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.     

Promotion of plasmalogen biosynthesis reverse lipid changes in a Barth Syndrome cell model

In Barth syndrome (BTHS) mutations in tafazzin leads to changes in both the quantities and the molecular species of cardiolipin (CL), which are the hallmarks of BTHS. Contrary to the well-established alterations in CL associated with BTHS; recently a marked decrease in the plasmalogen levels in Barth specimens has been identified. To restore the plasmalogen levels, the present study reports the effect of promotion of plasmalogen biosynthesis on the lipidome of lymphoblasts derived from Barth patients as well as on cell viability, mitochondria biogenesis, and mitochondrial membrane potential. High resolution ³¹P NMR phospholipidomic analysis showed an increase in the levels of plasmenylethanolamine (the major plasmalogen in lymphoblasts), which reached values comparable to the control and a compensatory decrease in the levels of its diacyl-PE counterpart. Importantly, ³¹P NMR showed a significant increase in the levels of CL, while not altering the levels of monolysocardiolipin. Mass spectrometry measurements showed that the promotion of plasmalogen biosynthesis did not change the molecular species profile of targeted phospholipids. In addition, promotion of plasmalogen biosynthesis did not impact on cellular viability, although it significantly decrease mitochondria copy number and restored mitochondrial membrane potential. Overall, the results showed the efficacy of the promotion of plasmalogen biosynthesis on increasing the CL levels in a BTHS cell model and highlight the potential beneficial effect of a diet supplemented with plasmalogen precursors to BTHS patients.     

De novo biosynthesis of the late endosome lipid, bis(monoacylglycero)phosphate

Bis(monoacylglycero)phosphate (BMP) is a unique lipid enriched in the late endosomes participating in the trafficking of lipids and proteins through this organelle. The de novo biosynthesis of BMP has not been clearly demonstrated. We investigated whether phosphatidylglycerol (PG) and cardiolipin (CL) could serve as precursors of de novo BMP synthesis using two different cellular models: CHO cells deficient in phosphatidylglycerophosphate (PGP) synthase, the enzyme responsible for the first step of PG synthesis; and human lymphoblasts from patients with Barth syndrome (BTHS), characterized by mutations in tafazzin, an enzyme implicated in the deacylation-reacylation cycle of CL. The biosynthesis of both PG and BMP was reduced significantly in the PGP synthase-deficient CHO mutants. Furthermore, overexpression of PGP synthase in the deficient mutants induced an increase of BMP biosynthesis. In contrast to CHO mutants, BMP biosynthesis and its fatty acid composition were not altered in BTHS lymphoblasts. Our results thus suggest that in mammalian cells, PG, but not CL, is a precursor of the de novo biosynthesis of BMP. Despite the decrease of de novo synthesis, the cellular content of BMP remained unchanged in CHO mutants, suggesting that other pathway(s) than de novo biosynthesis are also used for BMP synthesis.     

The enigmatic role of tafazzin in cardiolipin metabolism

The mitochondrial phospholipid cardiolipin plays an important role in cellular metabolism as exemplified by its involvement in mitochondrial energy production and apoptosis. Following its biosynthesis, cardiolipin is actively remodeled to achieve its final acyl composition. An important cardiolipin remodeling enzyme is tafazzin, of which several mRNA splice variants exist. Mutations in the tafazzin gene cause the X-linked recessive disorder Barth syndrome. In addition to providing an overview of the current knowledge in literature about tafazzin, we present novel experimental data and use this to discuss the functional role of the different tafazzin variants in cardiolipin metabolism in relation to Barth syndrome. We developed and performed specific quantitative PCR analyses of different tafazzin mRNA splice variants in 16 human tissues and correlated this with the tissue cardiolipin profile. In BTHS fibroblasts we showed that mutations in the tafazzin gene affected both the level and distribution of tafazzin mRNA variants. Transient expression of selected human tafazzin variants in BTHS fibroblasts showed for the first time in a human cell system that tafazzin lacking exon5 indeed functions in cardiolipin remodeling.     

