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.     

Human Trifunctional Protein Alpha Links Cardiolipin Remodeling to Beta-Oxidation

Cardiolipin (CL) is a mitochondrial membrane phospholipid which plays a key role in apoptosis and supports mitochondrial respiratory chain complexes involved in the generation of ATP. In order to facilitate its role CL must be remodeled with appropriate fatty acids. We previously identified a human monolysocardiolipin acyltransferase activity which remodels CL via acylation of monolysocardiolipin (MLCL) to CL and was identical to the alpha subunit of trifunctional protein (αTFP) lacking the first 227 amino acids. Full length αTFP is an enzyme that plays a prominent role in mitochondrial β-oxidation, and in this study we assessed the role, if any, which this metabolic enzyme plays in the remodeling of CL. Purified human recombinant αTFP exhibited acyl-CoA acyltransferase activity in the acylation of MLCL to CL with linoleoyl-CoA, oleoyl-CoA and palmitoyl-CoA as substrates. Expression of αTFP increased radioactive linoleate or oleate or palmitate incorporation into CL in HeLa cells. Expression of αTFP in Barth Syndrome lymphoblasts, which exhibit reduced tetralinoleoyl-CL, elevated linoleoyl-CoA acylation of MLCL to CL in vitro, increased mitochondrial respiratory Complex proteins and increased linoleate-containing species of CL. Knock down of αTFP in Barth Syndrome lymphoblasts resulted in greater accumulation of MLCL than those with normal αTFP levels. The results clearly indicate that the human αTFP exhibits MLCL acyltransferase activity for the resynthesis of CL from MLCL and directly links an enzyme of mitochondrial β-oxidation to CL remodeling.     

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.     

Cellular functions of cardiolipin in yeast

Cardiolipin (CL), the signature lipid of mitochondria, plays a critical role in mitochondrial function and biogenesis. The availability of yeast mutants blocked in CL synthesis has facilitated studies of the biological role of this lipid. Perturbation of CL synthesis leads to growth defects not only during respiratory growth but also under conditions in which respiration is not essential. CL was shown to play a role in mitochondrial protein import, cell wall biogenesis, aging and apoptosis, ceramide synthesis, and translation of electron transport chain components. The genetic disorder Barth syndrome (BTHS) is caused by mutations in the tafazzin gene resulting in decreased total CL levels, accumulation of monolysocardiolipin (MLCL), and decreased unsaturated fatty acyl species of CL. The variation in clinical presentation of BTHS indicates that other physiological factors play a significant role in modifying the phenotype resulting from tafazzin deficiency. Elucidating the functions of CL is expected to shed light on the role of this important lipid in BTHS and other disorders of mitochondrial dysfunction.     

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.     

Mitochondrial monolysocardiolipin acyltransferase is elevated in the surviving population of H9c2 cardiac myoblast cells exposed to 2-deoxyglucose-induced apoptosis.

Cardiolipin (CL) is a major mitochondrial membrane phospholipid in the mammalian heart and the remodeling of CL is essential to maintain its unique unsaturated fatty acyl composition. We examined CL de novo biosynthesis and remodeling in the surviving population of H9c2 cardiac myoblast cells exposed to 2-deoxyglucose (2-DG). H9c2 cells were incubated in the absence or presence of 2-DG for 16 h with [1,3-3H]glycerol or [1-14C]linoleic acid (bound to albumin in a 1:1 molar ratio). Dead cells were removed and radioactivity was incorporated into CL. Its pool size, fatty acid composition, and the activities of the CL biosynthesis and remodeling enzymes were determined. The CL pool size, its fatty acid composition, and [1,3-3H]glycerol or [1-14C]linoleic acid incorporated into CL were unaltered in the surviving population of 2-DG-treated cells compared with controls. In addition, the activities of the CL de novo biosynthetic enzymes were unaltered. Cleaved caspase-3 and poly(ADP-ribose) polymerase were slightly elevated in the surviving population of 2-DG-treated cells compared with controls, indicating that apoptosis induction was occurring in these cells. Mitochondrial phospholipase A2 and monolysocardiolipin acyltransferase (MLCL AT) activities increased 33% (p < 0.05) and 63% (p < 0.05), respectively, in 2-deoxyglucose-treated cells compared with controls. In contrast, the activity of ALCAT1, an endoplasmic reticulum MLCL AT, decreased 77% (p < 0.05), but this was not due to a reduction in ALCAT1 mRNA expression. The mRNA expression of the Barth syndrome gene TAZ, encoding a mitochondrial CL transacylase, was unaltered in 2-DG treated cells. The increase in mitochondrial MLCL AT activity was due to an elevated expression in MLCL AT protein. Thus, an increase in MLCL AT activity and expression occurs to maintain the CL pool in the surviving population of H9c2 cells as a compensatory mechanism for the elevated phospholipase A2 activity seen in 2-DG-induced apoptosis. We hypothesize that increased mitochondrial MLCL AT activity and its expression, and hence, elevated CL resynthesis, may be a protective mechanism against monolysocardiolipin-mediated apoptosis.     

