Doxorubicin (DOX) is a potent chemotherapy agent, considered as a first-line treatment of various malignancies [1]. However DOX may cause heart injury similar to dilated cardiomyopathy, that can lead to heart failure [2]. Cardiotoxicity of anthracyclines may not be detected until several years after the treatment and may significantly impact a patient's survival and quality of life independently of the oncological prognosis [3]. Survivors of childhood cancer treated with DOX are at the greatest risk of cardiovascular morbidity and mortality. About 60% of pediatric cancer patients are treated with DOX-based chemotherapy [3] and 10% of these patients develop cardiomyopathy up to 15 years after the end of chemotherapy [4], [5], [6]. Despite the extensive published literature, the exact mechanism of DOX cardiotoxicity remains unclear. Several mechanisms have been described as critical mediators of DOX-induced cardiotoxicity, such as mitochondrial dysfunction, dysregulation of calcium and iron homeostasis, DNA damage and senescence [7]. There are currently no clinically verified, standardized procedure to monitor cardiotoxicity post-DOX therapy [8]. The pathological basis of DOX-related cardiac dysfunction, based on a limited number of autopsy studies and endomyocardial biopsy series, has not been completely defined [9], [10], [11], [12]. In addition, most of these studies lack control group of cancer patients not treated with DOX. In an experimental rat study, we have shown that DOX treatment resulted in downregulation of multiple transcripts associated with mitochondrial oxidative phosphorylation system (OXPHOS), increased inflammation, and dysregulation of calcium homeostasis in the heart [13].

Next generation sequencing (NGS) has enabled large-scale genetic, epigenetic and gene expression studies to uncover molecular mechanisms and identify biomarkers of various diseases, including cardiovascular diseases [14], [15], [16]. However, due to the tissue-specific nature of gene expression, a major limitation of RNA-sequencing in the investigation of cardiac diseases is the limited availability of cardiac tissue. Formalin-fixed, paraffin embedded (FFPE) tissues are routinely prepared from biopsies and stored for many years, providing source for retrospective analysis in clinical settings and basic research [17]. Staining of FFPE slides with hematoxylin and eosin (H&E) for histopathological assessment, or with specific antibodies for immunohistochemical analysis are commonly used in clinical diagnostics, and prediction of therapeutic response [18]. However, several reports showed that formalin fixation caused degradation of nucleic acids and induced sequence artefacts, limiting the use of FFPE tissue for genetic, epigenetic, and gene expression analyses [19], [20], [21]. In contrast to these challenges, other studies have reported that FFPE tissue was suitable for NGS-based gene expression analysis [22], [23], [24]. RNA sequencing of FFPE tissue samples has been successfully tested in biomarker discoveries and bioinformatics studies [25], [26], [27]. Several studies investigated the robustness of RNA-sequencing from FFPE tissue as well as the concordance between gene expression patterns in frozen and FFPE tissue and reported that FFPE tissue was suitable for NGS-based gene expression analysis [28], [29], [30].

In this study we examined RNA transcriptomic profile of FFPE heart samples of rats with experimentally induced- breast tumors, that had been treated chronically with DOX and compared the resulting dataset to microarray-based gene expression data obtained from fresh heart tissue of rats and to publicly available genomic data of cardiomyopathy.