Cardiolipin Species Research Articles

Metabolic and non-metabolic effects of cardiac-specific and inducible deletion of the AMPKalpha2 in female and male mice

Mitochondrial dysfunction plays a major role in the Heart Failure (HF) pathophysiology. The AMP activated protein kinase (AMPK) is activated by a high AMP-ADP/ATP ratio and regulates a number of metabolic pathways. Many studies have highlighted a protective role of AMPK in HF, but its relevance to cardiac tissue, its metabolic part and its sex-specificity are not well established. The aim of this study is to determine the role of AMPK in the healthy and failing heart in male and female mice. We developed and validated a mouse strain with an adult-inducible cardiac-specific deletion of AMPKα2, the major cardiac isoform, using the Cre-Lox system (40 mg/kg tamoxifen injection on two consecutive days at adult age). At four months after the deletion, cardiac contractility, morphology and metabolism were studied in control and KO mice from both sexes. We observed only in male KO mice a decrease of left ventricular ejection fraction (− 10%), an increase of the total fibrosis (+ 64%) and defects in mitochondrial structures. Male KO mice also showed a reduced (− 28%) mitochondrial respiration via complex I associated with a different cardiolipin species distribution. Our results reveal in adult healthy hearts, a sex-specificity in the effects of AMPKα2 deletion, leading to impaired contractile function related to metabolic and non-metabolic alterations only in male mice.

Double bonds of unsaturated fatty acids differentially regulate mitochondrial cardiolipin remodeling

BackgroundSupplemented fatty acids can incorporate into cardiolipin (CL) and affect its remodeling. The change in CL species may alter the mitochondrial membrane composition, potentially disturbing the mitochondrial structure and function during inflammation.MethodTo investigate the effect of the unsaturation of fatty acids on CL, we supplemented macrophage-like RAW264.7 cells with 18-carbon unsaturated fatty acids including oleic acid (OA, 18:1), linoleic acid (LA, 18:2), α-linolenic acid (ALA, 18:3), γ-linolenic acid (GLA, 18:3), and stearidonic acid (SDA, 18:4). Mitochondrial changes in CL were measured through mass spectrometry.ResultOur data indicated that OA(18:1) was the most efficient fatty acid that incorporated into CL, forming symmetrical CL without fatty acid elongation and desaturation. In addition, LA(18:2) and ALA(18:3) were further elongated before incorporation, significantly increasing the number of double bonds and the chain length of CL. GLA and SDA were not optimal substrates for remodeling enzymes. The findings of RT-qPCR experiments revealed that none of these changes in CL occurred through the regulation of CL remodeling- or synthesis-related genes. The fatty acid desaturase and transportation genes—Fads2 and Cpt1a, respectively—were differentially regulated by the supplementation of five unsaturated 18-carbon fatty acids.ConclusionsThe process of fatty acid incorporation to CL was regulated by the fatty acid desaturation and transportation into mitochondria in macrophage. The double bonds of fatty acids significantly affect the incorporation process and preference. Intact OA(18:1) was incorporated to CL; LA(18:2) and ALA(18:3) were desaturated and elongated to long chain fatty acid before the incorporation; GLA(18:3) and SDA(18:4) were unfavorable for the CL incorporation.

The Differentiation of Spinal Cord Motor Neurons is Associated with Changes of the Mitochondrial Phospholipid Cardiolipin

Motor neuron damage caused by diseases, traumatic insults or de-afferentation of the spinal cord is often incurable due to the poor intrinsic regenerative capacity. Moreover, regenerated peripheral nerves often do not reach normal functionality. Here, we investigated cardiolipin in the process of neuro-differentiation, since cardiolipin is closely linked to the mitochondrial energy supply in cells. The NSC-34 hybrid cell line, produced by fusing neuroblastoma cells with primary spinal cord motor neurons, was used, since it shares several morphological and physiological characteristics with mature primary motor neurons. Their neuro-differentiation was supported by switching from normal to differentiation medium or by fatty acid supplementation. Differentiation was evaluated by measuring neurite-sprouting parameters and PPARα expression. Cellular fatty acid distribution was analyzed to indicate changes in lipid metabolism during differentiation. Cardiolipin was characterized by acyl-chain composition and the distribution of molecular cardiolipin species. Both, the switch from normal to differentiation medium as well as the administration of palmitic and oleic acid promoted neuro-differentiation. Stimulated differentiation was accompanied by changes in cardiolipin content and composition. The positive correlation between neuro-differentiation and concentration of those molecular cardiolipin species containing palmitic and oleic acid implied a link between differentiation of NSC-34 cells and cardiolipin metabolism. We further demonstrated the impact of cellular lipid metabolism, and particularly cardiolipin metabolism, during and NSC-34 neuritogenesis. Thus, cardiolipin may represent a new therapeutic target for axon regeneration after peripheral nerve injuries or when axon sprouting is required to compensate for motor neuron loss in response to aging and/or disease.

