The biochemical basis for overlap of clinical features of LCHAD/TFP deficiency with mitochondrial respiratory chain defects: Implications for new therapeutic approaches

Presented By:

*Areeg El-Gharbawy1 MD, Bianca Seminotti1 PhD, Genevieve Sparagna 2PhD,Grant Hatch3 PhD, Shrabani Basu1MS, Anuradha Karunanidhi1 MS, Walid Al-Mohsen1PhD , Jerry Vockley1,4 MD, PhD and Yudong Wang1 PhD

  1. Department. of Pediatrics, Division of Medical Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA
  2. Department of Medicine, Division of Cardiology, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado
  3. Department of Biochemistry, University of Manitoba, Winnipeg, Manitoba, Canada
  4. Department of Human Genetics, University of Pittsburgh Graduate School of Public Health


Introduction  Patients with long chain 3- hydroxy-acyl CoA dehydrogenase (LCHAD) and Trifunctional Protein (TFP; a 4a4b heteroctamer that includes LCHAD function) deficiencies, respiratory chain complex 1 defects (CI), and Barth syndrome (BS) share overlapping features including cardiomyopathy, fatigue, exercise intolerance, hypoglycemia and lactic acidosis during metabolic decompensation. This underscores the need to understand the functional relationship among mitochondrial bioenergetic pathways. While a multifunctional fatty acid oxidation (FAO) complex in which TFP physically interacts with CI in supercomplexes has been described, a link between FAO and cardiolipin remodeling through TFPα and monolysocardiolipin acetyl transferase (MLCLAT) has also been reported. Cardiolipin plays an important role in sustaining the integrity of the electron transport chain (ETC) by maintaining supercomplex stability in the inner mitochondrial membrane. We hypothesize that mutations in TFP disrupt its interaction with CI, and is associated with alterations in MLCL AT activity and cardiolipin, leading to supercomplex instability, and increased reactive oxygen species (ROS). We also hypothesize that defective cardiolipin in BS alters the TFP- CI interaction due to supercomplex instability.

Methods: Cells and tissues from patients with confirmed LCHAD, and TFP deficiencies, as well as BS were assessed for cardiolipin, ETC, and TFP. Mitochondrial extracts were subjected to blue native PAGE followed by SDS–PAGE and western blotting. Flow cytometry was used to measure ROS (Mito Sox Red) and mitochondrial proliferation (Mito Tracker Green). Oxygen consumption studies were performed using a Seahorse XF®96 Analyzer. Cardiolipin was studied using liquid chromatography mass spectrometry. TFPα and MLCLAT were quantitated using SDS-PAGE followed by western blotting, and measurement of MLCLAT activity.

Results: Fibroblasts from patients with LCHAD deficiency and BS both had a reduction in MLCLAT activity compared to control cells. There was evidence of altered cardiolipin content, destabilization of TFPα interaction with supercomplexes, increased ROS production, and mitochondrial proliferation compared to control.

Discussion: These findings suggest that studying alterations in the FAO- ETC- Cardiolipin interaction  increase our understanding of the pathophysiology of these disorders, providing impetus for development of new therapeutic approaches.

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