Abstract 12625: Mechanical Shear Stress Restores Mitochondrial Cytoarchitecture in Human Endothelial Cells Exposed to Angiotensin II by Modulating Activity of the Fission-Inducing Protein Dynamin Related Protein 1
Background: The mitochondria exist in a state of balance between fission (fragmentation) and fusion (joining of mitochondrial segments). Disease is characterized by excessive fission, leading to an overproduction of mitochondrial reactive oxygen species (ROS) which mediate pathological changes in vascular endothelium. Dynamin-related protein-1 (DRP-1) is the primary mediator of mitochondrial fission. The therapeutic benefit of exercise is largely due to increased endothelial shear; however the effects of shear stress on DRP-1 expression and mitochondrial structure are unknown.
Hypothesis: Shear stress reduces mitochondrial fission in cultured human umbilical vein endothelial cells (HUVECs) exposed to the fission-inducing stimulus angiotensin II (ANG II) by reducing DRP-1 expression and activity.
Methods: HUVECs were treated with MitoRFP for 24 hours to fluorescently label the mitochondria, then were exposed to ANG II (10 pM; 4 hours), fixed, and 25 random cells/group were imaged with a confocal microscope. Separate groups of cells were sheared (15 dyn/cm2; 24 hours) immediately following ANG II exposure using the Ibidi Pump System. To quantify mitochondrial fission, images were converted to binary color and ImageJ was used to count non-contiguous mitochondrial fragments. This value was divided by the number of pixels in the mitochondrial network and multiplied by 1000 to yield a Mitochondrial Fragmentation Index (MFI). Total DRP-1 expression and phosphorylation of the S616 activator and S637 suppressor sites were analyzed by Western blot.
Results: ANG II increased MFI (1.13±0.25 vs. 0.75±0.06), DRP-1 expression (+48%) and phosphorylation of the S616 activator site (+141%; p<0.05 for all) compared to static controls. Conversely, ANG II-treated cells sheared for 24 hours showed reduced mitochondrial fission (MFI = 0.50 vs. 0.75±0.06), DRP-1 expression (-24%) and phosphorylation at S616 (-11%) compared to control. ANG II treated cells exposed to shear also showed a robust increase (+528%) in phosphorylation of the S637 suppressor site of DRP-1.
Conclusions: High shear stress restores mitochondrial network integrity in HUVECs exposed to ANG II, suggesting exercise may promote vascular health by modulating mitochondrial fission.
Author Disclosures: M.J. Durand: None. D.D. Gutterman: Consultant/Advisory Board; Modest; General Electric. Consultant/Advisory Board; Significant; Impulse Dynamics.
- © 2014 by American Heart Association, Inc.