Cardiolipin Remodeling Research Articles

Mitochondrial bioenergetics and cardiolipin remodeling abnormalities in mitochondrial trifunctional protein deficiency.

Mitochondrial trifunctional protein (TFP) deficiency is an inherited metabolic disorder leading to a block in long-chain fatty acid β-oxidation. Mutations in HADHA and HADHB, which encode the TFP α and β subunits, respectively, usually result in combined TFP deficiency. A single common mutation, HADHA c.1528G>C (p.E510Q), leads to isolated 3-hydroxyacyl-CoA dehydrogenase deficiency. TFP also catalyzes a step in the remodeling of cardiolipin (CL), a phospholipid critical to mitochondrial membrane stability and function. We explored the effect of mutations in TFP subunits on CL and other phospholipid content and composition and the consequences of these changes on mitochondrial bioenergetics in patient-derived fibroblasts. Abnormalities in these parameters varied extensively among different fibroblasts, and some cells were able to maintain basal oxygen consumption rates similar to controls. Although CL reduction was universally identified, a simultaneous increase in monolysocardiolipins was discrepant among cells. A similar profile was seen in liver mitochondria isolates from a TFP-deficient mouse model. Response to new potential drugs targeting CL metabolism might be dependent on patient genotype.

ALCAT1-Mediated Pathological Cardiolipin Remodeling and PLSCR3-Mediated Cardiolipin Transferring Contribute to LPS-Induced Myocardial Injury.

Cardiolipin (CL), a critical phospholipid situated within the mitochondrial membrane, plays a significant role in modulating intramitochondrial processes, especially in the context of certain cardiac pathologies; however, the exact effects of alterations in cardiolipin on septic cardiomyopathy (SCM) are still debated and the underlying mechanisms remain incompletely understood. This study highlights a notable increase in the expressions of ALCAT1 and PLSCR3 during the advanced stage of lipopolysaccharide (LPS)-induced SCM. This up-regulation potential contribution to mitochondrial dysfunction and cellular apoptosis-as indicated by the augmented oxidative stress and cytochrome c (Cytc) release-coupled with reduced mitophagy, decreased levels of the antiapoptotic protein B-cell lymphoma-2 (Bcl-2) and lowered cell viability. Additionally, the timing of LPS-induced apoptosis coincides with the decline in both autophagy and mitophagy at the late stages, implying that these processes may serve as protective factors against LPS-induced SCM in HL-1 cells. Together, these findings reveal the mechanism of LPS-induced CL changes in the center of SCM, with a particular emphasis on the importance of pathological remodeling and translocation of CL to mitochondrial function and apoptosis. Additionally, it highlights the protective effect of mitophagy in the early stage of SCM. This study complements previous research on the mechanism of CL changes in mediating SCM. These findings enhance our understanding of the role of CL in cardiac pathology and provide a new direction for future research.

Overview of carboxyl‑terminal modulator protein 1 and its importance in various metabolic regulations (Review).

Acyl‑coenzyme A thioesterases (ACOTs) are crucial in mediating lipid metabolic functions, including energy expenditure, hepatic gluconeogenesis and neuronal function. The two distinct types are type I and II ACOTs, the latter of which are 'hotdog' fold superfamily members. Type II ACOTs include carboxyl‑terminal modulator protein 1 (CTMP1), also termed thioesterase superfamily member 4 (THEM4), and CTMP2, also termed THEM5. Due to their similar structural features and distinct sequence homology, CTMP1 and CTMP2 stand out from other type II ACOTs. CTMP1 was initially known as a protein kinase B (PKB) inhibitor that attenuates PKB phosphorylation. PKB is the central regulator of various cellular functions, including survival, proliferation, growth and metabolism. Therefore, by inhibiting PKB, CTMP1 can affect various cellular processes. Various other functions of CTMP1 have been revealed, including functions in cancer, brain injury, mitochondrial function and lipid metabolism. CTMP2 is a paralog of CTMP1 and was first identified as a cardiolipin remodeling factor involved in the development of fatty liver. As the functions of CTMP1 and CTMP2 were discovered separately, a review to summarize and connect these findings is essential. The current review delineates the intricate complexity of CTMP regulation across different metabolic pathways and encapsulates the principal discoveries concerning CTMP until the present day.

