Fig 2: HDAC8 selective inhibitor attenuates isoproterenol-induced cardiac hypertrophy in H9c2 cells (A) H9c2 cells were treated with various concentrations of PCI34051 for 24 h and cell viability was evaluated (n = 12). NS = not significant (B) H9c2 cells were seeded in 12-well plates and serum starved overnight. Cells were treated with isoproterenol (10 µM) for 24 h in the presence of PCI34051 (100 nM) or vehicle (DMSO). Phalloidin staining was performed to determine cell size. Representative images (B) and quantification of cell size (C) are shown (n = 88–142). Scale bar = 50 µm ***p < 0.001; ###p < 0.001 (D-F) H9c2 cells were serum starved overnight and treated with vehicle or isoproterenol (10 µM, 6 h) in the presence or absence of PCI34051 (10 or 100 nM, 5 h). mRNA expression levels of Nppa (D), Nppb (E), Myh7 (F) were determined using RT-PCR (n = 6–9). *p < 0.05, **p < 0.01, and ***p < 0.001; #p < 0.05, ##p < 0.01, and ###p < 0.001. Data are presented as mean ± S.E. Statistics: one-way ANOVA followed by Bonferroni post hoc tests.

Fig 3: Knockdown of HDAC8 reduces isoproterenol-induced cardiomyocyte hypertrophy in H9c2 cells. H9c2 cells were transfected with control or HDAC8 siRNA and then stimulated with isoproterenol (10 μM) for 9 h (A–C) mRNA expression levels of Hdac8, Nppa, and Nppb in these cells (n = 8–9). ***p < 0.001; ###p < 0.001; @@@ p < 0.001. NS = not significant (D-F) Representative western blot images (D) and quantification of HDAC8 (E) and BNP (F) protein levels (n = 5–6). ***p < 0.001; ###p < 0.001; @ p < 0.05 and @@@ p < 0.001 (G) Phalloidin-stained cells transfected with control or HDAC8 siRNA and then treated with isoproterenol (H) Quantification of cell size (n = 100–116). ###p < 0.001; @@@ p < 0.001. NS = not significant. Data are presented as mean ± S.E. Statistics: one-way ANOVA followed by Bonferroni post hoc tests.

Fig 4: Overexpression of HDAC8 induces cardiac hypertrophy in H9c2 cells (A) mRNA expression levels of HDAC8 in heart tissues from sham, ISP, and ISP + PCI34051 (30 mg/kg/day)-treated mice (n = 8). ***p < 0.001; ##p < 0.01 from one-way ANOVA followed by Bonferroni post hoc tests (B-C) Western blot analysis of HDAC8 and GAPDH (loading control) from the samples described in (A) (B) Representative blots (C) Quantification of HDAC8 (n = 4). **p < 0.01; ##p < 0.01 from one-way ANOVA followed by Bonferroni post hoc tests (D) H9c2 cells were transfected with pCMV6-HA-Myc or pCMV6-HDAC8-HA-Myc for 48 h Hdac8 mRNA expression levels. ***p < 0.001 from the Student’s t test (E) The transfected cells were fixed and stained using anti-HDAC8 antibody and Alexa Fluor 488 phalloidin. Scale bar = 50 μm. Red, green, and blue indicate HDAC8, actin filaments, and nuclei, respectively (F) Quantification of cell size (n = 70–75). ***p < 0.001 from the Student’s t test (G) Nppa and Nppb mRNA expression levels in these cells (n = 6). ***p < 0.001 from the Student’s t test (H) Western blot analysis for ANP, BNP, and HDAC8. Representative blots (I) Quantification of HDAC8, ANP, and BNP protein levels (n = 4). *p < 0.05, **p < 0.01, and ***p < 0.001 from the Student’s t test.

Fig 5: Relative changes in the protein levels of Troponin I, troponin C, and ANP in the heart of I. tridecemlineatus over the torpor-arousal cycle.Other information as in Fig. 2.