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.     

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.     

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.     

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.     

Linoleic acid supplementation of Barth syndrome fibroblasts restores cardiolipin levels: implications for treatment

The object of this study was to investigate whether the levels of cardiolipin in cultured skin fibroblasts of patients with Barth syndrome (BTHS) can be restored by addition of linoleic acid to growth media. To this end, fibroblasts from controls and BTHS patients were grown in the presence or absence of linoleic acid. High-performance liquid chromatography-electrospray ionization tandem mass spectrometry was used for quantitative and compositional analysis of cardiolipin. Incubation of cells from both BTHS and controls with different concentrations of linoleic acid led to a dose- and time-dependent increase of cardiolipin levels. The increased levels of cardiolipin in fibroblasts of BTHS patients after treatment with linoleic acid indicate that an increased amount of linoleic acid in the diet might be beneficial to BTHS patients.     

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.     

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.     

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.     

In vivo synthesis of monolysocardiolipin and cardiolipin by Acinetobacter baumannii phospholipase D and effect on cationic antimicrobial peptide resistance

Acinetobacter baumannii is an opportunistic pathogen, which has become a rising threat in healthcare facilities worldwide due to increasing antibiotic resistances and optimal adaptation to clinical environments and the human host. We reported in a former publication on the identification of three phopholipases of the phospholipase D (PLD) superfamily in A. baumannii ATCC 19606^T acting in concerted manner as virulence factors in Galleria mellonella infection and lung epithelial cell invasion. This study focussed on the function of the three PLDs. A Δpld1-3 mutant was defect in biosynthesis of the phospholipids cardiolipin (CL) and monolysocardiolipin (MLCL), whereas the deletion of pld2 and pld3 abolished the production of MLCL. Complementation of the Δpld1-3 mutant with pld1 restored CL biosynthesis demonstrating that the PLD1 is implicated in CL biosynthesis. Complementation of the Δpld1-3 mutant with either pld2 or pld3 restored MLCL and CL production leading to the conclusion that PLD2 and PLD3 are implicated in CL and MLCL production. Mutant studies revealed that two catalytic motifs are essential for the PLD3-mediated biosynthesis of CL and MLCL. The Δpld1-3 mutant exhibited a decreased colistin and polymyxin B resistance indicating a role of CL in cationic antimicrobial peptides (CAMPs) resistance.     

Cardiolipin-depleted bovine heart cytochrome c oxidase: binding stoichiometry and affinity for cardiolipin derivatives

Detergent-solubilized bovine heart cytochrome c oxidase requires 2 mol of tightly bound cardiolipin (CL) per mole of monomeric complex for functional activity. Four lines of evidence support this conclusion: (1) Phospholipid depletion shows that two tightly bound CL's must remain associated with cytochrome c oxidase in order to maintain full electron transport activity. (2) Removal of the two tightly bound CL's correlates with decreased activity that is restored by reassociation of 2 mol of exogenous CL. (3) CL-depleted cytochrome c oxidase has two high-affinity binding sites for 2-[14C]acetylcardiolipin (AcCL), Kd,app less than 0.1 microM, that are not present in enzyme containing endogenous CL. An additional 2-3 lower affinity AcCL binding sites, Kd,app = 4 microM, are present in the CL-depleted complex, but these sites are also present in enzyme containing endogenous CL. (4) CL, monolysocardiolipin (MLCL), and dilysocardiolipin (DLCL) compete for AcCL binding with approximately the same relative affinities as those measured by the restoration of electron transport activity (MLCL competes much better than DLCL). However, MLCL and DLCL are only 60% and 15% as effective as CL in restoring maximum activity when they are bound to the high-affinity sites. The binding specificity of CL, MLCL, DLCL, and some of their acylated derivatives indicates that the apolar tails are most important for binding, not the polar head group. The presence or absence of hydroxyl groups in CL, MLCL, or DLCL also has little effect upon binding affinities. Binding specificity clearly favors CL since phosphatidylglycerol, phosphatidic acid, and phosphatidylcholine each have very low affinity for the CL binding sites (Kd,app greater than 20 microM). We, therefore, conclude that restoration of activity to CL-depleted cytochrome c oxidase is highly specific and requires the reassociation of CL, or structurally similar compounds, with two high-affinity binding sites.     