Structural basis of complex I assembly and cardiolipin remodeling in mitochondria

Structural basis of complex I assembly and cardiolipin remodeling in mitochondria

Abstract Mo104: Cardiolipin-Metabolite Crosstalk in HFpEF

Background: Transition from fatty acid oxidation to glycolysis is linked to oncometabolism and cardiac hypertrophy. We hypothesize that such a metabolic shift is pivotal in driving HFpEF in 3 murine models. The citric acid cycle (CAC) enzyme αKGDH is dysregulated in HFpEF patient biopsies and Alport hearts. Therefore, we also hypothesize more generalized CAC dysregulation at αKGDH associated with HFpEF. Methods & Results: Alport and LDLR/P407 mice were generated on 129J background, and HFD/L-NAME HFpEF on C57Bl6N, (n= 5M,5F/group). Two groups of Alport and LDLR/P407 mice were fed a 2% αKG diet, and a 3rd subgroup of Alport mice injected with AAV9-CMV-αKGDH. PET-CT of 18 F-FDG showed significant increased cardiac glucose uptake in Alport and LDLR/P407 mice up to 8 wk of treatment. αKG diet conferred significantly increased cardiac glucose uptake and worse diastolic dysfunction in LDLR/P407 mice. αKGDH protein expression was reduced by ~40% (p<.05) in the HFD/L-NAME hearts. Targeted metabolomic analysis by LC-MS/MS indicated normalization of intermediate metabolites in LDLR/P407 hearts by αKG diet and significantly shortening life spans (p<.001). The αKG diet exacerbated adverse remodeling of cardiolipin (CL) in LDLR/P407 hearts. Expression of CL remodeling enzymes, CL Synthase (p<.05) and Tafazzin (p<.0001), were altered in the LDLR/P407 hearts. NADH increased in Alport and LDLR/P407 hearts suggesting decreased ATP availability and impaired oxidative phosphorylation. HFD/L-NAME HFpEF mice showed a possible block in CAC flux at αKGDH, with increased αKG and reduced succinyl-CoA. Acetyl-CoA and αKG were increased in Alport hearts. Serum metabolic signatures in 273 patients by 1 H NMR stratified into non-HF, pre-HFpEF, and HFpEF by the H2FPEF algorithm, indicated elevated serum αKG levels associated with patients with higher H2FPEF scores. Leucine is increased in LDLR/P407 and HFD/L-NAME hearts consistent with published findings in patients with HFpEF. These 2 models also have increased levels of purines. Increased G-6P in LDLR/P407 hearts supports increased glucose uptake. Glutamine is increased in HFD/L-NAME, consistent with published findings in patients with HFpEF. Conclusions: Dysregulated αKG and CL remodeling modulate oncometabolism and energy transduction that exacerbate HF in Alport, LDLR/P407, and HFD/L-NAME mice. Circulating αKG may constitute a marker of CAC dysfunction in HFpEF patients and αKGHD activation as a potential therapeutic goal.

Cardiolipin remodeling maintains the inner mitochondrial membrane in cells with saturated lipidomes

