and then treated with isoproterenol (10 μM). (E) The mRNA levels of Prkcg were determined by RT-PCR (n = 7). ***P < 0.001; ###P < 0.001; NS not…

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  Figure 7. PKCγ knockdown reduces isoproterenol-induced cardiac hypertrophy in H9c2 cells. (A‒E) H9c2 cells were transfected with empty vector or pCMV3-N-GFPSpark-PKCγ and then treated with protocatechuic acid (10 μM). (A) Representative western blot images showing PKCγ and BNP protein levels in pCMV3-N-GFPSpark-PKCγ-transfected cells. β-actin was used as a loading control. (B,C) Quantification of PKCγ and BNP protein levels (n = 6). ***P < 0.001; ###P < 0.001. (D,E) Representative images of cells transfected with pCMV3-N vector or pCMV3-N-GFPSpark-PKCγ, incubated with or without protocatechuic acid (10 μM), and then stained with anti-PKCγ antibody, Alexa Fluor 488 phalloidin, and DAPI. PKCγ-positive cells (red); nuclei (blue); actin filaments (green). Scale bar = 50 μm. (F,G) H9c2 cells were transfected with control or PKCγ siRNA, serum starved overnight, and then treated with isoproterenol (10 μM). The mRNA levels of Prkcg and Nppb were determined by RT-PCR (n = 6). **P < 0.01 and ***P < 0.001; ###P < 0.001; @@@P < 0.001. (I) Merged images of H9c2 cells transfected with PKCγ siRNA, incubated with or without isoproterenol (10 μM) for 24 h, and then stained with Alexa Fluor 488 phalloidin…

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  Figure 8. Protocatechuic acid does not regulate the expression of ROCK1 and Sp1 at the transcriptional level. (A,B) H9c2 cells were cultured with isoproterenol (10 μM) and then incubated with protocatechuic acid (10 μM) in the presence or absence of actinomycin D (2.5 μg/ml) for 6 h. The mRNA levels of Rock1 and Sp1 were quantified using RT-PCR and normalized to Gapdh. Data are presented as the mean ± S.E. Statistical analysis: one-way ANOVA followed by Bonferroni post hoc tests. ***P < 0.001; ###P < 0.001; NS, not significant. Graphs were prepared using GraphPad 5.0. (C) Schematic diagram of proposed mechanisms involved in isoproterenol-induced cardiac hypertrophy. Isoproterenol increases the expression of ROCK1, Sp1, and PKCγ. Sp1 increases the size of cardiomyocytes and induces PKCγ expression. Our results suggest that PKCγ is a downstream target of ROCK1 and Sp1. Protocatechuic acid treatment suppresses isoproterenol-induced cardiac hypertrophy via the downregulation of ROCK1–Sp1–PKCγ axis.