Abstract 15584: Mitotimer Protein Reveals Remarkable Macro and Micro Heterogeneity in the Heart; A Novel Tool for Determining Mitochondrial Turnover in the Heart
In order to study mitochondrial turnover at the level of a single mitochondrion, our laboratory has developed the MitoTimer protein. Timer is a mutant of DsRed fluorescent protein developed by Terskikh et al. The Timer protein transitions from green fluorescence to a more stable red conformation as it matures over a span of 48 h. Furthermore, the protein is very stable under physiological conditions, insensitive to variations in ionic strength, and changes in pH between 7.0 and 8.0. Notably, Timer maturation from green to red is significantly slowed in deoxygenated buffer, suggesting that molecular oxygen plays a part in fluorophore maturation. We created a construct that fused the Timer protein cDNA with the inner mitochondrial membrane signal sequence and placed it under the control of the cardiac-restricted α-myosin heavy chain promoter. This construct was used to create the α-MHC MitoTimer mice. Surprisingly, initial analysis of the hearts from these mice demonstrated a high degree of heterogeneity in the ratio of red-to-green fluorescence of MitoTimer in cardiac tissue, revealing regions of high and low oxygen tension. Further, individual mitochondria within cardiomyocytes display a higher red-to-green fluorescence relative to fluorescence of the other mitochondria in the cell, implying a block in import of newly synthesized MitoTimer caused by the lack of a high membrane potential, indicative of older, dysfunctional mitochondria. These mitochondria can be isolated and sorted from the heart by flow cytometry for further analysis. Initial studies suggest that these mice represent an elegant tool for the investigation of mitochondrial turnover in the heart.
Author Disclosures: A. Stotland: None. J. Ramil: None. R.A. Gottlieb: None.
- © 2015 by American Heart Association, Inc.