Cardiolipin (CL) is a unique, four-chain phospholipid synthesized in the inner mitochondrial membrane (IMM). The acyl chain composition of CL is regulated through a remodeling pathway, whose loss causes mitochondrial dysfunction in Barth syndrome (BTHS). Yeast has been used extensively as a model system to characterize CL metabolism, but mutants lacking its two remodeling enzymes, Cld1p and Taz1p, exhibit mild structural and respiratory phenotypes compared to mammalian cells. Here, we show an essential role for CL remodeling in the structure and function of the IMM in yeast grown under reduced oxygenation. Microaerobic fermentation, which mimics natural yeast environments, caused the accumulation of saturated fatty acids and, under these conditions, remodeling mutants showed a loss of IMM ultrastructure. We extended this observation to HEK293 cells, where phospholipase A2 inhibition by Bromoenol lactone resulted in respiratory dysfunction and cristae loss upon mild treatment with exogenous saturated fatty acids. In microaerobic yeast, remodeling mutants accumulated unremodeled, saturated CL, but also displayed reduced total CL levels, highlighting the interplay between saturation and CL biosynthesis and/or breakdown. We identified the mitochondrial phospholipase A1 Ddl1p as a regulator of CL levels, and those of its precursors phosphatidylglycerol and phosphatidic acid, under these conditions. Loss of Ddl1p partially rescued IMM structure in cells unable to initiate CL remodeling and had differing lipidomic effects depending on oxygenation. These results introduce a revised yeast model for investigating CL remodeling and suggest that its structural functions are dependent on the overall lipid environment in the mitochondrion.

Perturbations in mitochondrial metabolism associated with defective cardiolipin biosynthesis: An in-organello real-time NMR study.

Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases including cancer, cardiopathy, neurodegeneration, and heritable pathologies such as Barth syndrome. Cardiolipin, the signature phospholipid of the mitochondrion promotes proper cristae morphology, bioenergetic functions, and directly affects metabolic reactions carried out in mitochondrial membranes. To match tissue-specific metabolic demands, cardiolipin typically undergoes an acyl tail remodeling process with the final step carried out by the phospholipid-lysophospholipid transacylase tafazzin. Mutations in the tafazzin gene are the primary cause of Barth syndrome. Here, we investigated how defects in cardiolipin biosynthesis and remodeling impact metabolic flux through the tricarboxylic acid cycle and associated pathways in yeast. Nuclear magnetic resonance was used to monitor in real-time the metabolic fate of 13C3-pyruvate in isolated mitochondria from three isogenic yeast strains. We compared mitochondria from a wild-type strain to mitochondria from a Δtaz1 strain that lacks tafazzin and contains lower amounts of unremodeled cardiolipin, and mitochondria from a Δcrd1 strain that lacks cardiolipin synthase and cannot synthesize cardiolipin. We found that the 13C-label from the pyruvate substrate was distributed through about twelve metabolites. Several of the identified metabolites were specific to yeast pathways, including branched chain amino acids and fusel alcohol synthesis. Most metabolites showed similar kinetics amongst the different strains but mevalonate and α-ketoglutarate, as well as the NAD+/NADH couple measured in separate nuclear magnetic resonance experiments, showed pronounced differences. Taken together, the results show that cardiolipin remodeling influences pyruvate metabolism, tricarboxylic acid cycle flux, and the levels of mitochondrial nucleotides.

ETMR-20. CARDIOLIPIN ACYL CHAIN MODULATION AS A NOVEL MITOCHONDRIAL-FOCUSED THERAPEUTIC TARGET IN ETMR

Abstract Embryonal tumor with multilayered rosettes (ETMR) is a highly aggressive CNS neoplasm that occurs almost exclusively in infants and is associated with an extremely poor prognosis. Dysregulated mitochondrial bioenergetics and dynamics have been associated with the initiation and progression of diverse cancers and studies have linked metabolic rewiring to chemotherapy resistance. Cardiolipins are mitochondrial-specific lipids that reside in the mitochondrial membrane and their fatty acid composition has been extensively shown to regulate mitochondrial function and dynamics. Despite the known functional significance of cardiolipin structure, their role in mitochondrial regulation of brain tumors remains ill-defined. Using mass spectrometry imaging, we identified a shift to shorter acyl chain cardiolipins within the rapidly proliferating embryonal tumor cells in patient samples and patient-derived cell lines grown as 3D tumorspheres. Western blot analysis of the enzymes involved in cardiolipin synthesis and remodeling identified a significant increase in the expression of the cardiolipin remodeling enzyme, LCLAT1/ALCAT1. Orthogonal imaging techniques including immunohistochemistry, transmission electron microscopy and super-resolution microscopy correlated shorter acyl chain remodeling of cardiolipin with fragmented mitochondria (increased fission) and aberrant cristae structure. Further studies identified increased expression of the fission protein Drp1, decreased expression of respiratory chain enzymes and altered respiration in the embryonal tumor cells. Ongoing studies will utilize multiple methods to modulate cardiolipin acyl chain structure and use MS and MSI to correlate cardiolipin fatty acid structure to mitochondrial function and growth inhibition in the embryonal tumor cells.

