Mitigation of Doxorubicin Cardiotoxicity with Synergistic miRNA Combinations Identified Using Combinatorial Genetics en Masse (CombiGEM)

Background: Cardiomyocyte loss occurs in acute and chronic cardiac injury, including cardiotoxicity due to chemotherapeutic agents such as doxorubicin, and contributes to development of heart failure. There is a pressing need for cardiac-specific therapeutics that prevent the cardiac complications of chemotherapy without compromising efficacy by directly targeting cardiomyocyte loss. Objectives: We employed massively parallel combinatorial genetic screening to search for miRNA combinations that promote cardiomyocyte survival. Methods: Combinatorial genetics en masse (CombiGEM) screening in a cardiomyocyte cell line was followed by validation studies in the original cell type as well as primary cardiomyocytes to search for miRNA combinations that promote survival under various stress conditions. The top candidate combination was tested human iPSC-derived cardiomyocyte and in vivo mouse and developing zebrafish models of doxorubicin cardiotoxicity. RNA sequencing and in silico miRNA target prediction provided insight into possible mechanisms. Results: CombiGEM screens and validation experiments identified multiple miRNA combinations that protected cardiomyocytes from doxorubicin in vitro. The most effective miRNA combination (miR-222 + miR-455) appeared to act synergistically and had protective effects in both murine and zebrafish in vivo doxorubicin cardiotoxicity models. RNA sequencing data revealed synergistic and additive regulation of mechanistically relevant genes and biological processes, including mitochondrial homeostasis, cellular respiration, DNA replication/damage response, oxidative and ER stress, angiogenesis, muscle contraction/relaxation, and others. Conclusions: We identified miR-222 and miR-455 as a combination with potential therapeutic applications for cardioprotection. The results of this study further our knowledge of the cardiac effects of individual miRNAs and their combinations and demonstrate the value of the CombiGEM approach for discovery of cardioprotective combinatorial therapeutic candidates. To gain insight into potentially synergistic protective effects of miR-222 and miR-455 against doxorubicin cardiotoxicity, we transfected cultured neonatal rat ventricular myocytes with Ambion Pre-miR miRNA mimics of miR-222-3p (Thermofisher Scientific assay ID PM11376), miR-455-3p (assay ID PM12568), both miRNA mimics combined, or a negative control mimic (Cat # AM17110) at 5nM each. At 48h after transfection, cells were treated with 4mM doxorubicin or vehicle. RNA was extracted 24h later for RNA sequencing followed by differential expression and gene ontology analysis.