Effects of siRNA-dependent knock-down of cardiolipin synthase and tafazzin on mitochondria and proliferation of glioma cells

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function, and cell proliferation. Changes in CL are often paralleled by changes in the lipid environment of mitochondria that may contribute to mitochondrial function and proliferation. This study aimed to separate the effects of CL content and CL composition from cellular free fatty acid distribution on bioenergetics and proliferation in C6 glioma cells. To this end, cardiolipin synthase and the CL remodelling enzyme, tafazzin, were knocked-down by siRNA in C6 cells. After 72?h of cultivation, we analysed CL composition by means of LC/MS/MS, distribution of cellular fatty acids by means of gas chromatography, and determined oxygen consumption and proliferation. Knock-down of cardiolipin synthase affected the cellular CL content in the presence of linoleic acid (LA) in the culture medium. Knock-down of tafazzin had no consequence with respect to the pattern of cellular fatty acids but caused a decrease in cell proliferation. It significantly changed the distribution of molecular CL species, increased CL content, decreased oxygen consumption, and decreased cell proliferation when cultured in the presence of linoleic acid (LA). The addition of linoleic acid to the culture medium caused significant changes in the pattern of cellular fatty acids and the composition of molecular CL species. These data suggest that tafazzin is required for efficient bioenergetics and for proliferation of glioma cells. Supplementation of fatty acids can be a powerful tool to direct specific changes in these parameters.     

Cardiac and skeletal muscle defects in a mouse model of human Barth syndrome

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.     

Aberrant cardiolipin metabolism is associated with cognitive deficiency and hippocampal alteration in tafazzin knockdown mice

Cardiolipin (CL) is a key mitochondrial phospholipid essential for mitochondrial energy production. CL is remodeled from monolysocardiolipin (MLCL) by the enzyme tafazzin (TAZ). Loss-of-function mutations in the gene which encodes TAZ results in a rare X-linked disorder called Barth Syndrome (BTHS). The mutated TAZ is unable to maintain the physiological CL:MLCL ratio, thus reducing CL levels and affecting mitochondrial function. BTHS is best known as a cardiac disease, but has been acknowledged as a multi-syndrome disorder, including cognitive deficits. Since reduced CL levels has also been reported in numerous neurodegenerative disorders, we examined how TAZ-deficiency impacts cognitive abilities, brain mitochondrial respiration and the function of hippocampal neurons and glia in TAZ knockdown (TAZ kd) mice. We have identified for the first time the profile of changes that occur in brain phospholipid content and composition of TAZ kd mice. The brain of TAZ kd mice exhibited reduced TAZ protein expression, reduced total CL levels and a 19-fold accumulation of MLCL compared to wild-type littermate controls. TAZ kd brain exhibited a markedly distinct profile of CL and MLCL molecular species. In mitochondria, the activity of complex I was significantly elevated in the monomeric and supercomplex forms with TAZ-deficiency. This corresponded with elevated mitochondrial state I respiration and attenuated spare capacity. Furthermore, the production of reactive oxygen species was significantly elevated in TAZ kd brain mitochondria. While motor function remained normal in TAZ kd mice, they showed significant memory deficiency based on novel object recognition test. These results correlated with reduced synaptophysin protein levels and derangement of the neuronal CA1 layer in hippocampus. Finally, TAZ kd mice had elevated activation of brain immune cells, microglia compared to littermate controls. Collectively, our findings demonstrate that TAZ-mediated remodeling of CL contributes significantly to the expansive distribution of CL molecular species in the brain, plays a key role in mitochondria respiratory activity, maintains normal cognitive function, and identifies the hippocampus as a potential therapeutic target for BTHS.     

Cardiolipin remodeling and the function of tafazzin

Cardiolipin, the specific phospholipid of mitochondria, is involved in the biogenesis, the dynamics, and the supramolecular organization of mitochondrial membranes. Cardiolipin acquires a characteristic composition of fatty acids by post-synthetic remodeling, a process that is crucial for cardiolipin homeostasis and function. The remodeling of cardiolipin depends on the activity of tafazzin, a non-specific phospholipid–lysophospholipid transacylase. This review article discusses recent findings that suggest a novel function of tafazzin in mitochondrial membranes. By shuffling fatty acids between molecular species, tafazzin transforms the lipid composition and by doing so supports changes in the membrane conformation, specifically the generation of membrane curvature. Tafazzin activity is critical for the differentiation of cardiomyocytes, in which the characteristic cristae-rich morphology of cardiac mitochondria evolves. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Loss of tafazzin results in decreased myoblast differentiation in C2C12 cells: A myoblast model of Barth syndrome and cardiolipin deficiency