SS-31 treatment ameliorates cardiac mitochondrial morphology and defective mitophagy in a murine model of Barth syndrome

Barth syndrome (BTHS) is a lethal rare genetic disorder, which results in cardiac dysfunction, severe skeletal muscle weakness, immune issues and growth delay. Mutations in the TAFAZZIN gene, which is responsible for the remodeling of the phospholipid cardiolipin (CL), lead to abnormalities in mitochondrial membrane, including alteration of mature CL acyl composition and the presence of monolysocardiolipin (MLCL). The dramatic increase in the MLCL/CL ratio is the hallmark of patients with BTHS, which is associated with mitochondrial bioenergetics dysfunction and altered membrane ultrastructure. There are currently no specific therapies for BTHS. Here, we showed that cardiac mitochondria isolated from TAFAZZIN knockdown (TazKD) mice presented abnormal ultrastructural membrane morphology, accumulation of vacuoles, pro-fission conditions and defective mitophagy. Interestingly, we found that in vivo treatment of TazKD mice with a CL-targeted small peptide (named SS-31) was able to restore mitochondrial morphology in tafazzin-deficient heart by affecting specific proteins involved in dynamic process and mitophagy. This agrees with our previous data showing an improvement in mitochondrial respiratory efficiency associated with increased supercomplex organization in TazKD mice under the same pharmacological treatment. Taken together our findings confirm the beneficial effect of SS-31 in the amelioration of tafazzin-deficient dysfunctional mitochondria in a BTHS animal model.

In Vitro Hypoxia/Reoxygenation Induces Mitochondrial Cardiolipin Remodeling in Human Kidney Cells.

Renal ischemia/reperfusion is a serious condition that not only causes acute kidney injury, a severe clinical syndrome with high mortality, but is also an inevitable part of kidney transplantation or other kidney surgeries. Alterations of oxygen levels during ischemia/reperfusion, namely hypoxia/reoxygenation, disrupt mitochondrial metabolism and induce structural changes that lead to cell death. A signature mitochondrial phospholipid, cardiolipin, with many vital roles in mitochondrial homeostasis, is one of the key players in hypoxia/reoxygenation-induced mitochondrial damage. In this study, we analyze the effect of hypoxia/reoxygenation on human renal proximal tubule epithelial cell (RPTEC) cardiolipins, as well as their metabolism and mitochondrial functions. RPTEC cells were placed in a hypoxic chamber with a 2% oxygen atmosphere for 24 h to induce hypoxia; then, they were replaced back into regular growth conditions for 24 h of reoxygenation. Surprisingly, after 24 h, hypoxia cardiolipin levels substantially increased and remained higher than control levels after 24 h of reoxygenation. This was explained by significantly elevated levels of cardiolipin synthase and lysocardiolipin acyltransferase 1 (LCLAT1) gene expression and protein levels. Meanwhile, hypoxia/reoxygenation decreased ADP-dependent mitochondrial respiration rates and oxidative phosphorylation capacity and increased reactive oxygen species generation. Our findings suggest that hypoxia/reoxygenation induces cardiolipin remodeling in response to reduced mitochondrial oxidative phosphorylation in a way that protects mitochondrial function.

Tafazzin deficiency causes substantial remodeling in the lipidome of a mouse model of Barth Syndrome cardiomyopathy.