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.     

Tafazzin-dependent cardiolipin composition in C6 glioma cells correlates with changes in mitochondrial and cellular functions,and cellular proliferation

The mitochondrial phospholipid cardiolipin (CL) has been implicated with mitochondrial morphology, function and, more recently, with cellular proliferation. Tafazzin, an acyltransferase with key functions in CL remodeling determining actual CL composition, affects mitochondrial oxidative phosphorylation. Here, we show that the CRISPR-Cas9 mediated knock-out of tafazzin (Taz) is associated with substantial alterations of various mitochondrial and cellular characteristics in C6 glioma cells. The knock-out of tafazzin substantially changed the profile of fatty acids incorporated in CL and the distribution of molecular CL species. Taz knock-out was further associated with decreased capacity of oxidative phosphorylation that mainly originates from impaired complex I associated energy metabolism in C6 glioma cells. The lack of tafazzin switched energy metabolism from oxidative phosphorylation to glycolysis indicated by lower respiration rates, membrane potential and higher levels of mitochondria-derived reactive oxygen species but keeping the cellular ATP content unchanged. The impact of tafazzin on mitochondria was also indicated by altered morphology and arrangement in tafazzin deficient C6 glioma cells. In the cells we observed tafazzin-dependent changes in the distribution of cellular fatty acids as an indication of altered lipid metabolism as well as in stability/morphology. Most impressive is the dramatic reduction in cell proliferation in tafazzin deficient C6 glioma cells that is not mediated by reactive oxygen species. Our data clearly indicate that defects in CL phospholipid remodeling trigger a cascade of events including modifications in CL linked to subsequent alterations in mitochondrial and cellular functions.     

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.     

Deacylation on the matrix side of the mitochondrial inner membrane regulates cardiolipin remodeling

The mitochondrial-specific lipid cardiolipin (CL) is required for numerous processes therein. After its synthesis on the matrix-facing leaflet of the inner membrane (IM), CL undergoes acyl chain remodeling to achieve its final form. In yeast, this process is completed by the transacylase tafazzin, which associates with intermembrane space (IMS)-facing membrane leaflets. Mutations in TAZ1 result in the X-linked cardiomyopathy Barth syndrome. Amazingly, despite this clear pathophysiological association, the physiological importance of CL remodeling is unresolved. In this paper, we show that the lipase initiating CL remodeling, Cld1p, is associated with the matrix-facing leaflet of the mitochondrial IM. Thus monolysocardiolipin generated by Cld1p must be transported to IMS-facing membrane leaflets to gain access to tafazzin, identifying a previously unknown step required for CL remodeling. Additionally, we show that Cld1p is the major site of regulation in CL remodeling; and that, like CL biosynthesis, CL remodeling is augmented in growth conditions requiring mitochondrially produced energy. However, unlike CL biosynthesis, dissipation of the mitochondrial membrane potential stimulates CL remodeling, identifying a novel feedback mechanism linking CL remodeling to oxidative phosphorylation capacity.     

