[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 01/2012; 7(11):e48628. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Persistent pulmonary hypertension of the newborn (PPHN) results in right ventricular (RV) hypertrophy followed by right heart failure and an associated mitochondrial dysfunction. The phospholipid cardiolipin plays a key role in maintaining mitochondrial respiratory and cardiac function via modulation of the activities of enzymes involved in oxidative phosphorylation. In this study, changes in cardiolipin and cardiolipin metabolism were investigated during the development of right heart failure. Newborn piglets (<24 h old) were exposed to a hypoxic (10% O(2)) environment for 3 days, resulting in the induction of PPHN. Two sets of control piglets were used: 1) newborn or 2) exposed to a normoxic (21% O(2)) environment for 3 days. Cardiolipin biosynthetic and remodeling enzymes, mitochondrial complex II + III activity, incorporation of [1-(14)C]linoleoyl-CoA into cardiolipin precursors, and the tetralinoleoyl-cardiolipin pool size were determined in both the RV and left ventricle (LV). PPHN resulted in an increased heart-to-body weight ratio, RV-to-LV plus septum weight ratio, and expression of brain naturetic peptide in RV. In addition, PPHN reduced cardiolipin biosynthesis and remodeling in the RV and LV, which resulted in decreased tetralinoleoyl-cardiolipin levels and reduced complex II + III activity and protein levels of mitochondrial complexes II, III, and IV in the RV. This is the first study to examine the pattern of cardiolipin metabolism during the early development of both the RV and LV of the newborn piglet and to demonstrate that PPHN-induced alterations in cardiolipin biosynthetic and remodeling enzymes contribute to reduced tetralinoleoyl-cardiolipin and mitochondrial respiratory chain function during the development of RV hypertrophy. These defects in cardiolipin may play an important role in the rapid development of RV dysfunction and right heart failure in PPHN.
[Show abstract][Hide abstract] ABSTRACT: We examined if lipopolysaccharide (LPS) treatment of mice affected cardiolipin (CL) synthesis. Mice were injected i.p. with LPS, the liver harvested, and CL synthase (CLS) enzyme activity and its mRNA expression examined. Treatment of mice with LPS resulted in a 55% decrease (p < 0.01) in mRNA expression of murine CLS compared to controls, but CLS enzyme activity was unaltered. The pool size of liver CL and other phospholipids were unaltered by LPS treatment. A similar effect was observed in murine epidermal fat pad and in vitro in RAW mouse macrophages and in human HepG2 cells. LPS treatment of HepG2 cells transiently expressing a histidine-tagged human cardiolipin synthase-1 (hCLS1) reduced hCLS1 mRNA and newly synthesized CLS activity indicating that LPS inhibits production of newly synthesized hCLS1 via reduction in hCLS1 mRNA. The results clearly indicate that CLS mRNA levels cannot be correlated with CLS enzyme activity nor CL content in the LPS model of inflammation.
[Show abstract][Hide abstract] ABSTRACT: Here we report the identification of a previously uncharacterized human protein as the human monolysocardiolipin acyltransferase-1 (MLCL AT-1). Pig liver mitochondria were treated with n-butyl alcohol followed by Q-Sepharose chromatography, preparative gel electrophoresis, cytidine diphosphate-1,2-diacyl-sn-glycerol-Sepharose chromatography, and finally monolysocardiolipin-adriamycin-agarose affinity chromatography. Elution with either monolysocardiolipin or linoleoyl coenzyme A revealed a major band at 74 kDa with high specific activity (2,300 pmol/min/mg) for the acylation of monolysocardiolipin to cardiolipin using [1-(14)C]linoleoyl coenzyme A as substrate. Matrix-assisted laser desorption ionization time-of-flight-mass spectrometry analysis followed by search of the Mascot protein data base revealed peptide matches consistent with a 59-kDa protein identified as unknown human protein (GenBank(TM) protein accession number AAX93141; nucleotide accession number AC011742.3). The purified human recombinant MLCL AT-1 protein utilized linoleoyl coenzyme A > oleoyl coenzyme A > palmitoyl coenzyme A for the specific acylation of monolysocardiolipin to cardiolipin. Expression of MLCL AT-1 in HeLa cells increased mitochondrial monolysocardiolipin acyltransferase activity and [1-(14)C]linoleic acid incorporated into cardiolipin, whereas RNA interference knockdown of MLCL AT-1 in HeLa cells resulted in reduction in enzyme activity and [1-(14)C]linoleic acid incorporated into cardiolipin. In contrast, expression of MLCL AT-1 in HeLa cells did not alter [1-(14)C]oleic or [1-(14)C]palmitate incorporation into cardiolipin indicating in vivo specificity for the remodeling of cardiolipin with linoleate. Finally, expression of MLCL AT-1 in Barth syndrome lymphoblasts, which exhibit cardiolipin levels 20% that of normal lymphoblasts, increased mitochondrial monolysocardiolipin acyltransferase activity, [1-(14)C]linoleic acid incorporation into cardiolipin, cardiolipin mass, and succinate dehydrogenase (mitochondrial complex II) activity compared with mock-transfected Barth syndrome lymphoblasts. The results identify MLCL AT-1 as a human mitochondrial monolysocardiolipin acyltransferase involved in the remodeling of cardiolipin.
Journal of Biological Chemistry 09/2009; 284(44):30360-71. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.
