Fig. 1.
Therapeutic benefit of SAHA targeting the Ac/Dc axis. a Illustration of ISO-induced Takotsubo-like cardiomyopathy. In the pre-clinical model, mice were divided into four groups. Group 1 received IP saline and served as healthy controls. Group 2 served as a SAHA only control receiving IP injections every third day. The remaining groups (3 and 4) received subcutaneous ISO once daily for the first 5 days to induce TS-like cardiomyopathy. Group 4 commenced SAHA treatment on the day after ISO administration was concluded (REVersal). Mice were sacrificed 9 days following administration of the fifth ISO dose, on day 14. b Quantitative assessment of LV collagen content. Representative picrosirius red-stained images are shown. Data presented as the mean ± SEM. Control vs. ISO, exact P-value 0.0001 (***), ISO vs. REVersal, exact P-value 0.001 (**). Calculated using unpaired t-test. Picrosirius red staining also shown. Control (n = 5), SAHA (n = 3), ISO (n = 5) and Reversal (ISO/SAHA n = 5). c FPA-FTIR chemical images showing lipid and protein distribution using a Bruker Hyperion 2000 FTIR microscope equipped with a 64 × 64 element FPA detector and a 15× objective lens and acquired in transmission mode. d Comparison of FPA-FTIR (i) absorbance spectra and (ii) inverted second derivative spectra within the amide region (1800–1200 cm−1). e Apical LV tissues were assessed for TS-like response to SAHA treatment using Li-COR protein quantification, transcriptional expression index (TEI) determined by RNA-seq and chromatin modification by ChIP-seq. f Heatmap of cardiomyopathy pathways from multi-contrast GSEA derived from the Human Phenotype Ontology (HPO). Pathway significance is calculated by FDR P < 0.05 using multi-contrast analysis for ISO vs. healthy controls, SAHA vs. healthy controls, ISO/SAHA vs. healthy controls (REV), SAHA vs. ISO and ISO/SAHA vs. ISO (REV) (n = 3 each group). g Histone lysine map for the major sites assessed of H3 acetylation and methylation, these include the transcriptionally permissive marks H3K4me3, H3K9/14ac, and H3K36me3. The suppressive marks analysed include H3K9me1, H3K9me2, H3K9me3, H3K27me3. Lysine acetylation at positions K9 and K14 are linked in the schematic. h Protein levels of histone modification in apical LV tissue from ISO and SAHA-administered mice. Bar plots represent mean values of Li-COR Odyssey quantification of protein adjusted against total histone H3. Data are represented as the mean ± SEM. *P < 0.05, ** P < 0.01 vs. control, #P < 0.05. Calculated using one-way ANOVA (n = 3 each group). i Protein levels of histone-modifying enzymes (lysine acetyltransferases EP300 and CBP including the deacetyltransferases RYBP, HDAC1, HDAC2 and HDAC3) in apical LV tissue from ISO and SAHA-administered mice. Bar plots represent mean values of Li-COR Odyssey quantification of protein adjusted against total α-tubulin. Data are represented as the mean ± SEM. *P < 0.05, **P < 0.01 vs. control, #P < 0.05, ##P < 0.01, ###P < 0.001. Calculated using one-way ANOVA (n = 3 each group). j RNA-seq data derived from apical LV shows the transcriptional expression index (TEI) of genes in the major cardiomyopathy pathways identified above (f) in ISO-induced injury and influenced by SAHA treatment. Pathways include cardiac development, cardiomyopathy and heart failure, congestive heart failure, cardiomyopathy, cardiac scarring, collagen biosynthesis, extracellular matrix organisation, cardiac metabolism and genes associated with genetic variation for Takotsubo derived from SCAAR (Swedish Coronary Angiography and Angioplasty Registry). Highly connected pathways include electron transport chain (ETC), mitochondrial complex (MC), muscle contraction (MN) and collagen formation (CN). Gene–gene name (filtered by FDR P-value < 0.05, annotated to mm10 mouse genome); transcription expression index–median gene expression of transcripts in REV (n = 3, ISO/ SAHA) and ISO (n = 3) groups relative to healthy controls (n = 3) expressed as log2 fold-change. k Cardiac benefit of SAHA treatment in ISO-induced model of Takotsubo. Transcriptional expression index is shown for genes influenced by SAHA in the reversal model of ISO-induced cardiomyopathy. Genes implicated with Takotsubo-like injury are shown on the left of the axis (control relative to ISO-induced injury groups). Transcribed genes that shift to the right are closer to healthy control in the SAHA treatment group; REVersal (control vs. ISO/SAHA groups) n = 3/treatment group. l Heatmap of H3K9/14ac ChIP-seq signals relative to the TSS ( + /− 2500 bp) of genes identified above in panel j. Left insert columns show log2(fold-change) of H3K9/14ac signals for ISO vs. healthy controls, SAHA vs. healthy controls and (REV) ISO/SAHA vs. healthy controls. Elevated H3K9/14 acetylation (Ac) signals are shown in red on the colour gradient and the reduction of H3K9/14 acetylation or deacetylation (Dc) signals are shown in blue. No change in H3K9/14ac signal is denoted as white and grey is undetermined. Right insert columns show H3K9/14ac signals for SAHA vs. ISO and (REV) ISO/SAHA vs. ISO groups. Control (n = 5), ISO (n = 5), SAHA (n = 3) and REV (n = 5). m ChIP-seq contour profiles for H3K9/14ac relative to the TSS+/−5kb are shown for several representative genes and their associated pathways; Ptgs2 (cardiac development), mtNd5 (cardiomyopathy and heart failure), Hsd17b10 and Slc4a1 (cardiomyopathy), Car4 (cardiac scarring) and Adrb1 (Takotsubo SCAAR). Control (n = 5), ISO (n = 5), SAHA (n = 3) and REV (n = 5). n Proposed cardiac benefit of SAHA treatment in TS-like cardiomyopathy involves an Ac/Dc axis implicating the CBP/EP300 acetyltransferases and RYBP deacetyltransferase. SAHA treatment influences core genes associated with the Ac/Dc axis and cardiomyopathy. Combined ChIP-seq Ac/Dc profiles are shown for cardiomyopathy genes identified above in panels j–l (red Ac profile represents ISO vs. Control and blue Dc profile represents REV vs. ISO). Control (n = 5), ISO (n = 5), SAHA (n = 3) and REV (n = 5)