Defective Mitochondrial Cardiolipin Remodeling Dampens HIF-1? Expression in Hypoxia.
ABSTRACT: Mitochondria fulfill vital metabolic functions and act as crucial cellular signaling hubs, integrating their metabolic status into the cellular context. Here, we show that defective cardiolipin remodeling, upon loss of the cardiolipin acyl transferase tafazzin, decreases HIF-1? signaling in hypoxia. Tafazzin deficiency does not affect posttranslational HIF-1? regulation but rather HIF-1? gene expression, a dysfunction recapitulated in iPSC-derived cardiomyocytes from Barth syndrome patients with tafazzin deficiency. RNA-seq analyses confirmed drastically altered signaling in tafazzin mutant cells. In hypoxia, tafazzin-deficient cells display reduced production of reactive oxygen species (ROS) perturbing NF-?B activation and concomitantly HIF-1? gene expression. Tafazzin-deficient mice hearts display reduced HIF-1? levels and undergo maladaptive hypertrophy with heart failure in response to pressure overload challenge. We conclude that defective mitochondrial cardiolipin remodeling dampens HIF-1? signaling due to a lack of NF-?B activation through reduced mitochondrial ROS production, decreasing HIF-1? transcription.
Project description:Mitochondria fulfill vital metabolic functions and act as crucial cellular signaling hubs integrating their metabolic status into the cellular context. Here, we show that defective cardiolipin-remodeling, upon loss of the cardiolipin acyl transferase Tafazzin, mutes HIF-1a signaling in hypoxia. Tafazzin-deficiency does not affect posttranslational HIF-1a regulation but rather HIF-1a gene-expression, a dysfunction recapitulated in iPSCs-derived cardiomyocytes from Barth Syndrome patients with Tafazzin-deficiency. RNAseq analyses confirmed drastically altered signaling in Tafazzin mutant cells. In hypoxia, Tafazzin-deficient cells display reduced production of reactive oxygen species (ROS) perturbing NF-kB activation and concomitantly HIF-1a gene-expression. In agreement, Tafazzin-deficient mice hearts display reduced HIF-1a levels and undergo maladaptive hypertrophy with heart failure in response to pressure overload challenge. We conclude that defective mitochondrial cardiolipin-remodeling dampens HIF-1a signaling through inactivation of a non-canonical signaling pathway: Lack of NF-kB activation through reduced mitochondrial ROS production diminishes HIF-1a transcription. Overall design: Cells lacking tafazzin were subjected to hypoxia and compared to the ones at normoxia or isogenic WT cells either at hypoxia or nomoxia
Project description:Quantitative and qualitative alterations of mitochondrial cardiolipin have been implicated in the pathogenesis of Barth syndrome, an X-linked cardioskeletal myopathy caused by a deficiency in tafazzin, an enzyme in the cardiolipin remodeling pathway. We have generated and previously reported a tafazzin-deficient Drosophila model of Barth syndrome that is characterized by low cardiolipin concentration, abnormal cardiolipin fatty acyl composition, abnormal mitochondria, and poor motor function. Here, we first show that tafazzin deficiency in Drosophila disrupts the final stage of spermatogenesis, spermatid individualization, and causes male sterility. This phenotype can be genetically suppressed by inactivation of the gene encoding a calcium-independent phospholipase A(2), iPLA2-VIA, which also prevents cardiolipin depletion/monolysocardiolipin accumulation, although in wild-type flies inactivation of the iPLA2-VIA does not affect the molecular composition of cardiolipin. Furthermore, we show that treatment of Barth syndrome patients' lymphoblasts in tissue culture with the iPLA(2) inhibitor, bromoenol lactone, partially restores their cardiolipin homeostasis. Taken together, these findings establish a causal role of cardiolipin deficiency in the pathogenesis of Barth syndrome and identify iPLA2-VIA as an important enzyme in cardiolipin deacylation, and as a potential target for therapeutic intervention.
Project description:Cardiolipin (CL) is an inner mitochondrial membrane phospholipid which plays an important role in mitochondrial function. Perturbation in CL biosynthesis alters mitochondrial bioenergetics causing a severe genetic disorder commonly known as Barth syndrome. Barth syndrome patients are known to have a reduced concentration and altered composition of CL. Cardiolipin is also known to have a high affinity for the chemotherapeutic agent doxorubicin (Dox), resulting in an extensive mitochondrial accumulation of the drug. Our results indicate that B-lymphocytes from healthy individuals are more sensitive to Dox-induced oxidative stress and cellular toxicity compared to the B-lymphocytes from Barth syndrome as indicated by greater cell death and greater level of cleaved caspase-3 following Dox treatment. Barth lymphocytes, when compared to healthy lymphocytes, showed a greater basal level of mitochondrial reactive oxygen species (mito-ROS), yet exhibited a lower level of induced mito-ROS production in response to Dox. Significantly less ATP content and slightly greater OXPHOS protein levels were detected in healthy cells compared to Barth cells after Dox treatment. Consistent with greater mitochondrial ROS, treatment with Dox induced a higher level of lipid peroxidation and protein carbonylation in healthy lymphocytes compared to Barth lymphocytes. The final remodeling of CL during CL synthesis is catalyzed by the tafazzin protein. Knockdown of tafazzin gene in H9c2 cardiomyocytes using siRNA showed decreased oxidant-induced damage, as observed in Barth lymphocytes. Our findings demonstrate that a deficiency in CL might provide a therapeutic advantage in favor of oxidant-induced anticancer activities.
