Early transcriptional alteration of histone deacetylases in a murine model of doxorubicin-induced cardiomyopathy

Fig 1. Morphometric analysis of doxorubicin-treated mice. (A) Body weight at 10 weeks of age, prior to implantation of the Alzet pumps. (B) Body weight at the end of the trial (12 weeks of age) (C) tibia length (D) heart weight (E) heart weight to tibia length index. All values are mean ± SEM (n = 6 WT PBS, n = 6 WT doxorubicin), One-way ANOVA with Bonferroni post-hoc test: *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 2. Gross cardiac morphology of hearts treated with doxorubicin. Representative vinculin staining (green) in WT (PBS) mice (A) and WT doxorubicin (B). Nuclei (red) were visualized with draq5. Replacement fibrosis was detected in the doxorubicin treated hearts (D) but not in vehicle hearts (C), visualising with the anti-collagen VI antibody (green). Nuclei (blue) were visualised with DAPI, (E, F).

Fig 3. Re-activation of foetal gene markers in hearts treated with doxorubicin. Anp (atrial natriuretic peptide), Bmp-10 (brain morphogentic protein 10), Bnp (brain natriuretic protein), and Myh-7 (myosin heavy chain beta) were elevated in the hearts treated with doxorubicin. Bdnf (brain derived neurotophic factor) mRNA remained unchanged. All Taqman qPCR values were normalized to the geometric mean of three housekeeping genes: Actb, Cyc1 and Gapdh. Error bars are SEM (n = 6). One-way ANOVA with Bonferroni post-hoc test: *p < 0.05, **p < 0.01; ***p < 0.001.

Fig 4. Chronic administration of doxorubicin leads to a significant transcriptional deregulation of Hdacs. (A) Transcript levels of Hdac4, Hdac5, Hdac6, Hdac7, Hdac10 and Hdac11 were increased, while Hdac2 mRNA was significantly reduced, in the hearts of WT mice treated with doxorubicin. All Taqman qPCR values were normalized to the geometric mean of three housekeeping genes: Actb, Cyc1 and Gapdh. Error bars are ±SEM (n = 6). One-way ANOVA with Bonferroni post-hoc test: *p < 0.05, **p < 0.01; ***p < 0.001. (B) Representative western immunoblots of 20 μg of heart homogenates from WT doxorubicin and vehicle-treated mice (PBS). Protein levels of HDAC2 are significantly down-regulated; HDAC3 remains unchanged; HDAC4 and HDAC5 are significantly up-regulated. Relative expression levels of HDACs were obtained by normalisation to ATP5b in densitometry; values are mean ± SEM (n = 6). Student’s t test: *p < 0.05, **p < 0.01, ***p < 0.001.