A Canine Myocardial Slice Model Of Doxorubicin Induced Cardiotoxicity To Study Autophagy Modulation And Its Effect On Extracellular Vesicle Associated Mirna

Dogs with cancer treated with chemotherapy agents such as doxorubicin (DOX) develop cardiovascular toxicity, providing an opportunity to evaluate cardioprotective strategies in the setting of cancer treatment translatable to human disease. However, due to the lack of a suitable approach to culture primary adult canine cardiomyocytes, mechanistic interrogation of cardiotoxicity after cancer therapy remains a challenge. Our study thus aims to validate a canine myocardial slice culture model to study autophagy modulation and the role of extracellular vesicle (EV) associated miRNA in the setting of DOX induced cardiotoxicity. We hypothesize that induction of autophagy in canine myocardial tissue will reduce apoptosis, exert early changes to EVs, and ameliorate DOX cardiotoxicity. Left ventricular tissue from client-owned donated adult dog hearts was sectioned with a vibratome and viability of the cultured myocardial slices was evaluated by histology and MTT assay. Apoptosis was quantified by TUNEL, and autophagy by fluorescent LC3 protein puncta. Secreted EVs were isolated from cultured tissue by size exclusion chromatography and characterized by nanoparticle tracking analysis, transmission electron microscopy and immunoblot. Canine myocardial slices are consistently viable for 7 days in culture - cardiomyocyte morphology is maintained with low levels of apoptosis, and baseline autophagy is observed. Induction of autophagy with rapamycin treatment results in reduced apoptosis. Cardiac tissue derived extracellular vesicles showed typical size, morphology and enrichment of proteins including tetraspanin CD9 and will be evaluated for changes to EV miRNA profile with autophagy modulation. The canine myocardial slice model allows, for the first-time, the elucidation of complex cross-talk among apoptosis, autophagy, and EVs with molecular and cellular resolutions. Detecting early changes in canine cardiomyocyte autophagy to halt cardiac damage rather than managing its consequences is a paradigm shift translatable towards preventing chemotherapy associated cardiotoxicity.