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.

Lipid imaging for visualizing cilastatin amelioration of cisplatin-induced nephrotoxicity

Nephrotoxicity is a major limitation to cisplatin antitumor therapies. Cilastatin, an inhibitor of renal dehydropeptidase-I, was recently proposed as a promising nephroprotector against cisplatin toxicity, preventing apoptotic cell death. In this work, cilastatin nephroprotection was further investigated in a rat model, with a focus on its effect on 76 renal lipids altered by cisplatin, including 13 new cisplatin-altered mitochondrial cardiolipin species. Lipid imaging was performed with MALDI mass spectrometry imaging (MALDI-MSI) in kidney sections from treated rats. Cilastatin was proved to significantly diminish the lipid distribution alterations caused by cisplatin, lipid levels being almost completely recovered to those of control samples. The extent of recovery of cisplatin-altered lipids by cilastatin turned out to be relevant for discriminating direct or secondary lipid alterations driven by cisplatin. Lipid peroxidation induced by cisplatin was also shown to be reduced when cilastatin was administered. Importantly, significant groups separation was achieved during multivariate analysis of cortex and outer-medullary lipids, indicating that damaged kidney can be discerned from the nephroprotected and healthy groups and classified according to lipid distribution. Therefore, we propose MALDI-MSI as a powerful potential tool offering multimolecule detection possibilities to visualize and evaluate nephrotoxicity and nephroprotection based on lipid analysis.

Omega-3 and omega-6 fatty acid differentially impact cardiolipin remodeling in activated macrophage

BackgroundThe macrophage plays an important role in innate immunity to induce immune responses. Lipid replacement therapy has been shown to change the lipid compositions of mitochondria and potentially becomes an alternative to reduce the inflammatory response.MethodsWe examined the effects of omega-6 arachidonic acid (AA), omega-3 eicosapentaenoic acid (EPA), and omega-3 docosahexaenoic acid (DHA) supplementation on the activated the macrophage cell line RAW264.7 via KdO2-lipid A (KLA). The mitochondrial cardiolipin (CL) and monolysocardiolipin (MLCL) were analyzed by LC-MS.ResultsAfter macrophage activation by KLA, CL shifted to saturated species, but did not affect the quantity of CL. Inhibition of delta 6 desaturase also resulted in the same trend of CL species shift. We further examined the changes in CL and MLCL species induced by polyunsaturated fatty acid supplementation during inflammation. After supplementation of AA, EPA and DHA, the MLCL/CL ratio increased significantly in all treatments. The percentages of the long-chain species highly elevated and those of short-chain species reduced in both CL and MLCL.ConclusionsComparisons of AA, EPA and DHA supplementation revealed that the 20-carbon EPA (20:5) and AA (20:4) triggered higher incorporation and CL remodeling efficiency than 22-carbon DHA (22:6). EPA supplementation not only efficiently extended the chain length of CL but also increased the unsaturation of CL.

Mitochondrial bioenergetics and cardiolipin alterations in myocardial ischemia-reperfusion injury: implications for pharmacological cardioprotection.

Mitochondrial dysfunction plays a central role in myocardial ischemia-reperfusion (I/R) injury. Increased reactive oxygen species production, impaired electron transport chain activity, aberrant mitochondrial dynamics, Ca2+ overload, and opening of the mitochondrial permeability transition pore have been proposed as major contributory factors to mitochondrial dysfunction during myocardial I/R injury. Cardiolipin (CL), a mitochondria-specific phospholipid, plays a pivotal role in multiple mitochondrial bioenergetic processes, including respiration and energy conversion, in mitochondrial morphology and dynamics as well as in several steps of the apoptotic process. Changes in CL levels, species composition, and degree of oxidation may have deleterious consequences for mitochondrial function with important implications in a variety of pathophysiological conditions, including myocardial I/R injury. In this review, we focus on the role played by CL alterations in mitochondrial dysfunction in myocardial I/R injury. Pharmacological strategies to prevent myocardial injury during I/R targeting mitochondrial CL are also examined.