Project description:The phospholipid, cardiolipin, is essential for maintaining mitochondrial structure and optimal function. Cardiolipin-deficiency in humans, Barth syndrome, is characterized by exercise intolerance, dilated cardiomyopathy, neutropenia, and 3-methyl-glutaconic aciduria. The causative gene is the mitochondrial acyl-transferase, tafazzin, that is essential for remodeling acyl chains of cardiolipin. We sought to determine metabolic rates in tafazzin-deficient mice during resting and exercise, and investigate the impact of cardiolipin-deficiency on mitochondrial respiratory chain activities. Tafazzin-knockdown in mice markedly impaired oxygen consumption rates during an exercise, without any significant effect on resting metabolic rates. CL-deficiency resulted in significant reduction of mitochondrial respiratory reserve capacity in neonatal cardiomyocytes that is likely to be caused by diminished activity of complex-III, which requires CL for its assembly and optimal activity. Our results may provide mechanistic insights of Barth syndrome pathogenesis.
Project description:Barth syndrome is an X-linked genetic disorder caused by mutations in the tafazzin (taz) gene and characterized by dilated cardiomyopathy, exercise intolerance, chronic fatigue, delayed growth, and neutropenia. Tafazzin is a mitochondrial transacylase required for cardiolipin remodeling. Although tafazzin function has been studied in non-mammalian model organisms, mammalian genetic loss of function approaches have not been used. We examined the consequences of tafazzin knockdown on sarcomeric mitochondria and cardiac function in mice. Tafazzin knockdown resulted in a dramatic decrease of tetralinoleoyl cardiolipin in cardiac and skeletal muscles and accumulation of monolysocardiolipins and cardiolipin molecular species with aberrant acyl groups. Electron microscopy revealed pathological changes in mitochondria, myofibrils, and mitochondrion-associated membranes in skeletal and cardiac muscles. Echocardiography and magnetic resonance imaging revealed severe cardiac abnormalities, including left ventricular dilation, left ventricular mass reduction, and depression of fractional shortening and ejection fraction in tafazzin-deficient mice. Tafazzin knockdown mice provide the first mammalian model system for Barth syndrome in which the pathophysiological relationships between altered content of mitochondrial phospholipids, ultrastructural abnormalities, myocardial and mitochondrial dysfunction, and clinical outcome can be completely investigated.
Project description:Barth syndrome is a mitochondrial myopathy resulting from mutations in the tafazzin (<i>TAZ</i>) gene encoding a phospholipid transacylase required for cardiolipin remodeling. Cardiolipin is a phospholipid of the inner mitochondrial membrane essential for the function of numerous mitochondrial proteins and processes. However, it is unclear how tafazzin deficiency impacts cardiac mitochondrial metabolism. To address this question while avoiding confounding effects of cardiomyopathy on mitochondrial phenotype, we utilized <i>Taz</i>-shRNA knockdown (<i>Taz<sup>KD</sup></i> ) mice, which exhibit defective cardiolipin remodeling and respiratory supercomplex instability characteristic of human Barth syndrome but normal cardiac function into adulthood. Consistent with previous reports from other models, mitochondrial H<sub>2</sub>O<sub>2</sub> emission and oxidative damage were greater in <i>Taz<sup>KD</sup></i> than in wild-type (WT) hearts, but there were no differences in oxidative phosphorylation coupling efficiency or membrane potential. Fatty acid and pyruvate oxidation capacities were 40-60% lower in <i>Taz<sup>KD</sup></i> mitochondria, but an up-regulation of glutamate oxidation supported respiration rates approximating those with pyruvate and palmitoylcarnitine in WT. Deficiencies in mitochondrial CoA and shifts in the cardiac acyl-CoA profile paralleled changes in fatty acid oxidation enzymes and acyl-CoA thioesterases, suggesting limitations of CoA availability or "trapping" in <i>Taz<sup>KD</sup></i> mitochondrial metabolism. Incubation of <i>Taz<sup>KD</sup></i> mitochondria with exogenous CoA partially rescued pyruvate and palmitoylcarnitine oxidation capacities, implicating dysregulation of CoA-dependent intermediary metabolism rather than respiratory chain defects in the bioenergetic impacts of tafazzin deficiency. These findings support links among cardiolipin abnormalities, respiratory supercomplex instability, and mitochondrial oxidant production and shed new light on the distinct metabolic consequences of tafazzin deficiency in the mammalian heart.
