Role of peroxisomes in compensatory metabolic adaptation in Barth syndrome cardiomyopathy

Objective: The heart is one of the most energy consuming organs in the human body and is critically dependent on mitochondrial oxidative phosphorylation to match the energy demand. Cardiolipin is an essential phospholipid of mitochondrial membranes. An inherited defect in the enzyme Tafazzin, involved in cardiolipin biogenesis, causes Barth syndrome (BTHS), associated with defects in energy provision and redox homeostasis. We have shown earlier that dysfunctional mitochondria induce the ER resident integrated stress response signaling (ISR) pathway, which induces a substantial remodeling of cardiac metabolism in BTHS. Recent data indicate that alterations are not only confined to mitochondria, but also include peroxisomes. We find peroxisomal catalase upregulated in a BTHS mouse model with a knockdown of Tafazzin gene expression but the role of catalase in detoxifying ROS in BTHS is unclear. Peroxisomal alterations also include a deregulation of plasmalogen biogenesis. Plasmalogens are phospholipids located in the ER membrane, which are produced by peroxisomes. Plasmalogens are prone to oxidation due to their structure. The project aims to elucidate the function of plasmalogens in BTHS. Specifically, it seeks to determine whether plasmalogens serve as sacrificial antioxidants, shielding cells from oxidative damage, or if their heightened oxidation contributes to disease progression.

Methods: Mouse model with an inducible knock-down of Tafazzin gene expression and a MEF line with a CRISPR/Cas9 mediated knock-out of the Tafazzin gene. siRNA mediated knockdown of Catalase and Gnpat, the enzyme catalyzing peroxisomal plasmalogen biosynthesis. Cells were analyzed by immunofluorescence, western blot quantitative PCR.

Results: Peroxisomal catalase is upregulated in Tafazzin deficient mouse model and MEFs as shown by qPCR, western blot and immunofluorescence. Survival of Taz^KO MEFs was reduced, when peroxisomal catalase was inhibited. Uncontrolled membrane peroxidation can induce cell death. Taz^KO cells showed a predisposition to apoptosis including upregulation of pro-apoptotic PUMA and cleavage of caspase 3. It remains unclear whether membrane peroxidation also contributes to stress signaling. Two enzymes of plasmalogen biosynthesis, Fatty Acyl-CoA Reductase 1 (FAR1) and Glycerophosphate O-Acyltransferase (GNPAT) were upregulated in Tafazzin deficient mouse model and MEF cells. We tested, if plasmalogen administration has a beneficial effect on ROS compensation. Interestingly, we did not find a beneficial role of plasmalogen administration to Taz^KO MEFs. We also did not find adverse effects upon knock down of plasmalogen biosynthesis in Taz^KO MEFs. Surprisingly, we found that reducing plasmalogen biosynthesis rather alleviates stress signaling in Taz^KO MEFs. Here we test, if siRNA mediated knockdown of enzymes of plasmalogen biosynthesis affect the induction of the ER stress response pathway. This study addresses for the first time the role of plasmalogens in activating the ER stress response pathway.

Conclusion: Insights into the role of peroxisomal plasmalogens and catalase in compensating ROS and inducing stress response signaling pathways will enable the design of novel strategies alleviating BTHS cardiomyopathy.