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.

Genetic re-engineering of polyunsaturated phospholipid profile of Saccharomyces cerevisiae identifies a novel role for Cld1 in mitigating the effects of cardiolipin peroxidation

Cardiolipin (CL) is a unique phospholipid localized almost exclusively within the mitochondrial membranes where it is synthesized. Newly synthesized CL undergoes acyl remodeling to produce CL species enriched with unsaturated acyl groups. Cld1 is the only identified CL-specific phospholipase in yeast and is required to initiate the CL remodeling pathway. In higher eukaryotes, peroxidation of CL, yielding CLOX, has been implicated in the cellular signaling events that initiate apoptosis. CLOX can undergo enzymatic hydrolysis, resulting in the release of lipid mediators with signaling properties. Our previous findings suggested that CLD1 expression is upregulated in response to oxidative stress, and that one of the physiological roles of CL remodeling is to remove peroxidized CL. To exploit the powerful yeast model to study functions of CLD1 in CL peroxidation, we expressed the H. brasiliensis Δ12-desaturase gene in yeast, which then synthesized poly unsaturated fatty acids(PUFAs) that are incorporated into CL species. Using LC-MS based redox phospholipidomics, we identified and quantified the molecular species of CL and other phospholipids in cld1Δ vs. WT cells. Loss of CLD1 led to a dramatic decrease in chronological lifespan, mitochondrial membrane potential, and respiratory capacity; it also resulted in increased levels of mono-hydroperoxy-CLs, particularly among the highly unsaturated CL species, including tetralinoleoyl-CL. In addition, purified Cld1 exhibited a higher affinity for CLOX, and treatment of cells with H2O2 increased CLD1 expression in the logarithmic growth phase. These data suggest that CLD1 expression is required to mitigate oxidative stress. The findings from this study contribute to our overall understanding of CL remodeling and its role in mitigating oxidative stress.

Cardiolipin synthases of Escherichia coli have phospholipid class specific phospholipase D activity dependent on endogenous and foreign phospholipids

E. coli has three Cls-isoenzymes for cardiolipin (CL) synthesis but the differences between these three enzymes remain unresolved. All three Cls enzymes contain the phospholipase D (PLD) characteristic HKD motive and synthesize CL using PLD activity. Here, using LC-MS we show the effect of overexpressing or deletion of the three individual Cls enzymes on the lipidome, which included changes in lipid class distribution and CL species profiles. We demonstrate, for the first time, that overexpression of only ClsB resulted in the appreciable synthesis of a variety of phosphatidylalcohols, thereby establishing a ‘classic’ PLD activity for this enzyme: phospholipid headgroup exchange. Endogenous E. coli lipids and primary alcohols were substrates for this trans-phosphatidylation reaction. Furthermore, we show that endogenous levels of ClsA mediated a similar trans-phosphatidylation reaction to form phosphatidylalcohols, however this reaction was dependent on the presence of the foreign phospholipid class phosphatidylcholine (PC). This allows us to clarify the different specificities of the cardiolipin synthases.

Systems Analyses Reveal Physiological Roles and Genetic Regulators of Liver Lipid Species.

The genetics of individual lipid species and their relevance in disease is largely unresolved. We profiled a subset of storage, signaling, membrane, and mitochondrial liver lipids across 385 mice from 47 strains of the BXD mouse population fed chow or high-fat diet and integrated these data with complementary multi-omics datasets. We identified several lipid species and lipid clusters with specific phenotypic and molecular signatures and, in particular, cardiolipin species with signatures of healthy and fatty liver. Genetic analyses revealed quantitative trait loci for 68% of the lipids (lQTL). By multi-layered omics analyses, we show the reliability of lQTLs to uncover candidate genes that can regulate the levels of lipid species. Additionally, we identified lQTLs that mapped to genes associated with abnormal lipid metabolism in human GWASs. This work provides a foundation and resource for understanding the genetic regulation and physiological significance of lipid species.

