Effects of recombinant GDF15 pretreatment on mitochondrial damage and cytotoxicity induced by doxorubicin in human stem cell-derived cardiomyocytes

Background/Introduction: Cardiotoxicity is a major adverse effect of doxorubicin and other anthracyclines used in anticancer chemotherapy. While dexrazoxane remains the only agency-approved cardioprotective prophylaxis against anthracycline cardiotoxicity, its potential for secondary malignancy has limited its wider clinical use. Growth differentiation factor 15 (GDF15) is a pleiotropic metabolic cytokine that has been shown to reduce the pathophysiologic effects of hypertensive and ischemic cardiomyopathy in animal models but its cardioprotective effects are unclear in anthracycline cardiotoxicity. Purpose: We aimed to determine if recombinant GDF15 can protect human embryonic stem cell-derived cardiomyocytes (hESC-CM) against doxorubicin, and define the dose response and pretreatment timing that provide optimal prophylactic cytoprotection. Methods: Cardiac differentiation of H7 hESCs was performed as described previously. The cultured hESC-CMs exhibited a beating phenotype with over 91% of cells expressing cardiac troponin T on flow cytometric analysis (Fig 1A). hESC-CM viability was evaluated using the PrestoBlue assay (Fig 1B). Dose and time response curves for GDF15 pretreatment were generated to identify the optimal concentration and pretreatment timing based on cell viability in response to doxorubicin (1.5 µM) exposure. Mitochondrial structure (network morphology) and function (mitochondrial membrane potential) were quantitatively analysed using MitoTracker Deep Red (MTDR) and tetramethylrhodamine ethyl ester (TMRE) dyes, respectively. Cell apoptosis was assessed with Hoechst 33342 nuclear staining. Cell viability, mitochondrial structure and function, and apoptosis were compared between doxorubicin-exposed hESC-CMs pretreated with GDF15 and dexrazoxane (200 µM for 1 hour). Results: Doxorubicin at 1.5 µM reduced hESC-CM viability by 22% (Fig 1C & 1D), disrupted mitochondrial network (Fig 1E & 1F), decreased mitochondrial membrane potential (Fig 1E & 1G), and led to nuclear condensation and fragmentation suggestive of apoptosis (Fig 1E & 1H). Significant cytoprotective effects of GDF15 were seen starting at 6 hours of pretreatment, and peaked at 24 hours (Fig 1D). Optimal attenuation of cytotoxic effects on hESC-CMs were seen at a GDF15 concentration of 300 ng/ml applied 24 hours before addition of doxorubicin (Fig 1C & 1D). Protection from doxorubicin-induced cytotoxicity was seen at incremental GDF15 concentrations ranging from 100 to 1,000 ng/ml. No significant difference was observed between dexrazoxane and GDF15 in their cytoprotective effects on cell viability; mitochondrial network integrity, morphology and membrane potential; andapoptosis (Fig 2A–2G). Conclusion(s): Recombinant GDF15 pretreatment protected hESC-CMs from doxorubicin to a similar degree as dexrazoxane.