Role of peroxisomes in compensatory metabolic adaptation in Barth syndrome cardiomyopathy

Background: Barth syndrome (BTHS) is an extremely rare X-linked genetic disorder due to mutations in the Tafazzin gene. It encodes an enzyme responsible for the remodelling and maturation of cardiolipin, a phospholipid integral to mitochondrial membranes. A disturbance in the cardiolipin pool results in compromised mitochondrial integrity, leading to energy deficits with heightened oxidative stress. The heart is one of the most energetically demanding organs in the body, and relies heavily on mitochondrial oxidative phosphorylation, rendering it vulnerable to these energetic and structural defects. We have previously demonstrated that mitochondria from individuals with BTHS stimulate the integrated stress response pathway (ISR) of the endoplasmic reticulum associated with the remodelling of cardiac metabolism. While mitochondrial dysfunction is central to BTHS, recent findings suggest that peroxisomal adaptations may also play a significant role in managing oxidative stress. We found upregulation of the peroxisomal enzyme catalase in a mouse model of BTHS and a Tafazzin deficient cell line, consistent with a compensatory response against reactive oxygen species (ROS). Additionally, disruptions in plasmalogen biosynthesis, a lipid metabolism pathway involving peroxisomes, point to further cellular adaptations to counter oxidative stress. This study aims to explore these peroxisomal pathways to uncover metabolic adaptations in BTHS beyond mitochondrial dysfunction, which could be important for the resilience of cells in BTHS. Here, we outline the specific contribution of peroxisomal catalase and plasmalogen biosynthesis on the pathophysiology of BTHS to it's ROS detoxification, cell survival under conditions of stress and modulation of oxidative stress

Methods: With a Tafazzin-knockdown mouse model and a CRISPR/Cas9 knockout MEF cell line, we studied the responses of peroxisomal enzymes under oxidative stress conditions. Catalase and glycerophosphate O-acyltransferase (GNPAT), which is a key enzyme in plasmalogen biosynthesis, were knocked down by siRNA transfection. After catalase and GNPAT knockdown, ROS levels and indicators of apoptosis and cellular responses against stress were analyzed by western blotting, immunofluorescence, and quantitative PCR.

Results: Our findings show a marked upregulation of peroxisomal catalase in Taz^KO cells, which correlates with enhanced cell survival under oxidative stress. Inhibition of catalase provoked further accumulation of ROS, caspase-3 cleavage, and cell death, suggesting a compensatory role in mitigating oxidative stress. Interestingly, upregulated plasmalogen biosynthesis in Taz^KO cells did not affect ROS, indicating that the plasmalogens do not act as antioxidants directly. Knockdown of GNPAT was accompanied by a decrease in ISR signalling, indicating a novel role of plasmalogens in mediating a compensatory cellular stress response pathways.

Conclusion: Our results suggest a compensatory role of peroxisomes in BTHS. Peroxisomal catalase could represent an antioxidant response against ROS and may dampen oxidative stress. The findings also illustrate a role for plasmalogen biosynthesis in modulating stress responses rather than acting as direct antioxidants. These findings underscore the contribution of peroxisomal function to BTHS pathophysiology and indicate that targeting peroxisomal pathways may unveil novel therapeutic interventions to reduce oxidative injury in BTHS cardiomyopathy.