2357 Lost and found: Detection of brain cardiolipins in plasma after cardiac arrest

OBJECTIVES/SPECIFIC AIMS: Neurological injury remains as the main limiting factor for overall recovery after cardiac arrest (CA). Currently available indicators of neurological injury are inadequate for early prognostication after return of spontaneous circulation (ROSC). High diversification of brain mitochondrial cardiolipins (CL) makes them unique candidates to quantify brain injury and to predict prognosis early after ROSC. METHODS/STUDY POPULATION: CL content in plasma in 39 patients within 6 hours of ROSC and 10 healthy subjects as well as CL content in human heart and brain specimens were quantified using a high-resolution liquid chromatography mass spectrometry method. The quantities of brain-type CL species were correlated with clinical parameters of brain injury severity permitting derivation of a cerebral CL score (C-score) using linear regression. C-score and a single CL species (70:5) were evaluated in patients with varying neurological injury and outcome. Using a rat model of CA, CL was quantified in the plasma and brain of rats using similar methods and results compared with the controls. RESULTS/ANTICIPATED RESULTS: We found that brain and the heart fell on extreme ends of the CL diversity spectrum with 26 species of CL exclusively present in human brain not heart. Nine of these 26 species were present in plasma within 6 hours of ROSC with quantities correlating with greater brain injury. The C-score correlated with early neurologic injury and predicted discharge neurologic/functional outcome. CL (70:5) emerged as a potential point-of-care marker that alone was predictive of injury severity and outcome nearly as well as C-score. Using a rat CA model we showed a significant reduction in hippocampal CL content corresponding to CL released from the brain into systemic circulation. C-score was significantly increased in 10 minute Versus 5 minute no-flow CA and naïve controls. DISCUSSION/SIGNIFICANCE OF IMPACT: CA results in appearance and accumulation of CL in plasma, proportional to injury severity. Quantitation of brain-type CL species in plasma can be used to prognosticate neurological injury within 6 hours after ROSC.

Expression of human monolysocardiolipin acyltransferase-1 improves mitochondrial function in Barth syndrome lymphoblasts

The mitochondrial polyglycerophospholipid cardiolipin (CL) is remodeled to obtain specific fatty acyl chains. This is predominantly accomplished by the transacylase enzyme tafazzin (TAZ). Barth syndrome (BTHS) patients with TAZ gene mutations exhibit impaired TAZ activity and loss in mitochondrial respiratory function. Previous studies identified monolysocardiolipin acyltransferase-1 (MLCL AT-1) as a mitochondrial enzyme capable of remodeling CL with fatty acid. In this study, we analyzed what relationship, if any, exists between TAZ and MLCL AT-1 with regard to CL remodeling and whether transfection of BTHS lymphoblasts with an MLCL AT-1 expression construct improves mitochondrial respiratory function. In healthy lymphoblasts, reduction in TAZ expression through TAZ RNAi transfection resulted in a compensatory increase in MLCL AT-1 mRNA, protein, and enzyme activity, but CL mass was unaltered. In contrast, BTHS lymphoblasts exhibited decreased TAZ gene and protein expression but in addition decreased MLCL AT-1 expression and CL mass. Transfection of BTHS lymphoblasts with MLCL AT-1 expression construct increased CL, improved mitochondrial basal respiration and protein leak, and decreased the proportion of cells producing superoxide but did not restore CL molecular species composition to control levels. In addition, BTHS lymphoblasts exhibited higher rates of glycolysis compared with healthy controls to compensate for reduced mitochondrial respiratory function. Mitochondrial supercomplex assembly was significantly impaired in BTHS lymphoblasts, and transfection of BTHS lymphoblasts with MLCL AT-1 expression construct did not restore supercomplex assembly. The results suggest that expression of MLCL AT-1 depends on functional TAZ in healthy cells. In addition, transfection of BTHS lymphoblasts with an MLCL AT-1 expression construct compensates, but not completely, for loss of mitochondrial respiratory function.

