Al-Walid Mohsen PhD1,2*, Anuradha Karunanidhi MS1, Yu Leng Phua PhD1, and Jerry Vockley MD, PhD1,2
1Department of Pediatrics and 2Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA, USA,
*Correspondence email: firstname.lastname@example.org
Background: Long-chain 3-ketoacyl-CoA thiolase (LCKAT) catalyzes the last step of long-chain FAO and limiting its activity is expected to cause sequential accumulation of intermediates of the upstream reactions. Trimetazidine (TMZ) is a modulator of LCKAT and the resulting pathway product re-binding is hypothesized to have a chaperone effect conferring stability to mutant enzymes upstream from LCKAT. Among the results of biomarkers we have previously monitored to validate this hypothesis are an increase in mutant enzyme activity paralleling an increase in antigen signal and decrease in alternative metabolites. In this study, fibroblast cells from patient with severe very long chain acyl-CoA dehydrogenase activity were treated with TMZ, and O2 consumption, and ATP production, and fatty acid utilization were monitored.
Methods: Fibroblasts from a VLCAD deficient patient, FB671 (p.L540P) was used in a Seahorse Mito Stress assay to monitor O2 consumption, ATP production, and fatty acid utilization in the presence of various concentrations of TMZ. Cells were incubated in the presence of TMZ 48 hr prior to running the assay. Glucose, Gln, and pyruvate were omitted from the Seahorse assay to test the effect of TMZ on fatty acid consumption.
Results: Basal and maximal O2 consumption, ATP production, and fatty acid utilization increased in the presence of TMZ with maximum effect at 0.5 and 2.5 μM, consistent with our previously reported increase in enzyme activity and in situ antigen signal and decreased specific acylcarnitine species.
Discussion: The increase in O2 consumption and calculated ATP production in the presence of TMZ in the absence of the standard assay energy sources implies an increase in endogenous fatty acid consumption. Exogenous long chain fatty acid consumption tested increased as well. The results confirm the efficacy of TMZ in previous patient cell studies as a viable therapy for VLCAD deficiency and other FAO disorders.