Access Type

Open Access Dissertation

Date of Award

January 2023

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Miriam M. Greenberg

Abstract

Barth syndrome (BTHS) is a rare genetic disease that results from mutations in the TAFAZZIN gene, which encodes the cardiolipin (CL) remodeling enzyme tafazzin (Taz). The mechanisms linking perturbation of CL remodeling and the pathological features of BTHS are not understood. We have recently reported that intermediary metabolism is perturbed in BTHS, and activity of the metabolic gatekeeper enzyme pyruvate dehydrogenase (PDH) is deficient in BTHS models. The mechanism whereby PDH is regulated by Taz is unknown, and this knowledge gap represents an obstacle to the development of therapeutics to treat BTHS. Using an established C2C12 myoblast model of BTHS, TAZ-KO, I identified important mechanisms by which CL regulates PDH function. CL regulates PDH activity through activation of PDK4, and this is mediated by hyperactivation of AMPK activity, which facilitates FOXO1 nuclear translocation and subsequent increase in PDK4 expression in TAZ-KO cells. PDK4 upregulation leads to alterations in fuel utilization, consistent with observed metabolic changes in BTHS patients, including disrupted glucose and fatty acid oxidation and increased fatty acid accumulation in the heart and plasma. In addition, CL regulates PDH activity by facilitating PDP1-mediated dephosphorylation of PDH. Of different CL species tested in isolated TAZ-KO mitochondria, only tetralinoleoyl-CL (TLCL) rescues PDH activity. Interestingly, when phosphatase activity is inhibited, the PDH activity was not rescued by CL. Additionally, PDP1 activity in TAZ-KO cells and TLCL interacted with PDP1 in vitro. These findings suggest that PDP1 is required for CL-activation of PDH activity. It is likely that TLCL serves as a scaffold for concurrent binding of PDP1 and PDH, thereby facilitating their interaction. This is supported by the observed dose-dependency of TLCL rescue. PDP1 activity is also likely reduced by the finding that mitochondrial calcium levels are decreased in TAZ-KO cells, as PDP1 activity is dependent on calcium. TAZ-KO cells exhibit decreased mitochondrial calcium levels and supplementation with calcium lactate (CaLac) rescues PDH activity and oxygen consumption rate (OCR). The perturbation in mitochondrial calcium levels is further reflected in changes observed in ER-MT contacts (MAMs), which play an important role in channeling calcium. Components of MAMs, including IP3R, VDAC1, GRP75 and PDK4, are increased in TAZ-KO cells and mouse TAZ-KO heart tissues. Overall, I proposed two mechanisms whereby CL regulates PDH, including regulation of PDK4 via FOXO1-AMPK and interaction with PDP1 or/and modifying mitochondrial calcium levels. These findings provide insight into the regulatory mechanisms of PDH activity by CL, linking CL function to substrate metabolism and mitochondrial calcium regulation coordinating with ER.

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