P-294 - Detection of brain cardiolipins in plasma after cardiac arrest

Neurological injury resulting from dysfunction of brain mitochondria limits recovery after cardiac arrest (CA). Available indicators of neurological injury are inadequate for early prognostication after return of spontaneous circulation (ROSC). High diversification and essentiality of brain mitochondrial cardiolipins (CL) makes them unique candidates to quantify brain injury and predict prognosis early after ROSC. Using high-resolution liquid chromatography mass spectrometry, we determined CL content in plasma from 39 patients within 6 h of ROSC, 10 healthy controls and human heart and brain tissue. Cerebral score (C-score) was derived from brain-specific CL. Using a rat model of CA, CL was quantified in plasma and brain and results were compared with the controls. Brain and heart fell on extreme ends of CL diversity spectrum. Nine of 26 brain-specific CL were detected in plasma and correlated with brain injury. The C-score correlated with early neurologic injury and predicted neurologic outcome. In a rat CA model, a significant reduction in hippocampal CL content corresponded to CL released from the brain into systemic circulation. In conclusion brain-specific CL species accumulate in plasma after CA and can be used to predict injury severity and outcome.

Mitochondria, Temperature, and the Pace of Life.

Life history strategies, physiological traits, and behavior are thought to covary along a "pace of life" axis, with organisms at the fast end of this continuum having higher fecundity, shorter lifespan, and more rapid development, growth, and metabolic rates. Countergradient variation represents a special case of pace of life variation, in which high-latitude organisms occupy the fast end of the continuum relative to low-latitude conspecifics when compared at a common temperature. Here, we use Atlantic killifish (Fundulus heteroclitus) to explore the role of mitochondrial properties as a mechanism underlying countergradient variation, and thus variation in the pace of life. This species is found along the Atlantic coast of North America, through a steep latitudinal thermal gradient. The northern subspecies has faster development, more rapid growth, higher routine metabolic rate, and higher activity than the southern subspecies when compared at a common temperature. The northern subspecies also has greater mitochondrial respiratory capacity in the liver, although these differences are not evident in other tissues. The increased respiratory capacity of liver mitochondria in northern fish is associated with increases in the activity of multiple electron transport complexes, which largely reflects an increase in the amount of inner mitochondrial membrane per mitochondrion in the northern fish. There are also differences in the lipid composition of liver mitochondrial membranes, including differences in cardiolipin species, which could also influence respiratory capacity. These data suggest that variation in mitochondrial properties could, at least in part, underlie variation in the pace of life in Atlantic killifish.

Chlorella diet alters mitochondrial cardiolipin contents differentially in organs of Danio rerio analyzed by a lipidomics approach.

The zebrafish (Danio rerio) is an important and widely used vertebrate model organism for the study of human diseases which include disorders caused by dysfunctional mitochondria. Mitochondria play an essential role in both energy metabolism and apoptosis, which are mediated through a mitochondrial phospholipid cardiolipin (CL). In order to examine the cardiolipin profile in the zebrafish model, we developed a CL analysis platform by using liquid chromatography-mass spectrometry (LC-MS). Meanwhile, we tested whether chlorella diet would alter the CL profile in the larval fish, and in various organs of the adult fish. The results showed that chlorella diet increased the chain length of CL in larval fish. In the adult zebrafish, the distribution patterns of CL species were similar between the adult brain and eye tissues, and between the heart and muscles. Interestingly, monolyso-cardiolipin (MLCL) was not detected in brain and eyes but found in other examined tissues, indicating a different remodeling mechanism to maintain the CL integrity. While the adult zebrafish were fed with chlorella for four weeks, the CL distribution showed an increase of the species of saturated acyl chains in the brain and eyes, but a decrease in the other organs. Moreover, chlorella diet led to a decrease of MLCL percentage in organs except the non-MLCL-containing brain and eyes. The CL analysis in the zebrafish provides an important tool for studying the mechanism of mitochondria diseases, and may also be useful for testing medical regimens targeting against the Barth Syndrome.

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.

Mitochondrial targeted peptides preserve mitochondrial organization and decrease reversible myocardial changes in early swine metabolic syndrome.

