A strategy to protect the heart against doxorubicin induced cardiotoxicity

Kok CY1, Rao R1, Ghossein G1, Igoor S1, Skelton R1, Chong J1,2 and Kizana E1,2

Center for Heart Research, The Westmead Institute for Medical Research, The University of Sydney. Department of Cardiology, Westmead Hospital..

Doxorubicin is an anti-cancer drug used in treating a variety of malignancies. However, its major adverse effect is cardiotoxicity, which is dose dependent and can be either acute or chronic. Doxorubicin causes injury by DNA damage, formation of free reactive oxygen radicals and induction of apoptosis. Our aim is to induce expression of the multiple drug transporter gene (MRP1) in cardiomyocytes derived from human iPS cells (iPSC-CM), to determine whether this will allow cells to effectively remove doxorubicin. To determine the dose of lentiviral vector required for efficient gene delivery to iPSC-CM, a dose titration of GFP vector (LV.GFP) was performed. We found that an MOI of 20 was sufficient for transduction of >90% of cells. Having determined the optimal dose, we then generated a lentivirus vector for inducing expression of MRP1 (LV.MRP1) and validated its function in iPSC-CM by qPCR and western blot. We successfully showed increase of MRP1 mRNA and protein in transduced cells. The activity of the overexpressed MRP1 was also tested, by quantifying the amount of dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. In conclusion, we have optimised the conditions for gene delivery to human iPSC-CM in vitro. We have also shown that we can successfully over-express MRP1 protein in iPSC-CM, with functional transporter activity. The next step is to determine the dose of doxorubicin which induces cell toxicity, and then to assess the protective effect of MRP1 in those cells.