Tafazzin Mutation Affecting Cardiolipin Leads to Increased Mitochondrial Superoxide Anions and Mitophagy Inhibition in Barth Syndrome

Tafazzin is a phospholipid transacylase that catalyzes the remodeling of cardiolipin, a mitochondrial phospholipid required for oxidative phosphorylation. Mutations of the tafazzin gene cause Barth syndrome, which is characterized by mitochondrial dysfunction and dilated cardiomyopathy, leading to premature death. However, the molecular mechanisms underlying the cause of mitochondrial dysfunction in Barth syndrome remain poorly understood. We again highlight the fact that the tafazzin deficiency is also linked to defective oxidative phosphorylation associated with oxidative stress. All the mitochondrial events are positioned in a context where mitophagy is a key element in mitochondrial quality control. Here, we investigated the role of tafazzin in mitochondrial homeostasis dysregulation and mitophagy alteration. Using a HeLa cell model of tafazzin deficiency, we show that dysregulation of tafazzin in HeLa cells induces alteration of mitophagy. Our findings provide some additional insights into mitochondrial dysfunction associated with Barth syndrome, but also show that mitophagy inhibition is concomitant with apoptosis dysfunction through the inability of abnormal mitochondrial cardiolipin to assume its role in cytoplasmic signal transduction. Our work raises hope that pharmacological manipulation of the mitophagic pathway together with mitochondrially targeted antioxidants may provide new insights leading to promising treatment for these highly lethal conditions.

COmplexome Profiling ALignment (COPAL) reveals remodeling of mitochondrial protein complexes in Barth syndrome

ABSTRACTMotivationComplexome profiling combines native gel electrophoresis with mass spectrometry to obtain the inventory, composition and abundance of multiprotein assemblies in an organelle. Applying complexome profiling to determine the effect of a mutation on protein complexes requires separating technical and biological variations from the variations caused by that mutation.ResultsWe have developed the COmplexome Profiling ALignment (COPAL) tool that aligns multiple complexome profiles with each other. It includes the abundance profiles of all proteins on two gels, using a multidimensional implementation of the dynamic time warping algorithm to align the gels. Subsequent progressive alignment allows us to align multiple profiles with each other. We tested COPAL on complexome profiles from control mitochondria and from Barth syndrome (BTHS) mitochondria, which have a mutation in tafazzin gene that is involved in remodelling the inner mitochondrial membrane phospholipid cardiolipin. By comparing the variation between BTHS mitochondria and controls with the variation among either, we assessed the effects of BTHS on the abundance profiles of individual proteins. Combining those profiles with gene set enrichment analysis allows detecting significantly affected protein complexes. Most of the significantly affected protein complexes are located in the inner mitochondrial membrane (MICOS, prohibitins), or are attached to it (the large ribosomal subunit).Availability and implementationCOPAL is written in Python and is available from gttp://github.com/cmbi/[email protected]

A novel intronic splice site tafazzin gene mutation detected prenatally in a family with Barth syndrome

Barth syndrome (BTHS) is a rare X-linked disease characterized by dilated cardiomyopathy, proximal skeletal myopathy and cyclic neutropenia. It is caused by various mutations in the tafazzin (TAZ) gene located on Xq28 that results in remodeling of cardiolipin and abnormalities in mitochondria stability and energy production. Here we report on a novel c.285-1G>C splice site mutation in intron 3 of the TAZ gene that was detected prenatally.

Mitochondria-Targeted Antioxidant Prevents Cardiac Dysfunction Induced by Tafazzin Gene Knockdown in Cardiac Myocytes

Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS) have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress.

Barth syndrome mutations that cause tafazzin complex lability

Deficits in mitochondrial function result in many human diseases. The X-linked disease Barth syndrome (BTHS) is caused by mutations in the tafazzin gene TAZ1. Its product, Taz1p, participates in the metabolism of cardiolipin, the signature phospholipid of mitochondria. In this paper, a yeast BTHS mutant tafazzin panel is established, and 18 of the 21 tested BTHS missense mutations cannot functionally replace endogenous tafazzin. Four BTHS mutant tafazzins expressed at low levels are degraded by the intermembrane space AAA (i-AAA) protease, suggesting misfolding of the mutant polypeptides. Paradoxically, each of these mutant tafazzins assembles in normal protein complexes. Furthermore, in the absence of the i-AAA protease, increased expression and assembly of two of the BTHS mutants improve their function. However, the BTHS mutant complexes are extremely unstable and accumulate as insoluble aggregates when disassembled in the absence of the i-AAA protease. Thus, the loss of function for these BTHS mutants results from the inherent instability of the mutant tafazzin complexes.

Molecular Studies of Neutropenia in Barth Syndrome.

The Barth syndrome is a rare mitochondrial disease that causes dilated cardiomyopathy, skeletal myopathy, and severe neutropenia. It has been reported that at least two patients with Barth syndrome died from septicemia attributed to neutropenia. The disease is an X-linked autosomal recessive disorder caused by mutations in the G4.5 or TAZ (tafazzin) gene, which introduce truncations, substitution, or alternative splice sites. Hematological investigations revealed normal chemotaxis, but the presence cytoplasmic vacuoles in myeloid cells and maturation arrest of neutrophil development at the myelocyte stage. Thus, a defect in neutrophil formation appears to be the primary reason for neutropenia and the susceptibility to infections in the Barth syndrome patients. Recent studies were mostly focused on cardiac, genetic and metabolic abnormalities associated with the disorder. However, the neutropenia aspect of the Barth syndrome remains unclear. The TAZ gene mutations appear to truncate the tafazzin protein likely resulting in the loss of function. Although a drosophila model of the Barth syndrome was recently reported, the cellular or mouse models of this disorder are not available yet. Thus, the link between TAZ mutations and severe neutropenia remains unknown. We hypothesized that TAZ mutations, which lead to the loss of function of tafazzin protein, trigger impaired cell survival and reduced production of neutrophils and their neutrophil precursors, thus resulting in severe neutropenia in Barth syndrome. To test this hypothesis, we attempted to knock-down the expression of the tafazzin gene in human myeloid progenitor cells using TAZ-specific shRNA. Four shRNA sequences specific to exons 4 through 7 were used for transfection of human myeloid progenitor cells that were later examined by flow cytometry and Western blot analyses. At least 2 of the shRNA constructs resulted in a substantial down-regulation in the expression level of the tafazzin protein in transfected myeloid progenitor cells as determined by Western blot. Flow cytometry analyses revealed that the knock-down of TAZ gene expression was associated with approximately 40–50% increase in proportion of apoptotic annexin-positive cells compared with cells transfected with control scrambled shRNA. These data suggest that the loss of function of TAZ gene is cytotoxic to hematopoietic cells and that severe neutropenia is due to the accelerated apoptosis of myeloid progenitor cells in patients with Barth syndrome. Further studies needed to elucidate the specific signaling pathways and to identify potentially therapeutic agents capable of controlling accelerated apoptosis of myeloid cells in Barth syndrome.