Cardiac Metabolic Pathways Affected in the Mouse Model of Barth Syndrome

Cardiolipin (CL) is a mitochondrial phospholipid essential for electron transport chain (ETC) integrity. CL-deficiency in humans is caused by mutations in the tafazzin (Taz) gene and results in a multisystem pediatric disorder, Barth syndrome (BTHS). It has been reported that tafazzin deficiency destabilizes mitochondrial respiratory chain complexes and affects supercomplex assembly. The aim of this study was to investigate the impact of Taz-knockdown on the mitochondrial proteomic landscape and metabolic processes, such as stability of respiratory chain supercomplexes and their interactions with fatty acid oxidation enzymes in cardiac muscle. Proteomic analysis demonstrated reduction of several polypeptides of the mitochondrial respiratory chain, including Rieske and cytochrome c1 subunits of complex III, NADH dehydrogenase alpha subunit 5 of complex I and the catalytic core-forming subunit of F₀F₁-ATP synthase. Taz gene knockdown resulted in upregulation of enzymes of folate and amino acid metabolic pathways in heart mitochondria, demonstrating that Taz-deficiency causes substantive metabolic remodeling in cardiac muscle. Mitochondrial respiratory chain supercomplexes are destabilized in CL-depleted mitochondria from Taz knockdown hearts resulting in disruption of the interactions between ETC and the fatty acid oxidation enzymes, very long-chain acyl-CoA dehydrogenase and long-chain 3-hydroxyacyl-CoA dehydrogenase, potentially affecting the metabolic channeling of reducing equivalents between these two metabolic pathways. Mitochondria-bound myoglobin was significantly reduced in Taz-knockdown hearts, potentially disrupting intracellular oxygen delivery to the oxidative phosphorylation system. Our results identify the critical pathways affected by the Taz-deficiency in mitochondria and establish a future framework for development of therapeutic options for BTHS.     

Blocking phosphatidylglycerol degradation in yeast defective in cardiolipin remodeling results in a new model of the Barth syndrome cellular phenotype

Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant pgc1Δtaz1Δ of Saccharomyces cerevisiae deficient in phosphatidylglycerol-specific phospholipase C and tafazzin as a new yeast model of BTHS. Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies.     

The cardiolipin transacylase, tafazzin, associates with two distinct respiratory components providing insight into Barth syndrome

Mutations in the mitochondrial cardiolipin (CL) transacylase, tafazzin (Taz1p), result in the X-linked cardioskeletal myopathy, Barth syndrome (BTHS). The mitochondria of BTHS patients exhibit variable respiratory defects and abnormal cristae ultrastructure. The biochemical basis for these observations is unknown. In the absence of its target phospholipid, CL, a very large Taz1p complex is missing, whereas several discrete smaller complexes are still observed. None of the identified Taz1p complexes represents Taz1p homodimers. Instead, yeast Taz1p physically assembles in several protein complexes of distinct size and composition. The ATP synthase and AAC2, both required for oxidative phosphorylation, are identified in separate stable Taz1p complexes. In the absence of CL, each interaction is still detected albeit in reduced abundance compared with when CL is present. Taz1p is not necessary for the normal expression of AAC2 or ATP synthase subunits or assembly of their respective complexes. In contrast, the largest Taz1p complex requires assembled ATP synthase and CL. Mitochondria in Δtaz1 yeast, similar to ATP synthase oligomer mutants, exhibit altered cristae morphology even though ATP synthase oligomer formation is unaffected. Thus, the Taz1p interactome defined here provides novel insight into the variable respiratory defects and morphological abnormalities observed in mitochondria of BTHS patients.     

Monolysocardiolipin in cultured fibroblasts is a sensitive and specific marker for Barth Syndrome

Barth Syndrome (BTHS) is an X-linked recessive disorder that results in abnormal metabolism of the mitochondrial phospholipid cardiolipin (CL). CLs are decreased and monolysocardiolipins (MLCLs), intermediates in CL metabolism, are increased in a variety of tissues. Measurement of decreased CL levels in skin fibroblasts has previously been proposed as a diagnostic test for BTHS. We investigated whether elevated MLCL is specific for BTHS and whether the MLCL-to-CL ratio is a more sensitive and specific marker for BTHS. We measured CLs and MLCLs in skin fibroblasts from 5 BTHS patients, 8 controls, and 14 patients with biochemical and clinical findings similar to those in BTHS (group D), using high performance liquid chromatography-mass spectrometry. Our results showed a clear decrease of CL in combination with a marked increase of MLCL in fibroblasts from BTHS patients when compared with controls. MLCL/CL ratios ranged from 0.03-0.12 in control fibroblasts and from 5.41-13.83 in BTHS fibroblasts. In group D, the MLCL/CL ratio range was 0.02-0.06. We therefore conclude that elevations of MLCLs are specific for BTHS and that the MLCL/CL ratio in fibroblasts is a better diagnostic marker than CL alone. We also report the finding of two novel mutations in the TAZ gene that cause BTHS.     