NAD supplementation improves mitochondrial performance of cardiolipin mutants

Cardiolipin (CL) deficiency causes mitochondrial dysfunction and aberrant metabolism that are associated in humans with the severe disease Barth syndrome (BTHS). Several metabolic abnormalities are observed in BTHS patients and model systems, including decreased oxidative phosphorylation, reduced tricarboxylic acid (TCA) cycle flux, and accumulated lactate and D-β-hydroxybutyrate, which strongly suggests that nicotinamide adenine dinucleotide (NAD) redox metabolism may be altered in CL-deficient cells. In this study, we identified abnormal NAD⁺ metabolism in multiple BTHS model systems and demonstrate that supplementation of NAD⁺ precursors such as nicotinamide mononucleotide (NMN) improves mitochondrial function. Improved mitochondrial function in the Drosophila model was associated with restored exercise endurance, which suggests a potential therapeutic benefit of NAD⁺ precursor supplementation in the management of BTHS patients.     

Monolysocardiolipin: improved preparation with high yield

A simple, high-yielding preparation of monolysocardiolipin (MLCL) by phospholipase A2 hydrolysis of cardiolipin (CL) in methanol on a semi-preparative scale is described. In methanol, phospholipase A2 preferentially hydrolyzes CL to MLCL. This selectivity results in ～80% yield of MLCL. The synthesized MLCL and dilysocardiolipin were characterized by NMR and ESI-MS/MS. Only the sn-2 position of CL was hydrolyzed by phospholipase A2 in methanol.     

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.     

Dietary macronutrients modulate the fatty acyl composition of rat liver mitochondrial cardiolipins

The interaction of dietary fats and carbohydrates on liver mitochondria were examined in male FBNF1 rats fed 20 different low-fat isocaloric diets. Animal growth rates and mitochondrial respiratory parameters were essentially unaffected, but mass spectrometry-based mitochondrial lipidomics profiling revealed increased levels of cardiolipins (CLs), a family of phospholipids essential for mitochondrial structure and function, in rats fed saturated or trans fat-based diets with a high glycemic index. These mitochondria showed elevated monolysocardiolipins (a CL precursor/product of CL degradation), elevated ratio of trans-phosphocholine (PC) (18:1/18:1) to cis-PC (18:1/18:1) (a marker of thiyl radical stress), and decreased ubiquinone Q₉; the latter two of which imply a low-grade mitochondrial redox abnormality. Extended analysis demonstrated: i) dietary fats and, to a lesser extent, carbohydrates induce changes in the relative abundance of specific CL species; ii) fatty acid (FA) incorporation into mature CLs undergoes both positive (>400-fold) and negative (2.5-fold) regulation; and iii) dietary lipid abundance and incorporation of FAs into both the CL pool and specific mature tetra-acyl CLs are inversely related, suggesting previously unobserved compensatory regulation. This study reveals previously unobserved complexity/regulation of the central lipid in mitochondrial metabolism.     

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.     

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.     

Cardiolipin and monolysocardiolipin analysis in fibroblasts, lymphocytes, and tissues using high-performance liquid chromatography-mass spectrometry as a diagnostic test for Barth syndrome

Barth syndrome (BTHS) is an X-linked recessive disorder caused by mutations in the tafazzin (or TAZ) gene and is clinically characterized by (cardio)myopathy, neutropenia, and growth abnormalities. Biochemical abnormalities include decreased levels of the mitochondrial phospholipid cardiolipin, increased levels of monolysocardiolipin, and a lower degree of unsaturation of the (monolyso)cardiolipin acyl chains. Diagnostic testing for BTHS is routinely performed by TAZ gene sequencing, and recently a BTHS screening method in bloodspots has been developed, but both methods have important limitations. Because a validated confirmatory method is not yet available, we set up and validated a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for BTHS in cultured fibroblasts, lymphocytes, and skeletal muscle based on cardiolipin, monolysocardiolipin, and the monolysocardiolipin/cardiolipin ratio. In addition, we performed retrospective analysis of 121 muscle samples of patients with myopathy of which mitochondrial origin was presumed, and we identified one patient with cardiolipin abnormalities similar to BTHS patients. Molecular analysis revealed a bona fide mutation in the TAZ gene. We conclude that (monolyso)cardiolipin analysis by HPLC-MS not only is a powerful tool to diagnose patients with clinical signs and symptoms of BTHS but also should be used in patients suffering from mitochondrial myopathies with unknown etiology.