Project description:The tafazzin (TAZ) gene is highly conserved from yeast to humans, and the yeast taz1 null mutant shows alterations in cardiolipin (CL) metabolism, mitochondrial dysfunction and stabilization of supercomplexes similar to those found in Barth syndrome, a human disorder resulting from loss of tafazzin. We have previously shown that the yeast tafazzin mutant taz1Delta, which cannot remodel CL, is ethanol-sensitive at elevated temperature. In the current report, we show that in response to ethanol, CL mutants taz1Delta as well as crd1Delta, which cannot synthesize CL, exhibited increased protein carbonylation, an indicator of reactive oxygen species (ROS). The increase in ROS is most likely not due to defective oxidant defence systems, as the CL mutants do not display sensitivity to paraquat, menadione or hydrogen peroxide (H2O2). Ethanol sensitivity and increased protein carbonylation in the taz1Delta mutant but not in crd1Delta can be rescued by supplementation with oleic acid, suggesting that oleoyl-CL and/or oleoyl-monolyso-CL enables growth of taz1Delta in ethanol by decreasing oxidative stress. Our findings of increased oxidative stress in the taz1Delta mutant during respiratory growth may have important implications for understanding the pathogenesis of Barth syndrome.
Project description:Barth syndrome is a complex metabolic disorder caused by mutations in the mitochondrial transacylase tafazzin. Recently, an inducible tafazzin shRNA knockdown mouse model was generated to deconvolute the complex bioenergetic phenotype of this disease. To investigate the underlying cause of hemodynamic dysfunction in Barth syndrome, we interrogated the cardiac structural and signaling lipidome of this mouse model as well as its myocardial bioenergetic phenotype. A decrease in the distribution of cardiolipin molecular species and robust increases in monolysocardiolipin and dilysocardiolipin were demonstrated. Additionally, the contents of choline and ethanolamine glycerophospholipid molecular species containing precursors for lipid signaling at the sn-2 position were altered. Lipidomic analyses revealed specific dysregulation of HETEs and prostanoids, as well as oxidized linoleic and docosahexaenoic metabolites. Bioenergetic interrogation uncovered differential substrate utilization as well as decreases in Complex III and V activities. Transgenic expression of cardiolipin synthase or iPLA2? ablation in tafazzin-deficient mice did not rescue the observed phenotype. These results underscore the complex nature of alterations in cardiolipin metabolism mediated by tafazzin loss of function. Collectively, we identified specific lipidomic, bioenergetic, and signaling alterations in a murine model that parallel those of Barth syndrome thereby providing novel insights into the pathophysiology of this debilitating disease.
Project description:Tafazzin (TAZ) is a phospholipid transacylase that catalyzes the remodeling of cardiolipin, a mitochondrial phospholipid required for oxidative phosphorylation. Mutations of TAZ 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. Here we investigated the role of TAZ in regulating mitochondrial function and mitophagy. Using primary mouse embryonic fibroblasts (MEFs) with doxycycline-inducible knockdown of Taz, we showed that TAZ deficiency in MEFs caused defective mitophagosome biogenesis, but not other autophagic processes. Consistent with a key role of mitophagy in mitochondria quality control, TAZ deficiency in MEFs also led to impaired oxidative phosphorylation and severe oxidative stress. Together, these findings provide key insights on mitochondrial dysfunction in Barth syndrome, suggesting that pharmacological restoration of mitophagy may provide a novel treatment for this lethal condition.
Project description:Mitochondria constantly adapt to the metabolic needs of a cell. This mitochondrial plasticity is critical to T cells, which modulate metabolism depending on antigen-driven signals and environment. We show here that de novo synthesis of the mitochondrial membrane-specific lipid cardiolipin maintains CD8<sup>+</sup> T cell function. T cells deficient for the cardiolipin-synthesizing enzyme PTPMT1 had reduced cardiolipin and responded poorly to antigen because basal cardiolipin levels were required for activation. However, neither de novo cardiolipin synthesis, nor its Tafazzin-dependent remodeling, was needed for T cell activation. In contrast, PTPMT1-dependent cardiolipin synthesis was vital when mitochondrial fitness was required, most notably during memory T cell differentiation or nutrient stress. We also found CD8<sup>+</sup> T cell defects in a small cohort of patients with Barth syndrome, where TAFAZZIN is mutated, and in a Tafazzin-deficient mouse model. Thus, the dynamic regulation of a single mitochondrial lipid is crucial for CD8<sup>+</sup> T cell immunity.