Identifying common pathways for doxorubicin and carfilzomib-induced cardiotoxicities: transcriptomic and epigenetic profiling

Figures

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  Fig. 1. Human cardiomyocytes (HCM-ls) cell viability treated with Doxorubicin and Carfilzomib. (A) Using Cell Titre Glow, a dose response curve of HCM-ls viability treated with Doxorubicin and Carfilzomib for 72 h, EC50 was determined to be approximately 1 µM for both compounds (n = 3); (B) Using Lactate dehydrogenase (LDH) assay, a dose response curve of HCM-ls Doxorubicin and Carfilzomib-induced cytoxicity was determined (n = 3).

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  Fig. 2. Doxorubicin-induced DNA methylation changes in HCM-ls. (A) Volcano plot of the significantly different DMPs. 89 DMPs passed the significance threshold of 5.97E–5 (purple) of which 66/89 (74.2%) were hypermethylated while 23/89 (25.8%) were hypomethylated. 100,563 DMPs passed the less stringent threshold of P < 0.05 (green) with 54,734 (54.4%) being hypermethylated whilst 45,829 (45.6%) were hypomethylated. (B) Representative image of the probes that passed the significance threshold of 5.97E–5 and the enrichment in the corresponding genomic regions. (C) Top 15 Gene Ontology (GO) terms associated with hypermethylated probes. (D) Top 15 GO terms associated with hypomethylated probes. (E) Top 10 KEGG pathways associated with differentially methylated probes (P < 0.05).

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  Fig. 3. Carfilzomib-induced DNA methylation changes in HCM-ls (A) Volcano plot of the significantly different DMPs. 58 DMPs passed the significance threshold of 5.97E–5 (purple) of which 17/58 (29.3%) were hypermethylated while 41/58 (70.7%) were hypomethylated. 65,538 DMPs passed the less stringent threshold of P < 0.05 (green) with 22,185 (33.9%) being hypermethylated whilst 43,353 (66.1%) were hypomethylated. (B) Representative image of the probes that passed the significance threshold of 5.97E–5 and the enrichment in the corresponding genomic regions. (C) Top 15 GO terms associated with hypermethylated probes. (D) Top 15 GO terms associated with hypomethylated probes. (E) Top 10 KEGG pathways associated with differentially methylated probes (P < 0.05).

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  Fig. 4. Overlapping changes in DNA methylation that were observed for both Doxorubicin and Carfilzomib treated HCM-ls. (A) Representative diagram of the genes that are differentially methylated and in the same direction for both Doxorubicin and Carfilzomib treated HCM-ls (P < 0.05). There are 10,270 DMPs (6,623 DMPs hypomethylated by both treatments and 3,647 DMPs hypermethylated by both treatments; outlined in red) that are associated with chemotherapy damage to the cells regardless of the drug that was used. (B) Top 15 GO terms associated with the hypermethylated probes. (C) Top 15 GO terms associated with the hypomethylated probes. (D) The top 10 dysregulated KEGG pathways.

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  Fig. 5. Doxorubicin-induced gene expression changes across the transcriptome in HCM-ls. (A) Smear plot of the significant differentially expressed genes (FDR < 0.05, red) vs the insignificant genes (black) for Doxorubicin treated cells. 6,197/11,011 (56.3%) were upregulated, 4,814/11,011 (43.7%) were downregulated. (B) Heatmap of the top 50 differentially expressed genes for the Dox vs Vehicle comparison, generated with edgeR version 3.38.4 (https://bioconductor.org/packages/release/bioc/html/edgeR.html). (C) 15 top GO terms associated with upregulated genes. (D) 15 top GO terms associated with downregulated genes. (E) The top 10 dysregulated KEGG pathways. GO terms: Gene Ontology Biological Process (GO BP), Gene Ontology Cellular Component (GO CC), and Gene Ontology Molecular Function (GO MF).

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  Fig. 6. Carfilzomib-induced gene expression changes across the transcriptome in HCM-ls. (A) Smear plot of the significant differentially expressed genes (FDR < 0.05, red) vs the insignificant genes (black) for Carfilzomib treated cells. 6,070/10,618 (57.2%) were upregulated, 4,548/10,618 (42.8%) were downregulated. (B) Heatmap of the top 50 differentially expressed genes for the Cz vs Vehicle comparison, generated with edgeR version 3.38.4 (https://bioconductor.org/packages/release/bioc/html/edgeR.html). (C) Top 15 GO terms associated with upregulated genes. (D) Top 15 GO terms associated with downregulated genes. (E) The top 10 dysregulated KEGG pathways.

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  Fig. 7. Overlapping changes to gene expression that were observed for both Doxorubicin and Carfilzomib treated HCM-ls. (A) Representative diagram of the genes that are both differentially expressed in the same direction and at an FDR < 0.05. There are 5,643 DEGs (2,320 DEGs downregulated by both treatments and 3,323 DEGs upregulated by both treatments) that are associated with chemotherapy damage to the cells regardless of the drug that was used. (B) Top 15 GO terms associated with the upregulated probes. (C) Top 15 GO terms associated with the downregulated probes. (D) The top 10 dysregulated KEGG pathways.