Assessment of mitochondrial bioenergetics in fatty acid oxidation deficient fibroblasts
Bianca Seminotti1*, PhD, Al-Walid Mohsen1, PhD, Guilhian Leipnitz1, PhD, Anuradha Karunanidhi1, MSc, Vera Y. Roginskaya2, MSc, Bennett Van Houten2, PhD, Peter Wipf3, PhD, Jerry Vockley1, MD, PhD.
1Division Medical Genetics, Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USA
2 Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA
3 Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA
*bianca.seminotti@chp.edu, +1 (412) 692-9133
The mitochondrial fatty acid β-oxidation (FAO) pathway is an essential supplier of acetyl- CoA, the major source of reducing equivalents that drives ATP synthesis. Inherited defects have been described for most of the genes of FAO proteins, including medium-chain acyl- CoA dehydrogenase (MCAD) and very long-chain acyl-CoA dehydrogenase (VLCAD) deficiencies. These are characterized by hypoglycemia in both disorders, and rhabdomyolysis and cardiomyopathy in VLCAD deficiency. However, cellular pathophysiology leading to these problems remains unclear.
In this study, MCAD and VLCAD deficient fibroblasts were cultured in the absence of glucose for 48-72 h to shift cellular metabolism to fatty acids as their energy source. Oxygen consumption rate was monitored by a Seahorse XF analyzer. Mitochondrial mass and superoxide production were examined by flow cytometry. ATP production was measured by luminometry and protein immunocontent by western blotting.
MCAD and VLCAD deficient cells showed decreased basal respiration, reserve capacity and ATP production. We also observed absence of VLCAD protein, increased VDAC1 protein and a decrease of the complex I subunit ND6 in VLCAD deficient fibroblasts. Superoxide production was increased in VLCAD deficient fibroblasts, which was prevented by the mitochondrially targeted ROS scavenger JP4-039, but not by bezafibrate and N- acetylcysteine. Mitochondrial mass was increased in both cell lines.
Our findings identify a secondary decrease respiratory chain function in MCAD and VLCAD deficient fibroblasts that may play a role in increasing cellular pathophysiology beyond that expected on the basis of reduction of fatty acid oxidation. These pathophysiological manifestations suggest new possibilities for therapeutic strategies for FAO disorders.