Barth Syndrome (BTHS) is a rare X-linked disease, characterized clinically by cardiomyopathy, skeletal myopathy, neutropenia, and growth retardation. BTHS is caused by mutations in the phospholipid acyltransferase tafazzin (Gene: TAFAZZIN, TAZ). Tafazzin catalyzes the final step in the remodeling of cardiolipin (CL), a glycerophospholipid located in the inner mitochondrial membrane. As the phospholipid composition strongly determines membrane properties, correct biosynthesis of CL and other membrane lipids is essential for mitochondrial function. Mitochondria provide 95% of the energy demand in the heart, particularly due to their role in fatty acid oxidation. Alterations in lipid homeostasis in BTHS have an impact on mitochondrial membrane proteins and thereby contribute to cardiomyopathy. We analyzed a transgenic TAFAZZIN-knockdown (TAZ-KD) BTHS mouse model and determined the distribution of 193 individual lipid species in TAZ-KD and WT hearts at 10 and 50weeks of age, using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Our results revealed significant lipid composition differences between the TAZ-KD and WT groups, indicating genotype-dependent alterations in most analyzed lipid species. Significant changes in the myocardial lipidome were identified in both young animals without cardiomyopathy and older animals with heart failure. Notable alterations were found in phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), lysophosphatidylcholine (LPC) and plasmalogen species. PC species with 2-4 double bonds were significantly increased, while polyunsaturated PC species showed a significant decrease in TAZ-KD mice. Furthermore, Linoleic acid (LA, 18:2) containing PC and PE species, as well as arachidonic acid (AA, 20:4) containing PE 38:4 species are increased in TAZ-KD. We found higher levels of AA containing LPE and PE-based plasmalogens (PE P-). Furthermore, we are the first to show significant changes in sphingomyelin (SM) and ceramide (Cer) lipid species Very long-chained SM species are accumulating in TAZ-KD hearts, whereas long-chained Cer and several hexosyl ceramides (HexCer) species accumulate only in 50-week-old TAZ-KD hearts These findings offer potential avenues for the diagnosis and treatment of BTHS, presenting new possibilities for therapeutic approaches.

Dietary linoleic acid supplementation fails to rescue established cardiomyopathy in Barth syndrome

Barth syndrome (BTHS) is a mitochondrial lipid disorder caused by mutations in TAFAZZIN (TAZ), required for cardiolipin (CL) remodeling. Cardiomyopathy is a major clinical feature, with no curative therapy. Linoleic acid (LA) supplementation is proposed to ameliorate BTHS cardiomyopathy by enhancing linoleoyl group incorporation into CL. While the beneficial effect of dietary LA supplementation in delaying the development of BTHS cardiomyopathy has been recently tested, its potential to reverse established BTHS cardiomyopathy remains unclear. Our study revealed that LA supplementation cannot rescue established BTHS cardiomyopathy in mice, highlighting the importance of early initiation of LA supplementation for maximum benefits.

An upstream open reading frame (5′-uORF) links oxidative stress to translational control of ALCAT1 through phosphorylation of eIF2α

Acyl-CoA:lysocardiolipin acyltransferase 1 (ALCAT1) is an enzyme that promotes mitochondrial dysfunction by catalyzing pathological remodeling of cardiolipin. Upregulation of ALCAT1 protein expression by oxidative stress is implicated in the pathogenesis of age-related metabolic diseases, but the underlying molecular mechanisms remain elusive. In this study, we identified a highly conserved upstream open reading frame (uORF) at the 5′-untranslated region (5′-UTR) of ALCAT1 mRNA as a key regulator of ALCAT1 expression in response to oxidative stress. We show that the uORF serves as a decoy that prevents translation initiation of ALCAT1 under homeostatic condition. The inhibitory activity of the uORF on ALCAT1 mRNA translation is mitigated by oxidative stress but not ER stress, which requires the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Consequently, ablation of uORF or eIF2α phosphorylation at Ser51 renders ALCAT1 protein expression unresponsive to induction by oxidative stress. Taken together, our data show that the uORF links oxidative stress to translation control of ALCAT1 mRNAs through phosphorylation of eIF2α at Ser51.

