HDAC inhibition via suberoylanilide hydroxamic acid ameliorates doxorubicin-induced cardiotoxicity

Figures

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  Fig. 1. Doxorubicin promotes Topo IIb binding at MEF2-associated genomic regions and activates MEF2-dependent transcription. A Heatmaps of topoisomerase IIb (Topo IIb) ChIP-seq enrichment in control (Ctrl) and doxorubicin-treated (Doxo) NRVMs at differentially enriched peaks (Doxo vs. Ctrl, two averaged replicates/treatment, FDR < 0.1). Rows correspond to genomic regions. These regions were plotted in the same order for Ctrl (first panel), doxorubicin-treated cells (second panel), and for the difference Ctrl subtracted from Doxo (Doxo-Ctrl) in the third panel. The rightmost heatmap shows MEF2-ChIP-seq enrichment over the same ordered set of regions. B Average Topo IIb ChIP-seq enrichment over all differentially enriched peaks in Ctrl and Doxo-treated NRVMs (±2 kb around the peak). C Distribution of Doxo-induced Topo IIb peaks to genomic locations. The pie chart shows the respective fraction of peaks located in promoters/transcriptional start sites (TSS), intergenic regions, introns, or 3′ UTRs. D Scatterplot highlighting the differential Topo IIb-bound regions after Doxo treatment with the highest fold change (FC) Doxo/Ctrl (y-axis). The x-axis indicates the genomic distance of the peak…

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  Fig. 2. SAHA attenuates doxorubicin-induced MEF2 activation by promoting nuclear HDAC4 retention and disrupting HDAC4 - 14-3-3 binding. A Dose response of suberoylanilide hydroxamic acid (SAHA) on myocyte enhancer factor 2 (MEF2) activity measured via luciferase assays. P values were calculated via a two-tailed Mann–Whitney test. Bars show mean ± SEM of 14–17 measurements per group from three independent experiments. Exact p values for the indicated comparisons were: Ctrl vs 1 µM: p < 0.0001, Ctrl vs 3 µM: p < 0.0001. B Doxorubicin (300 nM) increases MEF2 activity, which is attenuated by co-treatment with SAHA (3 µM), one-way ANOVA followed by Bonferroni’s multiple comparisons test. Bars show values for luciferase signal (mean ± SEM) of 12 measurements per treatment group from three independent experiments. Exact p values for the indicated comparisons were: MEF2-Luc vs. MEF2-Luc + Doxo, p < 0.0001; MEF2-Luc + Doxo vs. MEF2-Luc + Doxo + SAHA, p < 0.0001. C siRNA-driven knockdown of HDAC1-11 in NRVMs as indicated. MEF2 activity is shown as luciferase signal. Nine to ten measurements/group from two independent experiments. Data are presented as mean ± SEM. Statistical significance is…

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  Fig. 3. SAHA protects against chronic doxorubicin-induced cardiac dysfunction, fibrosis and transcriptional remodeling in vivo. A Graphical illustration of the in vivo model of doxorubicin (Doxo) and SAHA applications in 10-week-old female and male C57BL/6J mice as indicated. 12 mice (6 female and 6 male) were initially allocated to each treatment group. (NaCl, n = 12; Doxo, n = 12; SAHA, n = 12; Doxo + SAHA, n = 11). B Heart weight (HW) normalized to tibia length (TL). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparisons test. Exact p values for the indicated comparisons were: NaCl vs. Doxo, p = 0.0015; NaCl vs. SAHA, p = 0.8200; SAHA vs. Doxo + SAHA, p = 0.0131; Doxo vs. Doxo + SAHA, p = 0.6199. C Body weight (BW) at endpoint. Data are presented as box plots, where the center line indicates the median, the box represents the interquartile range (25th−75th percentiles), whiskers indicate the minimum and maximum values, and individual data points are shown. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparisons test. Exact p values for the indicated…

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  Fig. 4. Class IIa HDAC inhibition with TMP195 does not protect against chronic doxorubicin-induced cardiotoxicity in vivo. A Graphical illustration of the in vivo model of Doxorubicin (Doxo) and TMP195 (class IIa HDAC inhibitor) applications in 10-week-old female and male C57BL/6J mice. 12 mice (6 female and 6 male) were initially allocated to each treatment group. B Heart weight (HW) of the animals that survived (NaCl (n = 12), Doxo (n = 10), TMP195 (n = 11), Doxo+TMP195 (n = 6)) normalized to tibia length (TL). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparisons test. C Body weight (BW) at endpoint of survived animals (NaCl (n = 12), Doxo (n = 10), TMP195 (n = 11), Doxo+TMP195 (n = 6). Data are presented as box plots, where the center line indicates the median, the box represents the interquartile range (25th−75th percentiles), whiskers indicate the minimum and maximum values, and individual data points are shown. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparisons test. Exact p values for the indicated comparisons were: NaCl vs. Doxo, p = 0.0011; NaCl…

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  Fig. 5. Cardiomyocyte-specific HDAC4-deletion attenuates SAHA-mediated protection against doxorubicin-induced cardiotoxicity. A Graphical illustration of the in vivo experimental design showing 10–13-week-old female and male wild-type (WT) littermate controls and HDAC4-cKO mice on a C57BL/6N genetic background. WT littermate controls were treated with NaCl (n = 5; 2 females, 3 males), doxorubicin (Doxo; n = 7; 2 females, 5 males), or Doxo in combination with SAHA (n = 8; 5 females, 3 males), and HDAC4-cKO mice were treated with NaCl (n = 5; 3 females, 2 males), Doxo (n = 7; 3 females, 4 males), or Doxo+ SAHA (n = 7; 4 females, 3 males). B Kaplan–Meier survival analysis shows a significant difference in survival probability between the treatment groups (log-rank test for trend, p = 0.0097). C Heart weight (HW) of the animals that survived normalized to tibia length (TL); WT NaCl (n = 5); WT Doxo (n = 7); WT Doxo+SAHA (n = 7); HDAC4-cKO NaCl (n = 5); HDAC4-cKO Doxo (n = 5); HDAC4-cKO Doxo+SAHA (n = 4); in WT squares: fl−/fl− cre+, triangles: fl+/fl+ cre−. Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Bonferroni’s multiple comparisons…