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.     

Mitochondrial mislocalization and altered assembly of a cluster of Barth syndrome mutant tafazzins

None of the 28 identified point mutations in tafazzin (Taz1p), which is the mutant gene product associated with Barth syndrome (BTHS), has a biochemical explanation. In this study, endogenous Taz1p was localized to mitochondria in association with both the inner and outer mitochondrial membranes facing the intermembrane space (IMS). Unexpectedly, Taz1p does not contain transmembrane (TM) segments. Instead, Taz1p membrane association involves a segment that integrates into, but not through, the membrane bilayer. Residues 215-232, which were predicted to be a TM domain, were identified as the interfacial membrane anchor by modeling four distinct BTHS mutations that occur at conserved residues within this segment. Each Taz1p mutant exhibits altered membrane association and is nonfunctional. However, the basis for Taz1p dysfunction falls into the following two categories: (1) mistargeting to the mitochondrial matrix or (2) correct localization associated with aberrant complex assembly. Thus, BTHS can be caused by mutations that alter Taz1p sorting and assembly within the mitochondrion, indicating that the lipid target of Taz1p is resident to IMS-facing leaflets.     

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.     

Loss of tafazzin in yeast leads to increased oxidative stress during respiratory growth

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.     

Monolysocardiolipins accumulate in Barth syndrome but do not lead to enhanced apoptosis

Barth syndrome (BTHS) is an X-linked recessive disorder that is biochemically characterized by low cellular levels of the mitochondrial phospholipid cardiolipin (CL). Previously, we discovered that the yeast disruptant of the TAZ ortholog in Saccharomyces cerevisiae not only displays CL deficiency but also accumulates monolysocardiolipins (MLCLs), which are intermediates in CL remodeling. Therefore, we set out to investigate whether MLCL accumulation also occurs in BTHS. Indeed, we observed MLCL accumulation in heart, muscle, lymphocytes, and cultured lymphoblasts of BTHS patients; however, only very low levels of these lysophospholipids were found in platelets and fibroblasts of these patients. Although the fatty acid composition of the MLCLs was different depending on the tissue source, it did parallel the fatty acid composition of the (remaining) CLs. The possible implications of these findings for the two reported CL remodeling mechanisms, transacylation and deacylation/reacylation, are discussed. Because MLCLs have been proposed to be involved in the initiation of apoptosome-mediated cell death by the sequestration of the proapoptotic protein (t)BH3-interacting domain death agonist (Bid) to the mitochondrial membrane, we used control and BTHS lymphoblasts to investigate whether the accumulation of MLCLs results in higher levels of apoptosis. We found no differences in susceptibility to death receptor-mediated apoptosis or in cellular distribution of Bid, cytochrome c, and other parameters, implying that MLCL accumulation does not lead to enhanced apoptosis in cultured BTHS lymphoblasts.     

Cardiolipin fingerprinting of leukocytes by MALDI-TOF/MS as a screening tool for Barth syndrome

Barth syndrome (BTHS), an X-linked disease associated with cardioskeletal myopathy, neutropenia, and organic aciduria, is characterized by abnormalities of card­iolipin (CL) species in mitochondria. Diagnosis of the disease is often compromised by lack of rapid and widely available diagnostic laboratory tests. The present study describes a new method for BTHS screening based on MALDI-TOF/MS analysis of leukocyte lipids. This generates a “CL fingerprint” and allows quick and simple assay of the relative levels of CL and monolysocardiolipin species in leukocyte total lipid profiles. To validate the method, we used vector algebra to analyze the difference in lipid composition between controls (24 healthy donors) and patients (8 boys affected by BTHS) in the high-mass phospholipid range. The method of lipid analysis described represents an important additional tool for the diagnosis of BTHS and potentially enables therapeutic monitoring of drug targets, which have been shown to ameliorate abnormal CL profiles in cells.