Decreased pyruvate dehydrogenase activity in Tafazzin-deficient cells is caused by dysregulation of pyruvate dehydrogenase phosphatase 1 (PDP1)

Cardiolipin (CL), the signature lipid of the mitochondrial inner membrane, is critical for maintaining optimal mitochondrial function and bioenergetics. Disruption of CL metabolism, caused by mutations in the CL remodeling enzyme TAFAZZIN, results in the rare and life-threatening disorder Barth syndrome (BTHS). While the clinical manifestations of BTHS, such as dilated cardiomyopathy and skeletal myopathy, point to defects in mitochondrial bioenergetics, the disorder is also characterized by broad metabolic dysregulation, including abnormal levels of metabolites associated with the tricarboxylic acid (TCA) cycle. In line with this, recent studies have identified inhibition of pyruvate dehydrogenase (PDH), the gatekeeper enzyme for TCA cycle carbon influx, as a key deficiency in various BTHS model systems. However, the molecular mechanisms linking aberrant CL remodeling, particularly the primary, direct consequence of reduced tetralinoleoyl-CL (TLCL) levels, to PDH activity deficiency are not yet understood. This knowledge gap has limited our understanding of lipid-mediated metabolic regulation in BTHS and hindered the development of effective treatment strategies. In the current study, we provide evidence that remodeled TLCL promotes PDH function by directly binding to and enhancing the activity of PDH phosphatase 1 (PDP1). This is supported by our findings that TLCL uniquely activates PDH in a dose-dependent manner, TLCL binds to PDP1 in vitro, TLCL-mediated PDH activation is attenuated in the presence of phosphatase inhibitor, and PDP1 activity is decreased in Tafazzin-knockout (TAZ-KO) C2C12 myoblasts. Additionally, we observed decreased mitochondrial calcium levels in TAZ-KO cells, which may affect the calcium-sensitive activity of PDP1. Treating TAZ-KO cells with calcium lactate (CaLac) increases mitochondrial calcium and restores PDH activity and mitochondrial oxygen consumption rate. Based on our findings, we conclude that reduced mitochondrial calcium levels and decreased binding of PDP1 to TLCL contribute to decreased PDP1 activity in TAZ-KO cells.

Cyclohexylalanine-Containing α-Helical Amphipathic Peptide Targets Cardiolipin, Rescuing Mitochondrial Dysfunction in Kidney Injury.

Mitochondrial dysfunction is linked to degenerative diseases, resulting from cardiolipin (CL)-induced disruption of cristae structure in the inner mitochondrial membrane (IMM); therefore, preserving cristae and preventing CL remodeling offer effective strategies to maintain mitochondrial function. To identify reactive oxygen species (ROS)-blocking agents against mitochondrial dysfunction, a library of cyclohexylamine-containing cell-penetrating α-helical amphipathic "bundle" peptides were screened. Among these, CMP3013 is selectively bound to abnormal mitochondria, preserving the cristae structure impaired by mitochondria-damaging agents. With a stronger affinity for CL compared with other IMM lipid components, CMP3013 exhibited high selectivity. Consequently, it protected cristae, reduced ROS production, and enhanced adenosine triphosphate (ATP) generation. In mouse models of acute kidney injury, a 1 mg/kg dose of CMP3013 demonstrated remarkable efficacy, highlighting its potential as a therapeutic agent for mitochondrial dysfunction-related disorders. Overall, CMP3013 represents a promising agent for mitigating mitochondrial dysfunction and associated diseases.

A patatin-like phospholipase is important for mitochondrial function in malaria parasites.

For their proliferation within red blood cells, malaria parasites depend on a functional electron transport chain (ETC) within their mitochondrion, which is the target of several antimalarial drugs. Here, we have used gene disruption to identify a patatin-like phospholipase, PfPNPLA2, as important for parasite replication and mitochondrial function in Plasmodium falciparum. Parasites lacking PfPNPLA2 show defects in their ETC and become hypersensitive to mitochondrion-targeting drugs. Furthermore, PfPNPLA2-deficient parasites show differences in the composition of their cardiolipins, a unique class of phospholipids with key roles in mitochondrial functions. Finally, we demonstrate that parasites devoid of PfPNPLA2 have a defect in gametocyte maturation, underlining the importance of a functional ETC for parasite transmission to the mosquito vector.