The mechanisms responsible for cardiac damage in the early stages of metabolic syndrome (MetS) remain unknown. Mitochondria are intimately associated with cellular myofibrils, with the cytoskeleton functioning as a linkage coordinator, and closely associated to the calcium release sites of the sarcoplasmic reticulum (SR). We hypothesized that early MetS is characterized by mitochondria-related myocardial damage, associated with altered cytoskeletal-mitochondria-SR interaction. Domestic pigs were studied after 16 weeks of diet-induced MetS, MetS treated for the last 4 weeks with the mitochondrial-targeted peptide elamipretide (ELAM; 0.1 mg/kg SC q.d), or Lean controls (n = 6/group). Cardiac remodeling and function were assessed by fast comuted tomography. Myocardial mitochondrial structure, SR-mitochondria interaction, calcium handling, cytoskeletal proteins, oxidative stress, and apoptosis were studied ex-vivo. MetS pigs developed hyperlipidemia, hypertension, and insulin resistance, yet cardiac function was preserved. MetS-induced mitochondrial disorganization, decreased (C18:2)4 cardiolipin, disrupted ATP/ADP balance, and decreased cytochrome-c oxidase (COX)-IV activity. MetS also increased mitochondrial hydrogen peroxide (H2O2) production, decreased nicotinamide adenine dinucleotide phosphate (NADPH)/NADP and GSH/GSSG, and decreased myocardial desmin and β2 tubulin immunoreactivity, and impaired SR-mitochondrial interaction and mitochondrial calcium handling, eliciting myocardial oxidative stress and apoptosis. ELAM improved mitochondrial organization and cardiolipin species profile, restored ATP/ADP ratio and COX-IV activity, decreased H202 production, and improved generation of NADPH and GSH. ELAM also improved cytoskeletal-mitochondria-SR interaction and mitochondrial calcium handling, attenuating oxidative stress, and apoptosis. Disorganization of cardiomyocyte cytoskeletal-mitochondria-SR network is associated with cardiac reversible changes in early MetS, preceding overt cardiac dysfunction. These findings may introduce novel therapeutic targets for blunting cardiac damage in early MetS.

Role of Acyl Chain Composition of Phosphatidylcholine in Tafazzin-Mediated Remodeling of Cardiolipin in Liposomes.

Remodeling of the acyl chain compositions of cardiolipin (CL) species by the transacylase tafazzin is an important process for maintaining optimal mitochondrial functions. The results of mechanistic studies on the tafazzin-mediated transacylation from phosphatidylcholine (PC) to monolyso-CL (MLCL) in artificial lipid membranes are controversial. The present study investigated the role of the acyl chain composition of PC in the Saccharomyces cerevisiae tafazzin-mediated remodeling of CL by examining the structural factors responsible for the superior acyl donor ability of dipalmitoleoyl (16:1) PC over dipalmitoyl (16:0) PC. To this end, we synthesized systematic derivatives of dipalmitoleoyl PC; for example, the location of the cis double bond was migrated from the Δ9-position toward either end of the acyl chains (the Δ5- or Δ13-position), the cis double bond in the sn-1 or sn-2 position or both, was changed to a trans form, and palmitoleoyl and palmitoyl groups were exchanged in the sn-1 and sn-2 positions, maintaining similar PC fluidities. Analyses of the tafazzin-mediated transacylation from these PCs to sn-2'-MLCL(18:1-18:1/18:1-OH) in the liposomal membrane revealed that tafazzin strictly discriminates the molecular configuration of the acyl chains of PCs, including their glycerol positions (sn-1 or sn-2); however, the effects of PC fluidity on the reaction may not be neglected. On the basis of the findings described herein, we discuss the relevance of the so-called thermodynamic remodeling hypothesis that presumes no acyl selectivity of tafazzin.

Role of Cardiolipin in Mitochondrial Signaling Pathways.

The phospholipid cardiolipin (CL) is an essential constituent of mitochondrial membranes and plays a role in many mitochondrial processes, including respiration and energy conversion. Pathological changes in CL amount or species composition can have deleterious consequences for mitochondrial function and trigger the production of reactive oxygen species. Signaling networks monitor mitochondrial function and trigger an adequate cellular response. Here, we summarize the role of CL in cellular signaling pathways and focus on tissues with high-energy demand, like the heart. CL itself was recently identified as a precursor for the formation of lipid mediators. We highlight the concept of CL as a signaling platform. CL is exposed to the outer mitochondrial membrane upon mitochondrial stress and CL domains serve as a binding site in many cellular signaling events. During mitophagy, CL interacts with essential players of mitophagy like Beclin 1 and recruits the autophagic machinery by its interaction with LC3. Apoptotic signaling pathways require CL as a binding platform to recruit apoptotic factors such as tBid, Bax, caspase-8. CL required for the activation of the inflammasome and plays a role in inflammatory signaling. As changes in CL species composition has been observed in many diseases, the signaling pathways described here may play a general role in pathology.