HDAC inhibitors arrest cardiac fibroblast cell cycle progression via upregulation of p57. Cultured NRVFs were synchronized by serum starvation for 24 hours and subsequently stimulated with FBS (20%) in the absence or presence of DMSO vehicle, MGCD0103 (1 μM), TSA (200 nM), or apicidin (3 μM). At 32 hours after FBS treatment, cells were stained with propidium iodide and cell cycle progression was quantified by flow cytometry. (A) Representative histograms of propidium iodide–stained cells. (B) Quantification of cells in the S phase of the cell cycle (n = 3 plates of cells/condition). Data represent means ± S.E.M. (*P < 0.05 versus all other conditions). (C) qRT-PCR was performed with RNA isolated from synchronized ARVFs stimulated for 32 hours in the absence or presence of HDAC inhibitors, as described above. qRT-PCR of p57 was performed with three plates of cells/condition, and technical duplicates were run for each individual sample. Data represent means ± S.E.M. (*P < 0.05 versus vehicle-treated cells). MGCD, MGCD0103; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; Veh, vehicle.

Benzamide HDAC inhibitors stimulate PAI-1 protein expression in cardiac fibroblasts. (A) Immunoblot analysis of PAI-1 expression in cultured, passage 2 ARVFs treated with DMSO vehicle, MGCD0103 (1 μM), TSA (200 nM), or apicidin (1–3 μM) for 24 hours. (B) Treatment of neonatal rat ventricular cardiomyocytes for 48 hours with MGCD0103 in the absence or presence of the hypertrophic agonist PE (10 μM) failed to enhance PAI-1 expression. (C) The PAI-1 signal in (B) was quantified by densitometry and normalized to the loading control, calnexin. Data represent means ± S.E.M. (differences were not statistically significantly different). (D) Chemical structure of the benzamide class I HDAC inhibitor, MS-275. (E) Treatment of ARVFs with MGCD0103 (1 μM) or MS-275 (1 μM) for 24 hours led to increased PAI-1 protein expression. (F) Passage 0, 1, 2, or 3 ARVFs were treated with MGCD0103 (1 μM) for 16 hours. The ability of MGCD0103 to increase PAI-1 expression was unaffected by myofibroblast phenotype, as defined by the progressive increase in α-smooth muscle actin expression upon fibroblast passage. For all blots, each lane represents an independent plate of cells. (G) The PAI-1 signal in (F), as well as from other samples from an independent preparation of ARVFs (not shown), was quantified by densitometry and normalized to the loading control, calnexin (n = 5 plates of cells/condition). Data represent means ± S.E.M. (*P < 0.05 versus vehicle-treated cells). MGCD, MGCD0103; PE, phenylephrine; Veh, vehicle.

Differential effects of HDAC inhibitors on fibrosis-related gene expression. (A) qRT-PCR was performed with RNA isolated from cultured AMVFs treated with DMSO vehicle, MGCD0103 (1 μM), or TSA (200 nM) for 24 hours (n = 3 plates of cells/condition with technical duplicates run for each sample). Data represent means ± S.E.M. (*P < 0.05 versus all other conditions). (B) RNA was isolated from cultured AMVFs treated with DMSO vehicle or MGCD0103 (1 μM) for 24 hours (n = 3 plates of cells/condition). Pooled cDNA from each group was analyzed for expression of 84 fibrosis-related genes using the QIAGEN RT2 Profiler PCR Array for mouse fibrosis. Shown are the relative changes in mRNA expression between MGCD0103-treated and vehicle-treated cells for all genes that were found to have ≥2-fold change in gene expression. (C) qRT-PCR for MMP13 and Plau was performed with RNA from independent AMVFs to confirm PCR array results. Data shown are target gene expression relative to B2M expression. B2M, β-2 microglobulin; BMP, bone morphogenetic protein; Ccl, CC chemokine ligand; HGF, hepatocyte growth factor; IFN, interferon; IL, interleukin; ITGB, β integrin; MGCD, MGCD0103; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction; TIMP, tissue inhibitor of metalloproteinases; Veh, vehicle.

MGCD0103-induced PAI-1 expression is mediated by HDAC1 and HDAC2 inhibition. (A) Western blot analysis of PAI-1 expression in cultured ARVFs treated with the isoform-selective HDAC inhibitors BA-60 (HDAC1/2 inhibitor, 300 nM) and BRD3308 (HDAC3 inhibitor, 1 μM) for 24 hours. (B) The PAI-1 signal in (A) was quantified by densitometry and normalized to the loading control, calnexin. Data represent means ± S.E.M. (*P < 0.05 versus vehicle-treated cells). (C) AMVFs were infected with shControl or lentiviruses encoding shRNAs to target HDAC1 and/or HDAC2. After 96 hours of infection, cell homogenates were subjected to immunoblotting with antibodies specific for HDAC1, HDAC2, and PAI-1. Calnexin served as a loading control. Each lane represents an independent plate of cells. BA-60, biaryl-60; shControl, short-hairpin control lentivirus; shRNA, short-hairpin RNA; Veh, vehicle.

MGCD0103 promotes de novo synthesis of PAI-1. (A) qRT-PCR was performed with RNA isolated from cultured NRVFs pretreated with either DMSO vehicle or MGCD0103 for 4 hours followed by the addition of CHX at 30 μM for 18 hours (n = 4 plates of cells/condition). Data represent mean PAI-1:18S expression ± S.E.M. (*P < 0.05 compared with CHX alone). (B) Immunoblot analysis of PAI-1 expression from ARVFs treated as described above with CHX and MGCD0103. Each lane represents an independent plate of cells. MGCD, MGCD0103; qRT-PCR, quantitative reverse transcriptase polymerase chain reaction.

MGCD0103 does not activate the TGF-β/SMAD pathway. (A and B) ARVFs were treated with 1 μM MGCD0103 (A) or 5 ng/ml TGF-β (B) for the indicated times prior to cell homogenization and immunoblotting with antibodies specific for PAI-1, phospho-SMAD2/3, or total SMAD2/3. Calnexin served as a loading control. Each lane represents an independent plate of cells. MGCD, MGCD0103.