Cancer‐induced Metabolic Cardiotoxicity Characterized through Optical Metabolic Imaging

Abstract

Heart disease is a primary contributor to mortality in the world and progressively develops by advancing age, obesity, and other disease states. Pathological hypertrophy, atrophy, apoptotic cell death, and fibrosis can all be contributing factors to heart failure but diminished oxidative capacity seems to be a universal characteristic. New applications of two‐photon excited fluorescence microscopy (TPEF) can aid in the characterization of cardiac metabolic alterations. Specifically, NADH and FAD are fluorescent metabolic cofactors that play important roles in metabolic pathways of the cytoplasm and the mitochondria. The optical reduction‐oxidation (redox) ratio (ORR) of FAD/(FAD+NADH) provides a validated method to quantify the redox state of a cell thus characterizing reliance on oxidative metabolism versus glycolytic utilization.PurposeTo utilize the ORR as a non‐destructive, label free marker of cardiac metabolism in a mouse model of cancer‐induced heart disease and in‐vitro genetically altered cardiomyocytes.Methods1×106 Lewis Lung Carcinoma (LLC) cells were injected into the flank of C57BL6/J mice and the tumor was allowed to develop for 4 wks and compared to PBS injected control. TPEF was performed on 10μm sections of the left ventricle to quantify ORR. Further histological analysis (Sirius Red, TUNEL, 2HG) and immunoblot analysis (mitochondrial turnover markers, apoptosis signaling) were performed to verify and characterize this unique form of cardiomyopathy. To examine if changes in ORR are indicative of a transition from oxidative to glycolytic metabolism, experiments were performed using H9c2 cardiomyocytes transfected with shRNA targeting mitochondrial translation initiation factor 2 (mtIF2). Oxidative capacity was measured using bioenergetics flux analysis and TPEF to verify decreased oxidative capacity resulting from impaired mitochondrial mRNA translation products.ResultsLLC hearts demonstrated ~15% lower ORR (FAD/FAD+NADH) indicating greater glycolytic reliance compared to PBS controls. LLC hearts had ~50% greater content in markers of mitochondrial content (COX‐IV, VDAC) likely due to ~50% lower PINK1/Parkin‐mediated mitophagy markers while biogenesis proteins PGC‐1α, PPARα, and PPARδ were unaffected. H9c2 cells transfected with mtIF2 shRNA had decreased basal, maximal, and reserve oxygen consumption rates, greater AMPK phosphorylation and a ~40% lower ORR.ConclusionsThese results demonstrate how TPEF of the optical redox state can identify metabolic characteristics associated with cardiomyopathy as demonstrated using cancer‐induced cardiac atrophy and in vitro genetic alteration of cardiomyocytes. Further research could use this non‐destructive, label‐free approach to screen for drugs contributing to cardiotoxic effects of cancer treatments as well as pharmaceuticals that may attenuate the development of cardiomyopathies.Support or Funding InformationThis study was supported by Arkansas Bioscience Institute (NR, NPG), American College of Sports Medicine Foundation Doctoral Student Research Grant (DEL) and National Institutes of Health R15AR069913 (NPG).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.