Abstract 19140: Quantification of Mitochondrial Structure for the First Time in Intact Arteries
Introduction: Endothelial cell (EC) mitochondrial structure has only been evaluated in cultured cells as methods to label the mitochondria in intact arteries lack proper resolution and cell-specificity for high resolution imaging. Thus, the native structure of the EC mitochondrial network in intact arteries is unknown.
Objective: To quantify the mitochondrial structure of ECs in intact arteries vs. cultured cells.
Methods: Cre-stop mito-Dendra2 mice, expressing the fluorescent protein Dendra2 in the mitochondrial matrix only, were used to label EC mitochondria using Cre-recombinase under the control of the VE-cadherin promoter. Aortas were either fixed immediately with paraformaldehyde or incubated in normal (NG; 5 mM) or high glucose (HG; 33 mM) media for 24 h at 37 °C, cut open, mounted on a glass slide and fluorescent images were obtained on a Nikon A1 confocal microscope (ex 488 nm; em 550 nm) at 100X magnification. ImageJ was used to calculate form factor (FF; 1/circularity) and aspect ratio (AR) of the mitochondrial segments. Mitochondrial fission ratio (MFR) was calculated by counting non-contiguous mitochondrial particles and dividing by the number of pixels which composed the mitochondrial network. Primary aortic EC cultures (48 h in culture) were also generated to compare the mitochondrial structure of cultured ECs vs. intact arteries.
Results: Compared to cultured ECs, the mitochondrial network of ECs from the intact aorta was in a highly fragmented state (MFR: 8.09 ± 0.55 vs. 24.65 ± 2.76, respectively; p<0.001; 15 cells/group). The mitochondrial segments of ECs within the aorta were more circular in shape (FF: 3.6 ± 0.13 vs. 1.88 ± 0.11, respectively; p<0.001) and had less branching (AR: 3.01 ± 0.11 vs. 2.22 ± 0.05, respectively; p<0.001) compared to ECs in culture. HG exposure caused further fission compared to NG in the intact aorta (MFR: 37.52 ± 4.13 vs. 14.70 ± 1.23, respectively; p<0.001).
Conclusion: Using this novel mouse model, for the first time we were able to obtain high resolution images of EC mitochondrial structure in intact arteries. The mitochondrial network was significantly more fragmented in the intact aorta compared to ECs in culture, indicating that mitochondria assume a more elongated and branched phenotype in cultured cells.
Author Disclosures: M.J. Durand: None. K. Ait Aissa: None. D.D. Gutterman: None. A.M. Beyer: None.
- © 2016 by American Heart Association, Inc.