TCA Cycle Defects in Barth Syndrome

Barth syndrome (BTHS) is a genetic disorder caused by defective remodeling of the phospholipid cardiolipin (CL) due to mutations in the gene coding for tafazzin. The disorder is characterized by dilated cardiomyopathy and mitochondrial dysfunction. CL is a unique membrane lipid that has multiple cellular functions. CL that is synthesized de novo contains mostly saturated fatty acids. It is deacylated by phospholipase to monolysocardiolipin (MLCL), which is reacylated by the transacylase tafazzin to form CL that contains mostly unsaturated fatty acids. Tafazzin deficiency, the defect in BTHS, causes decreased total CL, an increased MLCL/CL ratio, and decreased unsaturated (remodeled) CL species. The mechanism linking CL deficiency to BTHS pathology is not clear. Our previous studies indicate that CL deficiency in the yeast CL synthase mutant (crd1Δ) leads to perturbation of iron sulfur (Fe‐S) biogenesis. This is characterized by decreased activity of Fe‐S requiring enzymes, including the TCA cycle enzymes aconitase and succinate dehydrogenase. As tafazzin mutant cells exhibit decreased levels of CL, we investigated the possibility that tafazzin deficiency as seen in BTHS leads to TCA cycle defects in mammalian cells. To this end, we constructed a CRISPR‐generated stable tafazzin knockout C2C12 myocyte cell line. A mass spectrometric analysis of C2C12 isolated mitochondria was performed using an ambient, solid‐state ionization method, matrix‐assisted ionization (MAI), in which the mitochondria, mixed with an organic MAI matrix, is directly introduced into a commercial electrospray ionization (ESI) skimmer cone to create multiply charged ions. Coupled with the ion mobility spectrometry capabilities of the Waters SYNAPT G2 mass spectrometer, MAI mass spectrometry was used to determine the MLCL/CL ratio of the C2C12 mitochondria. TAZ‐KO cell lines exhibited the increased MLCL/CL ratio characteristic of tafazzin‐deficient cells. Decreased aconitase activity is expected to lead to decreased synthesis of α‐ketoglutarate, which is utilized in the synthesis of glutamate. Therefore, we predicted that CL‐deficient cells would exhibit a deficiency in glutamate. To test this possibility, we carried out an unsupervised 1H NMR metabolomic analysis comparing TCA cycle metabolites and amino acids in CL deficient yeast (crd1Δ) and mammalian (tafazzin‐KO C2C12) cells to wild type cells. As predicted, glutamate levels were decreased in crd1Δ and tafazzin‐KO C2C12 cells. Consistent with these findings, the growth defect of crd1Δ cells is restored by supplementation with glutamate. The findings from this study will facilitate our understanding of highly conserved mechanisms that control mitochondrial metabolism, and offer the possibility of new treatments for BTHS and other cardiomyopathies and disorders of CL deficiency based on supplementation with simple metabolites. Support or Funding Information RO1 HL 117880 from the National Institute of Health (to Miriam L. Greenberg)