Abstract 2303 Cardiolipin at the epicenter of energy metabolism – implications for Barth syndrome

Cardiolipin (CL), the signature lipid of the mitochondrial membrane, is crucial for optimal mitochondrial function and bioenergetics. Perturbation of CL metabolism due to mutation of the TAFAZZIN (TAZ) gene leads to the life-threatening disorder, Barth syndrome (BTHS). TAZ encodes the transacylase tafazzin (Taz) responsible for incorporating unsaturated acyl chains into CL. While the clinical phenotypes of BTHS, including dilated cardiomyopathy and skeletal myopathy, point to mitochondrial bioenergetic defects, metabolic dysregulation is also a key pathological component. Taz-deficient cells exhibit abnormal levels of metabolites associated with the tricarboxylic acid (TCA) cycle. Consistent with this, we have identified inhibition of pyruvate dehydrogenase (PDH), the gatekeeper enzyme for TCA cycle carbon influx, as a key deficiency in various BTHS models. Further, we determined that deficient PDH activity can be rescued by supplementation of solubilized mitochondria with unsaturated CL, the primary product of CL remodeling in the heart and skeletal muscle. To elucidate the molecular mechanisms linking Taz deficiency to decreased PDH function, we utilized TAZ-knockout C2C12 mouse myoblasts (TAZ-KO) and cardiac and skeletal muscle tissue from TAZ-knockout mice. Our studies have identified two mechanisms linking perturbation of CL metabolism to decreased PDH function. PDH activity is known to be enhanced through dephosphorylation by PDH phosphatase 1 (PDP1) and inhibited through phosphorylation by PDH kinase 4 (PDK4). We have demonstrated that PDP1 directly and specifically binds with unsaturated CL, and the ability of unsaturated CL to restore PDH activity is attenuated by PDP1 inhibition. In TAZ-KO myoblasts, which have diminished levels of unsaturated CL, both the activity of PDP1 and its binding affinity for PDH are substantially reduced. Intriguingly, we have also shown that PDK4 protein levels are elevated in TAZ-KO myoblasts and mouse tissue. PDK4 levels are enhanced by the transcription factor forkhead box protein O1 (FOXO1), and our studies show that FOXO1 is enriched in the nuclei of TAZ-KO myoblasts, where it shows increased binding to the PDK4 promoter. Nuclear translocation of FOXO1 can be mediated through phosphorylation by the cellular energy sensor AMP-activated protein kinase (AMPK), and we have demonstrated that AMPK is hyperactivated in TAZ-KO myoblasts. Importantly, we found that inhibition of AMPK results in decreased nuclear enrichment of FOXO1, reduced PDK4 mRNA, and a concomitant decrease in PDH phosphorylation. In accordance with the role of PDH as a 'metabolic switch' regulating the source of substate utilized for intermediary metabolism, we have reported alterations in glucose homeostasis in TAZ-KO cells. Taken together, these findings suggest that deficient CL remodeling leads to bimodal inhibition of PDH activity resulting in altered metabolic substrate utilization. This work was supported by the National Institutes of Health grant numbers R01 HL117880 (to M.L.G.) and T32 GM142519 (providing support to T.R.E. through the Chemistry Biology Interface program at Wayne State University).