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.     

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.     

Proliferation of C6 glioma cells requires the phospholipid remodeling enzyme tafazzin independent of cardiolipin composition

The mitochondrial phospholipid (CL) has been linked to mitochondrial and cellular functions. It has been postulated that the composition of CL is of impact for mitochondrial energy metabolism and cell proliferation. Although a correlation between CL composition and proliferation could be demonstrated for several cell types, evidence for a causal relationship remains obscure. Here, we applied two independent approaches, i) supplementation of fatty acids and ii) knock-out of the phospholipid remodeling enzyme tafazzin, to manipulate CL composition and analyzed the response on proliferation of C6 glioma cells. Both strategies caused substantial changes in the distribution of cellular fatty acids as well as in the distribution of fatty acids incorporated in CL that were accompanied by changes of the composition of molecular CL species. These changes did not correlate with cell proliferation. However, knock-out of tafazzin caused dramatic reduction in proliferation of C6 glioma cells independent of CL composition. The mechanism of tafazzin-dependent restriction of proliferation remains unclear. Among the various fatty acids administered only palmitic acid restricted cell proliferation by induction of cell death.     

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.     

Identification of the Human Mitochondrial Linoleoyl-coenzyme A Monolysocardiolipin Acyltransferase (MLCL AT-1)

Here we report the identification of a previously uncharacterized human protein as the human monolysocardiolipin acyltransferase-1 (MLCL AT-1). Pig liver mitochondria were treated with n-butyl alcohol followed by Q-Sepharose chromatography, preparative gel electrophoresis, cytidine diphosphate-1,2-diacyl-sn-glycerol-Sepharose chromatography, and finally monolysocardiolipin-adriamycin-agarose affinity chromatography. Elution with either monolysocardiolipin or linoleoyl coenzyme A revealed a major band at 74 kDa with high specific activity (2,300 pmol/min/mg) for the acylation of monolysocardiolipin to cardiolipin using [1-¹⁴C]linoleoyl coenzyme A as substrate. Matrix-assisted laser desorption ionization time-of-flight-mass spectrometry analysis followed by search of the Mascot protein data base revealed peptide matches consistent with a 59-kDa protein identified as unknown human protein (GenBank^TM protein accession number {"type":"entrez-protein","attrs":{"text":"AAX93141","term_id":"62702215","term_text":"AAX93141"}}AAX93141; nucleotide accession number {"type":"entrez-nucleotide","attrs":{"text":"AC011742.3","term_id":"9887800","term_text":"AC011742.3"}}AC011742.3). The purified human recombinant MLCL AT-1 protein utilized linoleoyl coenzyme A > oleoyl coenzyme A > palmitoyl coenzyme A for the specific acylation of monolysocardiolipin to cardiolipin. Expression of MLCL AT-1 in HeLa cells increased mitochondrial monolysocardiolipin acyltransferase activity and [1-¹⁴C]linoleic acid incorporated into cardiolipin, whereas RNA interference knockdown of MLCL AT-1 in HeLa cells resulted in reduction in enzyme activity and [1-¹⁴C]linoleic acid incorporated into cardiolipin. In contrast, expression of MLCL AT-1 in HeLa cells did not alter [1-¹⁴C]oleic or [1-¹⁴C]palmitate incorporation into cardiolipin indicating in vivo specificity for the remodeling of cardiolipin with linoleate. Finally, expression of MLCL AT-1 in Barth syndrome lymphoblasts, which exhibit cardiolipin levels 20% that of normal lymphoblasts, increased mitochondrial monolysocardiolipin