Abstract 16676: A-mhc MitoTimer Mouse Reveals Metabolic Heterogeneity in the Heart

Abstract

Introduction and hypothesis: To study mitochondrial turnover in the heart, we developed MitoTimer, a fluorescent protein targeted to the mitochondrial matrix that matures from green to red fluorescence over time. We generated a mouse line where MitoTimer is expressed under the cardiac-restricted promoter and were able to observe newly-synthesized and quiescent mitochondria in cardiomyocytes. Surprisingly, the hearts exhibit considerable heterogeneity in green-to-red MitoTimer fluorescence at the level of cardiomyocytes as well as between the mitochondria within individual cells. We hypothesized that these discrete subpopulations of cells represent fibers engaged in specialized functions. Methods: To isolate cells from the heart for proteomic and biochemical analysis, we developed an isolation strategy utilizing flow-cytometry followed by sequential processing to separate cytosolic, myofilament and insoluble protein fractions for liquid chromatography/mass spectrometry. Results of the analysis were validated by biochemical techniques. Results: Analysis of cardiomyocyte sub-populations revealed substantial differences in their protein compositions. Green cells display a profile consistent with an upregulation of PPARA and KLF15 signaling pathways and a downregulation of insulin receptor signaling, suggesting a preference for fatty acid oxidation (FAO). Consistent with this analysis, Western blots revealed that green cells express higher levels of CD36 and Cpt1b proteins compared to red cells. Interestingly, maturation of Timer protein fluorescence to red is sensitive to changes in molecular oxygen concentration. Cells that exhibit higher utilization of FAO and thus a lower oxygen tension will have a decrease in MitoTimer protein maturation, resulting in a greater green fluorescence. These results suggest MitoTimer may be a useful not only as a marker of mitochondrial age, but also as a marker of metabolic substrate preference. Conclusion: Cardiomyocytes in the heart present a highly heterogeneous population, arranged in discrete fibers that have a higher preference for one metabolic substrate over another. We are currently investigating the physiological roles these fibers play in the heart.