Role of oxidative stress in doxorubicin-induced cardiotoxicity

Louisiana State Univ Hsc Shreveport, Shreveport LA

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Abstract

Doxorubicin (dox) is a widely used chemotherapy with dose-dependent cardiotoxicity. Genetic variability contributes to inter-patient variability in cardiotoxicity severity, with patients having particular genotypes conferring a 3-fold higher risk. Single-nucleotide polymorphisms (SNPs) that are associated with a higher risk of dox-cardiotoxicity in a particular group of patients do not show the same association in a different group of patients, emphasizing the role of the patient-dependent genetic landscape in dox-cardiotoxicity. On the other hand, we showed that oxidative stress is a major underlying mechanism of dox-cardiotoxicity. Notably, the oxidative stress severity is dependent on patient-specific genetic makeup. Our preliminary data identified a novel dox-cardiotoxicity-related genetic locus that encodes for a long non-coding RNA (lncRNA) EPHA2-AS1 and may regulate dox-cardiotoxicity via tuning oxidative stress in cardiomyocytes. Importantly, SNPs in EPHA2-AS1 are associated with altered EPHA2 expression, calcium levels, and hepatic inflammatory markers. EPHA2.AS1 is located on the antisense strand adjacent to EPH receptor A2 (EPHA2). EPHA2 promotes mitochondrial dysregulation and oxidative stress and is upregulated during the pathogenesis of multiple diseases. Therefore, we hypothesize that heart cells generated from patients with higher oxidative stress are more prone to severe dox-induced cardiotoxicity than those derived from patients with lower oxidative stress and that EPHA2.AS1 regulates EPHA2 in the human heart, which dysregulates cardiac mitochondria, exacerbating dox-induced cardiotoxicity. We will investigate the role of patient-specific cardiac regulation of oxidative stress in dox-induced cardiotoxicity. We will use a cohort of patient-specific human induced pluripotent stem cell-cardiomyocytes derived from patients (with low and high oxidative stress based on patient-specific genetic architecture) with and without dox-cardiotoxicity to examine ROS production, investigate their effect on dox-cardiotoxicity using our well-established biochemical and functional assays, reveal genes/pathways implicating patient-specific cardiac regulation using RNA-Seq, and screen for cardioprotective antioxidants in iPSC-cardiomyocytes and mice (Aim1). In Aim 2, we will define the role of EPHA2.AS1 in mitochondrial dysregulation-driven dox-cardiotoxicity. We will use CRISPR-Cas9-generated EPHA2.AS1 KO, OE, and isogenic iPSC-cardiomyocytes to assess its effect on the severity of dox-cardiotoxicity and on EPHA2 regulation and signaling. Furthermore, we will use CRISPR-Cas9 to introduce a potential candidate EPHA2.AS1 SNPs in isogenic iPSC-cardiomyocytes followed by characterization of mitochondria dysregulation-driven dox-cardiotoxicity. Moreover, we will use an Epha2 KO mouse to assess the implication of Epha2 in dox-cardiotoxicity in vivo. Successful completion of these aims will establish the implication of oxidative stress in dox-induced cardiotoxicity in a patient-specific manner, uncover novel patient-specific cardioprotective agents, and reveal a novel role for EPHA2.AS1 in the cardiac regulation of oxidative stress-induced cardiotoxicity.