acyltransferase activity, [1-¹⁴C]linoleic acid incorporation into cardiolipin, cardiolipin mass, and succinate dehydrogenase (mitochondrial complex II) activity compared with mock-transfected Barth syndrome lymphoblasts. The results identify MLCL AT-1 as a human mitochondrial monolysocardiolipin acyltransferase involved in the remodeling of cardiolipin.Cardiolipin (CL)² is a major phospholipid found in mammalian mitochondria with a multitude of biological functions (reviewed in Refs. 1–7). For example, CL is responsible for modulation of the activity of several mitochondrial enzymes involved in the generation of ATP (reviewed in Refs. 8, 9). In fact, it has been suggested that CL is the “glue” that holds the mitochondrial respiratory complex together (10). The role of CL in genetic diseases such as Barth syndrome, a rare X-linked genetic disorder, is beginning to emerge. Barth syndrome is the only known genetic disease in which the specific biochemical defect is a reduction in CL and accumulation of monolysocardiolipin (MLCL) caused by mutations in the TAZ gene (reviewed in Refs. 2, 7, 11, 12). In addition, the role that CL plays in apoptosis is now well documented (reviewed in Ref. 13). Thus, maintenance of the appropriate content and fatty acyl composition of CL in mitochondria is essential for proper cellular function.The molecular composition of CL appears to be important for the biological function of CL. In general, there is a selection of a particular kind of fatty acid as well as restriction of the number of fatty acid species (14). The major tetra-acyl molecular species found in rat liver (∼57% of total) and bovine heart (∼48% of total) are 18:2 in each of the four fatty acyl positions of the cardiolipin molecule. Remodeling of CL is essential to obtain this enrichment of CL with linoleate because CL synthase has no molecular species substrate specificity for cytidine-5′-diphosphate-1,2-diacyl-sn-glycerol (15). In addition, the species pattern of CL precursors is similar enough to imply that the enzymes of the CL synthetic pathway are not molecular species-selective (16). Alterations in the molecular composition of CL are associated with various disease states, including diabetes and Barth syndrome (17, 18).Remodeling of CL occurs via at least three enzymes. Mitochondrial CL was shown to be remodeled by a deacylation-reacylation cycle in which newly synthesized CL was rapidly deacylated to MLCL and then reacylated back to CL with linoleoyl-CoA (19). A mitochondrial MLCL acyltransferase (MLCL AT) activity was characterized and purified from pig liver mitochondria (20, 21). An acyl-CoA-dependent reacylation of MLCL to CL was shown to occur in rat liver microsomes (22). This enzyme was identified as acyllysocardiolipin acyltransferase-1 (ALCAT1) (23). Recently it was shown that ALCAT1 expression in endothelial and hematopoietic lineages resulted in elevated hematopoietic and endothelial genes and increased blast colonies and their progenies (24, 25). The opposite effect was observed with ALCAT1 small interfering RNA indicating that ALCAT1 may play a role in the early specification of hematopoietic and endothelial cells (24, 25). In addition to these mitochondrial and microsomal acyltransferase activities, mitochondrial CL may be remodeled by a mitochondrial CL transacylase reaction first described in rat liver (26). The Barth syndrome gene product tafazzin (TAZ) is a CL transacylase (27). Although TAZ specifically remodels mitochondrial CL with linoleic acid, TAZ alone cannot determine the fatty acid profile of mitochondrial CL (3). In this study, we identify a human protein, MLCL AT-1, with a linoleoyl coenzyme A-specific mitochondrial MLCL AT activity.     