Barth syndrome (BTHS) is an X-linked genetic disorder resulting from mutations in the tafazzin gene (TAZ), which encodes the transacylase that remodels the mitochondrial phospholipid cardiolipin (CL). While most BTHS patients exhibit pronounced skeletal myopathy, the mechanisms linking defective CL remodeling and skeletal myopathy have not been determined. In this study, we constructed a CRISPR-generated stable tafazzin knockout (TAZ-KO) C2C12 myoblast cell line. TAZ-KO cells exhibit mitochondrial deficits consistent with other models of BTHS, including accumulation of monolyso-CL (MLCL), decreased mitochondrial respiration, and increased mitochondrial ROS production. Additionally, tafazzin deficiency was associated with impairment of myocyte differentiation. Future studies should determine whether alterations in myogenic determination contribute to the skeletal myopathy observed in BTHS patients. The BTHS myoblast model will enable studies to elucidate mechanisms by which defective CL remodeling interferes with normal myocyte differentiation and skeletal muscle ontogenesis.     

Suppression of Tafazzin promotes thyroid cancer apoptosis via activating the JNK signaling pathway and enhancing INF2-mediated mitochondrial fission

Tafazzin has been found to be associated with tumor progression. Mitochondrial homeostasis regulates cancer cell viability and metastasis. However, the roles of Tafazzin and mitochondrial homeostasis in thyroid cancer have not been explored. The aim of our study is to investigate the influences of Tafazzin on thyroid cancer apoptosis with a focus on mitochondrial fission. Our results indicated that Tafazzin deletion induced death in thyroid cancer via apoptosis. Biological analysis demonstrated that mitochondrial stress, including mitochondrial bioenergetics disorder, mitochondrial oxidative stress, and mitochondrial apoptosis, was activated by Tafazzin deletion. Furthermore, we found that Tafazzin affected mitochondrial stress by triggering inverted formin 2 (INF2)-related mitochondrial fission. The loss of INF2 sustained mitochondrial function and promoted cancer cell survival. Molecular investigation illustrated that Tafazzin regulated INF2 expression via the JNK signaling pathway; moreover, the blockade of JNK prevented Tafazzin-mediated INF2 expression and improved cancer cell survival. Taken together, our results highlight the key role of Tafazzin as a master regulator of thyroid cancer viability via the modulation of INF2-related mitochondrial fission and the JNK signaling pathway. These findings defined Tafazzin deletion and INF2-related mitochondrial fission as tumor suppressors that act by promoting cancer apoptosis via the JNK signaling pathway, with potential implications for new approaches to thyroid cancer therapy.     

Barth syndrome, a human disorder of cardiolipin metabolism

Barth syndrome is an X-linked recessive disease caused by mutations in the tafazzin gene. Patients have reduced concentration and altered composition of cardiolipin, the specific mitochondrial phospholipid, and they have variable clinical findings, often including heart failure, myopathy, neutropenia, and growth retardation. This article provides an overview of the molecular basis of Barth syndrome. It is argued that tafazzin, a phospholipid acyltransferase, is involved in acyl-specific remodeling of cardiolipin, which promotes structural uniformity and molecular symmetry among the cardiolipin molecular species. Inhibition of this pathway leads to changes in mitochondrial architecture and function.     

Barth syndrome cells display widespread remodeling of mitochondrial complexes without affecting metabolic flux distribution

Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by cardiac and skeletal myopathy, neutropenia and growth abnormalities. The disease is caused by mutations in the tafazzin (TAZ) gene encoding an enzyme involved in the acyl chain remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, this leads to decreased levels of mature CL and accumulation of the intermediate monolysocardiolipin (MLCL). At a cellular level, this causes mitochondrial fragmentation and reduced stability of the respiratory chain supercomplexes. However, the exact mechanism through which tafazzin deficiency leads to disease development remains unclear. We therefore aimed to elucidate the pathways affected in BTHS cells by employing proteomic and metabolic profiling assays. Complexome profiling of patient skin fibroblasts revealed significant effects for about 200 different mitochondrial proteins. Prominently, we found a specific destabilization of higher order oxidative phosphorylation (OXPHOS) supercomplexes, as well as changes in complexes involved in cristae organization and CL trafficking. Moreover, the key metabolic complexes 2-oxoglutarate dehydrogenase (OGDH) and branched-chain ketoacid dehydrogenase (BCKD) were profoundly destabilized in BTHS patient samples. Surprisingly, metabolic flux distribution assays using stable isotope tracer-based metabolomics did not show reduced flux through the TCA cycle. Overall, insights from analyzing the impact of TAZ mutations on the mitochondrial complexome provided a better understanding of the resulting functional and structural consequences and thus the pathological mechanisms leading to Barth syndrome.