Premium
Downregulation of Fis1 in Endothelial Cells Does Not Impact Normal Endothelial Cell Mitochondrial Bioenergetics, Metabolism, or Mitochondrial Protein Content
Author(s) -
kakarla mamatha,
Wang Jingli,
Waters Heather,
Puppala V. krishna,
Tyagi Sudhi,
Hofeld Benjamin,
Ying Rong,
Anger Amberly,
Harwig Megan Cleland,
Egner John,
Hill Ronald Blake,
Widlansky Michael E.
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.03104
Subject(s) - fis1 , dnm1l , mitochondrial fission , microbiology and biotechnology , mitochondrial fusion , mfn2 , mitochondrion , atp–adp translocase , biology , transfection , mfn1 , dnaja3 , bioenergetics , mitochondrial membrane transport protein , chemistry , biochemistry , inner mitochondrial membrane , mitochondrial dna , gene
Our prior data suggest that knockdown of Fis1 protein improves vascular endothelial health by reversing endothelial dysfunction associated with dysglycemic conditions and reducing endothelial inflammation and permeability of the endothelium. Fis1 is an outer mitochondrial membrane protein that triggers mitochondrial fission when bound by dynamin‐related protein‐1. Fis1 is important to mitochondrial fission process specifically under pathophysiological conditions making Fis1 an attractive therapeutic target. We hypothesize that reducing Fis1 expression will not alter other mitochondrial proteins and mitochondrial bioenergetics under different glycemic conditions. We measured mitochondrial proteins, and mitochondrial bioenergetics in wild type immortalized human microvascular endothelial cells (HMEC‐1) and HMEC‐1 cells that were transfected with Fis1 siRNA (20 nM) or scrambled siRNA (20 nM). Upon transfection, cells were exposed to different glycemic conditions: normal (5mM), high (30mM for six hours), and low (2mM for two hours) glucose levels. Mitochondrial proteins expression in Fis1 siRNA HMEC‐1 cell lysates and scrambled control cell lysates were assessed under different glycemic conditions by high throughput immunodetection WES system. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in transfected cells under different glycemic conditions were measured by Seahorse mitochondrial and glycolytic stress tests. There were no significant differences in mitochondrial bioenergetics (OCR and ECAR; n=5, p>0.05), and expression of other mitochondrial proteins (MFF, MFN1, MFN2, Drp1, OPA1, Cyt‐c, TOMM20, MFF, pAMPKa, POLG, P62, NDUFB8, GABARAP) between siFis1 cells and scrambled controls in any of the glycemic conditions (n=4, p>0.05). There is significant increase in expression of other mitochondrial proteins (MFF, MFN1, Cyt‐c, TOMM20, MFF, pAMPKa, POLG, P62, NDUFB8, GABARAP) under low glucose conditions (n=4, p<0.05) compared to normal and high glucose conditions. Reducing the expression of Fis1 did not alter other mitochondrial proteins and mitochondrial bioenergetics that are critical for normal physiological processes. Therapeutic potential of Fis1 knockdown is enhanced by a lack of impact on endothelial cell metabolism and other mitochondrial protein expression. Support or Funding Information This project was supported by the National Institutes of Health, through Grant Number R01‐HL128240 and HL125409 .