Mitochondrial Dysfunction Lab Report

The corneal endothelium is metabolically highly active, and accordingly, endowed with a large number of mitochondria (**). Recent studies have claimed mitochondrial dysfunction in response to deliberate oxidative stress and in FECD (**). Although mitochondrial damage is correlated to cell death, its effects could be more subtle when the damage to the organelle is not severe. Specifically, when mitochondrial damage is not severe, the cellular functions could be impacted by ATP depletion. The latter can subvert activity of Na+/K+-ATPase (necessary for fluid pump activity) and integrity of the actin cytoskeleton (**) leading to compromised fluid pump and barrier functions, respectively. Thus, the goal of this experiment is to determine if the functional activity of mitochondria is in anyway compromised in …show more content…
For our experiments, HCEC cells will be seeded onto microplates from the manufacturer and grown to confluence. Then the cells will be treated with butyl hydroperoxide with and without n-acetylcysteine through standard injection ports. Assays will be initiated by replacing the growth medium with XF assay medium, which is formulated with unbuffered Dulbecco's modified Eagle's medium, 2 mM sodium pyruvate and 25 mM glucose, pH 7.4. The mitochondrial assays will include oligomycin (to inhibit ATP synthase activity), tri-fluorocarbonylcyanide phenylhydrazone (FCCP) (to uncouple mitochondrial oxidative phosphorylation), and rotenone and antimycin A (to inhibit electron transport in complex I and III, respectively). Subsequently, data can be analyzed to obtain mitochondrial reserve capacity among a host of many other metabolic parameters (**). After we establish baseline data with normal HCEC, we will repeat experiments with HCEC derived from FECD corneas. Our next step will be to assess if the mitochondrial inhibitors employed in the experiments above affect PAMR and TER (as measured by