A novel panel of Drosophila TAFAZZIN mutants in distinct genetic backgrounds as a resource for therapeutic testing.

Barth Syndrome is a rare, X-linked disorder caused by mutation of the gene TAFAZZIN (TAZ). The corresponding Tafazzin protein is involved in the remodeling of cardiolipin, a phospholipid with critical roles in mitochondrial function. While recent clinical trials have been promising, there is still no cure for Barth Syndrome. Because TAZ is highly conserved, multiple animal and cell culture models exist for pre-clinical testing of therapeutics. However, since the same mutation in different patients can lead to different symptoms and responses to treatment, isogenized experimental models can't fully account for human disease conditions. On the other hand, isogenized animal models allow for sufficient numbers to thoroughly establish efficacy for a given genetic background. Therefore, a combined method for testing treatments in a panel of isogenized cohorts that are genetically distinct from each other would be transformative for testing emerging pre-clinical therapies. To aid in this effort, we've created a novel panel of 10 Drosophila lines, each with the same TAZ mutation in highly diverse genetic backgrounds, to serve as a helpful resource to represent natural variation in background genetics in pre-clinical studies. As a proof of principle, we test our panel here using nicotinamide riboside (NR), a treatment with established therapeutic value, to evaluate how robust this therapy is across the 10 genetic backgrounds in this novel reference panel. We find substantial variation in the response to NR across backgrounds. We expect this resource will be valuable in pre-clinical testing of emerging therapies for Barth Syndrome.

B-170 Identifying Metabolic Phenotyping Variability in a Cellular Model of Barth Syndrome

Abstract Introduction Barth Syndrome is an X-linked multisystem disease caused by the mutation in the TAZ gene (TAZ, G 4.5, OMIM 300394) that encodes for acyltransferase taffazin. Taffazin is a mitochondrial protein highly expressed in heart and skeletal muscle and has a central role in cardiolipin remodeling process. TAZ is the only known single gene defect in cardiolipin remodeling, with more than a hundred TAZ mutations identified. However, no systematic correlation has been established between TAZ genotypes and clinical or biochemical phenotypes in Barth syndrome. There is a large phenotypic heterogeneity among affected individuals sometimes even within the same family. This raises a possibility of genetic and metabolic modifiers involved in TAZ clinical and metabolic phenotypes. The aim of this study is to apply LC-MS/MS untargeted and targeted metabolic analyses to explore possible correlations between two TAZ genotypes and metabolome in a cellular model of Barth Syndrome. Methods Mutant TAZ 3(c.647G>T), TAZ 4(c.778-24_778-7delinsA) fibroblast and matching by donors’ age and gender control fibroblast lines (healthy#1, healthy #2) were cultured under identical conditions. Cells (n = 6 for each genotype) were harvested and extracted followed Bligh and Dyer protocol. Extracted metabolites were analyzed by Agilent 6545 Q-TOF coupled with 1290 Infinity II HPLC. Chromatographic separation was achieved by using a Water Premier BEH Z-HILIC Column (2.5 µM, 2.1 × 150 mm) and Waters X-select HSS T3 (2.5 µM, 2.1 × 100 mm) columns for polar and non-polar metabolites respectively. Raw data was normalized with internal standard and total cellular protein amounts. Statistical and pathway enrichment analyses were performed with a chemometric platform Mass Profiler Professional (Agilent) and Metaboanalyst webtool. Results Data was treated first as for two separate genotypes (healthy vs TAZ) followed and compared to the analysis with four separate genotypes (healthy#1, healthy #2 TAZ c.647G>T, TAZ c.778-24_778-7delinsA). Metabolites contributing to discrimination of the genotypes based on metabolic profiles were identified by using a univariate analysis (P < 0.05) and a supervised PLSDA (VIP>1 for small polar metabolites and VIP > 3 for non-polar molecules). The differentially expressed metabolites included amino acids, acylcarnitine, Krebs cycle intermediates, ATP, 3-methylglutaconic acid, fatty acids and a variety of phospholipids. Pathway enrichment analysis revealed that several metabolic pathways were involved. Conclusions Our analyses reveal that genetic variations in TAZ gene cause the differences in the abundance of certain metabolites and affected pathways. This leads us to the conclusion that from the point of view of metabolic pathophysiology, TAZ should be considered beyond a single-gene defect and a more personalized approach is needed in search for potential therapies.

