Deep RNA-sequencing of cardiac tissue from early and late onset toxic cardiomyopathy patients suggest regulators of DNA methylation underlies late onset disease pathophysiology through epigenetic memory.. DIFFERENTIAL EXPRESSION OF EPIGENETIC MODIFIERS IN EARLY AND LATE CARDIOTOXIC HEART FAILURE REVEALS DNA METHYLATION AS A KEY REGULATOR OF CARDIOTOXICITY.

Anthracycline-induced cardiotoxicity is a well-known serious clinical entity. However, detailed mechanistic insights on how short-term administration of chemotherapy can lead to late and long-lasting cardiotoxicity, are still largely undiscovered. We hypothesize that chemotherapy provokes a long-lasting memory effect at the level of non-genetic DNA modifications which subsequently lead to cardiotoxicity even years after cessation of chemotherapy. We explored the temporal evolution of epigenetic modifiers in early and late cardiotoxicity due to anthracyclines by means of deep RNA-sequencing of human endomyocardial left ventricular biopsies and mass spectrometry of genomic DNA. Based on these findings, validation of differentially regulated genes was obtained by performing RT-qPCR. Finally, a proof-of-concept in vitro mechanistic study was performed to dissect some of the mechanistic aspects of epigenetic memory in anthracycline-induced cardiotoxicity. Correlation of expression of annotated genes between late and early onset cardiotoxicity revealed an R2 value of 0.98, demonstrating a total of 369 differentially expressed genes, of which 72% upregulated, and 28% of genes downregulated in later as compared to earlier onset cardiotoxicity. Gene ontology analysis showed significant enrichment of genes involved in the following pathways: oxidative phosphorylation, the oxidative stress response, FAS signaling, sarcomere organization, methyl-CpG DNA binding, chromatin remodeling, peptidase activator activity, regulation of transcription and positive regulation of apoptosis. Differential mRNA expression of genes involved in DNA methylation metabolism were confirmed by performing classical RT-qPCR in endomyocardial biopsies. In a larger biopsy cohort, it was shown that Tet2 expression was more abundantly expressed in cardiotoxicity biopsies versus control biopsies and versus niCMP patients. Moreover, an in vitro study was performed: following short-term doxorubicin treatment, H9c2 cells were cultured and passaged once they reached a confluency of 70-80%. When compared to vehicle-only treated cells, in doxorubicin-treated cells, three weeks after short term treatment, Nppa, Nppb, Tet1/2 and genes involved in active DNA demethylation were markedly upregulated. These alterations coincided with a loss of DNA methylation and a gain in hydroxymethylation, reflecting the epigenetic changes seen in the endomyocardial biopsies. Short-term administration of anthracyclines provokes long-lasting epigenetic modifications in cardiomyocytes both in vivo and in vitro, which explain in part the time lapse between the use of chemotherapy and the development of cardiotoxicity and, eventually, heart failure.