1Deutsches Herzzentrum der Charite (DHZC) Innere Medizin-Kardiologie Berlin, Deutschland
BACKGROUND: Cardiovascular diseases and cancer are the leading causes of death in the Western world. Doxorubicin is a highly effective anti-cancer drug of the anthracycline class. However, its applicability is limited by its dose-dependent cardiotoxic effect, which results in a potentially fatal cardiomyopathy in about 6%-9% of patients. Men are more susceptible to these adverse effects than women, but the underlying causes are poorly understood. A mediating effect of the endothelium on doxorubicin-induced cardiotoxicity (DIC) has been proposed. Here, induced pluripotent stem cells (iPSCs) are an ideal tool to investigate the molecular sex-specific effects of doxorubicin on cardiomyocytes and the endothelium’s modulating effects in the absence or presence of sex hormones.
AIMS: This study aims to elucidate sex differences in DIC by exposing male and female human iPSC-derived cardiomyocytes (iPSC-CMs) to doxorubicin and co-stimulating them with extracellular vesicles (EVs) from doxorubicin-treated iPSC-derived endothelial cells from isogenic donors.
METHODS: Human iPSC lines from five healthy male and female donors with Caucasian or Chinese genetic backgrounds were differentiated into cardiomyocytes and endothelial cells (ECs). IPSC-ECs were then stimulated with doxorubicin [1 μM] for 24 hours. The iPSC-EC supernatant was collected, and extracellular vesicles (iPSC-EC-EVs) were concentrated via tangential flow filtration. Then, iPSC-CMs were treated with doxorubicin [1 μM] for 24 hours with or without iPSC-EC-EVs. Then, cell death, intracellular calcium, and reactive oxygen species (ROS) were assessed via fluorescence microscopy. Mean fluorescence intensity (MFI) was measured with Fiji/ImageJ. Statistical analysis was performed with RStudio. To investigate potential mechanisms of action of iPSC-EC-EVs on iPSC-CMs, total RNA was isolated from EVs (treated ± doxorubicin), and microRNA-sequencing, alignment and differential expression analysis were performed using Bioconductor/RStudio.
RESULTS: Male iPSC-CMs displayed increased cell death compared to female iPSC-CMs only after combined treatment of doxorubicin with iPSC-EC-EVs [Live/Dead fixable Near-IR MFI: male 3,73±0,79 and female 2,66±0,88; p = 0.0059 with mean±SE, t-test]. Twenty-one microRNAs were differentially expressed in male vs female EVs from doxorubicin- vs control-treated iPSC-ECs with p<0.05 (e.g., miR-1290, miR-503-5p, miR-4508, etc.). Over-expression analysis, performed with the miEAA online tool (Aparicio-Puerta et al.), associated the microRNAs with the following GO terms: “extrinsic apoptotic signalling pathway” (Benjamini-Hochberg adjusted p=0.0063), “response to G1 DNA damage checkpoint signalling” (BH p=0.02), “response to oxygen radical” (BH p=0.03) and “telomere maintenance” (BH p=0.03) among others.
CONCLUSIONS: We demonstrate that a sex-specific iPSC model can mimic some of the clinically observed sex differences in DIC and show that iPSC-EC-EVs modulate the cardiotoxic effect of doxorubicin. The associated functions, e.g., apoptosis, ROS and DNA damage response, of the differentially expressed microRNAs in iPSC-EC-EVs also reflect known cellular responses to doxorubicin, further emphasising the applicability of our iPSC model. Some limitations that need to be addressed in the future are the relatively small number of iPSC lines and the effects of sex hormones, which have so far been ignored. Furthermore, iPSC-CM transcriptome changes need to be explored further.