B-161 Mass Spectrometric Assay for the Early Diagnosis of Barth Syndrome

Abstract Background Barth syndrome (BTHS) is an inborn error of phospholipid metabolism caused by pathogenic variants in the TAFAZZIN gene locus on a chromosome X. TAFAZZIN encodes a phospholipid transacylase enzyme, tafazzin, that promotes cardiolipin (CL) remodeling. Deficiency of tafazzin activity in BTHS results in accumulation of monolysocardiolipin (MLCL) and decreased abundance of mature CL, making an increased ratio of MLCL to CL as a diagnostic marker for BTHS. 52:2-MLCL to 72:8-CL in dried-blood spot (DBS) has been used for provisional identification BTHS using LC-MS/MS. A development of multiplexed method that targets several species of MLCL and CL can enhance the diagnosis of BTHS. Methods A targeted LC-MS/MS with scheduled multiple reaction monitoring (MRM) mode in negative electrospray ionization was developed using a triple quadrupole MS. DBS from patients with BTHS were obtained at Johns Hopkins University and provided by Barth Syndrome Foundation while control DBS were provided by Biochemical Genetics Laboratory at Mayo Clinic. Internal standards and chloroform:methanol (1:1, v/v) were added to DBS (1/8″ diameter) and sonicated. Supernatants were collected and dried under nitrogen gas. Followed by reconstitution in methanol, samples were subjected to LC-MS/MS analysis. Results More than 20 species of MLCL and CL species having various fatty acyl chains were detected and quantified. Remodeled CL species were decreased while MLCL and nascent CL species were found to be increased in known patients of BTHS (n = 8) compared to controls (n = 8). Notably, several ratios of lipids including 52:2-MLCL/72:8-CL and 54:2-MLCL/70:6-CL showed >300-fold elevation in the patients. Conclusion Various species of MLCL and CL that can be used to diagnose BTHS can be measured from an easily accessible specimen such as DBS using this method.

Phenotypic Characterization of Female Carrier Mice Heterozygous for Tafazzin Deletion.

Barth syndrome (BTHS) is caused by mutations in tafazzin resulting in deficits in cardiolipin remodeling that alter major metabolic processes. The tafazzin gene is encoded on the X chromosome, and therefore BTHS primarily affects males. Female carriers are typically considered asymptomatic, but age-related changes have been reported in female carriers of other X-linked disorders. Therefore, we examined the phenotype of female mice heterozygous for deletion of the tafazzin gene (Taz-HET) at 3 and 12 months of age. Food intakes, body masses, lean tissue and adipose depot weights, daily activity levels, metabolic measures, and exercise capacity were assessed. Age-related changes in mice resulted in small but significant genotype-specific differences in Taz-HET mice compared with their female Wt littermates. By 12 months, Taz-HET mice weighed less than Wt controls and had smaller gonadal, retroperitoneal, and brown adipose depots and liver and brain masses, despite similar food consumption. Daily movement, respiratory exchange ratio, and total energy expenditure did not vary significantly between the age-matched genotypes. Taz-HET mice displayed improved glucose tolerance and insulin sensitivity at 12 months compared with their Wt littermates but had evidence of slightly reduced exercise capacity. Tafazzin mRNA levels were significantly reduced in the cardiac muscle of 12-month-old Taz-HET mice, which was associated with minor but significant alterations in the heart cardiolipin profile. This work is the first to report the characterization of a model of female carriers of heterozygous tafazzin deficiency and suggests that additional study, particularly with advancing age, is warranted.