In vitro platform to model anthracycline-induced cardiotoxicity and evaluate cardioprotective agents in human -induced pluripotent stem cells-derived cardiomyocytes

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Abstract Background/Introduction Cancer is a leading cause of morbidity and mortality worldwide. Among the different types of treatment, chemotherapy is a common one for many types of cancers. Anthracyclines, especially doxorubicin (DOXO), are common and highly efficient chemotherapy drugs, which have significantly improved survival rates among patients with malignancies during the last decades. Unfortunately, the use of anthracyclines is limited by their cardiotoxic effects, known as anthracycline-induced cardiotoxicity (AIC), which affects approximately 5% to 25% of patients. To mitigate this risk, dexrazoxane (DRZ) is a potential cardioprotective drug, which is metabolized in vivo by the liver and kidneys into its active form, ADR-925, an iron chelator capable of reducing oxidative stress caused by anthracyclines. Despite its potential, the lack of knowledge regarding its efficacy, safety and action mechanisms still greatly limits its use. Purpose This study aimed to develop an in vitro platform for modelling AIC, evaluating cardioprotective agents, and identifying potential predictive markers for AIC. Methods Peripheral blood mononuclear cells were isolated for reprogramming into iPSC using the CytoTuneTMiPS 2.0 Sendai Reprogramming kit. The cells were characterised for pluripotency by flow cytometry and RT-PCR. The efficiency of the differentiation protocol was verified by the presence of cardiac troponin T by flow cytometry. Cell viability was assessed using a fluorescence-based assay (PrestoBlue Cell Viability Reagent) and the DNA damage was measured via the comet assay. Results The platform consisted of cardiomyocytes derived from human-induced pluripotent stem cells (CM-iPSC) obtained from a patient affected by AIC (CM-S) and a healthy donor (CM -CTRL). Pluripotency was confirmed by the presence of specific transcripts and the expression of pluripotency-associated proteins (OCT3/4, SOX2, NANOG). A highly efficient cardiac differentiation protocol was then developed, achieving differentiation rates exceeding 87% in both cell lineages. Cardiomyocytes were treated with DOXO and the cardioprotective agents DRZ and ADR-925. The CM-S cells presented greater sensitivity to DOXO (IC50 CM-S: 0.214 μM; IC50 CM-CTRL: 1.337 μM, p<0.000001). Co-treatment with ADR-925 provided significant cardioprotection to CM-S (IC50 CM-S DOXO + ADR 100μM: 0.299 μM, p<0.000001; n=5), whereas DRZ did not (IC50 CM-S DOXO + DRZ 100μM: 0.167 μM, p=0.0005; n=5). Furthermore, ADR-925 reduced DNA damage in CM-S, as demonstrated by a comet assay measuring DNA double-strand breaks (DOXO 0.1μM: 7,740 μm²; DOXO 0.1μM + ADR 100μM: 1,181 μm²; p<0.0001; n=21). Conclusion These findings suggest that ADR-925 provides cardioprotection against DOXO-induced damage.

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