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.     

Cardiolipin and apoptosis

Cardiolipin (CL) is recognized to be an essential phospholipid in eukaryotic energy metabolism so that physiological and pathological perturbations in its synthetic and catabolic pathways play key roles in maintaining mitochondrial structure and function, and ultimately cell survival. This review describes potential regulatory mechanisms in CL synthesis and the effects of de-acylation pathways on steady state levels of CL and its interaction with cytochrome c. The latter interaction is significant in the initiation of programmed cell death. Physiological factors that modify CL acylation include ageing, dietary influences and ischemia/reperfusion where the terminal events may be either necrosis or apoptosis. In various pathologies, phospholipase activity increases in response to production of peroxidized CL. The cell may use lysosomal or mitochondrial pathways for CL degradation. However, the manner by which CL and cytochrome c leave the mitochondria is not well understood. The lipid (CL)-bound form of cytochrome c is thought to initiate apoptosis via a lipid transfer step involving mitochondrially targeted Bid. A direct relationship between CL loss and cytochrome c release from the mitochondria has been identified as an initial step in the pathway to apoptosis. An absolute requirement for CL in the function of crucial mitochondrial proteins, e.g., cytochrome oxidase and the adenine nucleotide translocase, are likely additional factors impacting apoptosis and cellular energy homeostasis. This is reflected in the occurrence of both oncotic and apoptotic events in ischemia and reperfusion injury. Other potential clinical manifestations of perturbations of CL synthesis are discussed with particular emphasis on Barth Syndrome where a primary defect can be attributed to CL metabolism and is associated with dilated cardiomyopathy. Finally, the model of fatty acid induced apoptosis is used as a paradigm to our understanding of the temporal relationship between decreased mitochondrial CL, release of cytochrome c, and initiation of apoptosis.     

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.     

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.     

The influence of the acyl chain composition of cardiolipin on the stability of mitochondrial complexes; An unexpected effect of cardiolipin in α-ketoglutarate dehydrogenase and prohibitin complexes

The role of cardiolipin acyl chain composition in assembly/stabilization of mitochondrial complexes was investigated using three yeast deletion mutants (acb1Δ strain; taz1Δ strain; and acb1Δtaz1Δ strain). Deletion of the TAZ1 gene, involved in cardiolipin acyl chain remodeling, is known to increase the content of monolyso-cardiolipin (MLCL) at the expense of CL, and to decrease the unsaturation of the remaining CL. Deletion of the ACB1 gene encoding the acyl-CoA-binding protein, involved in fatty acid elongation, decreases the average length of the CL acyl chains. Furthermore, a TAZ1ACB1 double deletion mutant strain was used in this study which has both a decrease in the length of the CL acyl chains and an increase in MLCL. BN/SDS PAGE analysis revealed that cardiolipin is important for the prohibitin–m-AAA protease complex, the α-ketoglutarate dehydrogenase complex and respiratory chain supercomplexes. The results indicate that the decreased level of complexes in taz1Δ and acb1Δtaz1Δ mitochondria is due to a decreased content of CL or the presence of MLCL.     

Cardiolipin and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis

We previously reported that during death receptor-mediated apoptosis, cardiolipin (CL) relocates to the cell surface, where it reacts with autoantibodies from antiphospholipid syndrome sera. Here, we analysed the intracellular distribution of CL and its metabolites during the early phase of cell death signalling triggered by Fas stimulation in U937 cells and mouse liver. We found a redistribution of mitochondrial CL to the cell surface by using confocal microscopy and flow cytometry. Mass spectrometry revealed that CL and its metabolites relocated from mitochondria to other intracellular organelles during apoptosis, with a conversion into non-mitochondrial lipids. Concomitantly, cytosolic Bid relocated to the light membranes comprised in fraction P100, including the plasma membrane and associated vesicular systems. A direct Bid-CL interaction was demonstrated by the observation that CL and monolysoCL coimmunoprecipitated with Bid especially after Fas stimulation, suggesting a dynamic interaction of the protein with CL and its metabolites.