Epigenetic Mechanisms Underlying Anthracycline-Induced Cardiotoxicity

Abstract

We sought to develop a mouse model of pediatric anthracycline cardiotoxicity to test our hypothesis for an epigenetic mechanism (including epigenetic memory), which may be operating to explain delayed-onset anthracycline-induced cardiotoxicity. Three week-old male and female C57Bl/6J mice were administered 5 weekly injections with low-dose doxorubicin (1mg/kg) (n=24) or saline (n=8). Half of the doxorubicin-treated (DOX) mice (n=12) and all controls (n=8) were sacrificed 1 week after the last injection; the rest of DOX mice (n=12) were given 4 weeks of drug-free holiday before sacrifice. Echocardiographic measurements were obtained before the first injection, 1 week after the final injection, and before sacrifice. Gene expression profiles were assessed by RNA-sequencing, genome-wide DNA methylation by reduced representation bisulphite sequencing (RRBS), and chromatin accessibility by ATAC-seq. These analyses were performed on purified pools of isolated cardiac myocytes, procured using a published Langendorff-free method. With the selected dosing regimen, there was no death in either the DOX or control mice. Left ventricular chamber dimension and wall thickness as well as fractional shortening did not differ between DOX and controls, implying the lack of obvious functional insufficiency from this dose of DOX. Similarly, DOX mice did not differ in body weight, heart weights (HW), and HW-to-tibia length ratios compared with controls. Cardiomyocyte dimensions and myocardial interstitial fibrosis (using picrosirius red) were the same between groups. Despite the lack of echocardiographic and histological changes in DOX mice, RNA-sequencing analysis revealed profound and significant differentially expressed genes between the DOX mice and controls. Importantly, a significant proportion of downregulated and upregulated genes persisted, despite the period of drug-free holiday (Figure 1). Moreover, despite being taken off DOX, cardiomyocytes appeared to display new gene expression changes taking place even 4 weeks after stopping DOX. Detailed results of RNA-sequencing, RRBS and ATAC-Seq will be presented during the meeting. These results suggest that cardiomyocytes display DOX-related epigenetic alterations, which may implicate the basis for delayed-onset anthracycline-induced cardiotoxicity.