The Journal of Lipid Research 12/2008; 50(8):1600-8. · 4.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Biochemistry and Cell Biology 02/2008; 86(1):11-20. · 2.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fetal alcohol syndrome is a serious developmental disorder and exposure of the fetal heart to alcohol results in disturbances in the biochemistry of all cellular substructures. Mitochondrial effects include diminished respiratory function and physical alteration of the membrane secondary to interaction of ethanol with the hydrophobic region of the bilayer. Cardiolipin is a major mitochondrial membrane phospholipid in the heart and plays an important role in the function of mitochondrial enzymes involved in cellular respiration. We examined the activity of cardiac monolysocardiolipin acyltransferase, a key enzyme responsible for the molecular remodelling of cardiolipin with new fatty acids, in the newborn and adult rat and in new born rats that were exposed to alcohol in utero. Cardiac monolysocardiolipin acyltransferase activities were 57% lower (p < 0.05) in adult rats compared to newborn rats. Cardiac mitochondrial monolysocardiolipin acyltransferase activities were 36% lower (p < 0.05) in newborn rats that were exposed to alcohol in utero and this was due to reduced mitochondrial monolysocardiolipin acyltransferase expression. The results indicate that cardiac mitochondrial monolysocardiolipin acyltransferase activity declines during postnatal development in the rat and that in utero exposure to alcohol inhibits cardiac monolysocardiolipin acyltransferase activity and expression.
Proceedings of the Western Pharmacology Society 02/2007; 50:115-8.
[Show abstract][Hide abstract] ABSTRACT: CL (cardiolipin) is a major mitochondrial membrane phospholipid important for the regulation of mitochondrial function. We examined CL de novo biosynthesis and its resynthesis in isolated rat liver hepatocytes prepared 48 h subsequent to two-thirds PHx (partial hepatectomy). The pool size of CL and its de novo biosynthesis from [1,3-(3)H]glycerol were increased 3.3-fold (P<0.05) and 3.1-fold (P<0.05) respectively in hepatocytes prepared from PHx rats compared with sham-operated controls. The reason for the increased CL biosynthesis was a 65% increase (P<0.05) in enzymic activity in PGP-S (phosphatidylglycerolphosphate synthase), a key enzyme in de novo CL biosynthesis. The increase in PGP-S activity was due to a 3-fold increase (P<0.05) of hepatic PGP-S mRNA expression. The increase in de novo CL biosynthesis and pool size corresponded to a 2.3-fold increase (P<0.05) in the amount of [1-14C]linoleic acid incorporated into CL of hepatocytes prepared from PHx rats compared with sham-operated controls, indicating an increase in CL resynthesis. The activity of MLCL-AT (monolysocardiolipin acyltransferase), a rate-limiting enzyme of CL resynthesis, was increased by 43% (P<0.05) in hepatocytes prepared from PHx rats compared with sham-operated controls; this result would explain the increase in [1-14C]linoleic acid incorporation into CL. The increase in MLCL-AT activity was due to an increase in hepatic MLCL-AT protein expression. The results show that CL de novo biosynthesis and its resynthesis are increased during liver regeneration.
[Show abstract][Hide abstract] ABSTRACT: The role of peroxisome proliferator-activated receptor alpha (PPARalpha)-stimulated phospholipase A2 (PLA2) in cardiac mitochondrial cardiolipin (CL) biosynthesis was examined in both in vivo and in vitro models. Treatment of rat heart H9c2 cells with clofibrate increased the expression and activity of 14 kDa PLA2 but did not affect the pool size of CL. Clofibrate treatment stimulated de novo CL biosynthesis via an increase in phosphatidylglycerolphosphate (PGP) synthase activity, accounting for the unaltered CL content. Cardiac PLA2, PGP synthase, and CDP-1,2-diacyl-sn-glycerol synthase (CDS-2) activities and CDS-2 mRNA levels were elevated in mice fed clofibrate for 14 days compared with controls. In PPARalpha-null mice, clofibrate feeding did not alter cardiac PLA2, PGP synthase activities, or CDS-2 activity and mRNA level, confirming that these enzymes are regulated by PPARalpha activation. In contrast to mouse heart, clofibrate treatment did not affect the activity or mRNA levels of CDS-2 in H9c2 cells, indicating that CDS-2 is regulated differently in rat heart H9c2 cells in vitro and in mouse heart in vivo. These results clearly indicate that cardiac CL de novo biosynthesis is stimulated by PPARalpha activation in responsive rodent models and that CDS-2 is an example of an enzyme that exhibits alternative regulation in vivo and in cultured cell lines. This study is the first to demonstrate that CL de novo biosynthesis is regulated by PPARalpha activation.
The Journal of Lipid Research 03/2004; 45(2):244-52. · 4.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In mammalian tissues cardiolipin is rapidly remodeled by monolysocardiolipin acyltransferase subsequent to its de novo biosynthesis (Ma, B. J., Taylor, W. A, Dolinsky, V. W., and Hatch, G. M. (1999) J. Lipid Res. 40, 1837-1845). We report here the purification and characterization of a monolysocardiolipin acyltransferase activity from pig liver mitochondria. Monolysocardiolipin acyltransferase activity was purified over 1000-fold by butanol extraction, hydroxyapatite chromatography, and preparative SDS-PAGE. The purified 74-kDa protein catalyzed acylation of monolysocardiolipin to cardiolipin with [(14)C]linoleoyl coenzyme A. Photoaffinity labeling of the protein with 12-[(4-[(125)I]azidosalicyl)amino]dodecanoyl coenzyme A indicated coenzyme A was bound at its active site and photoaffinity cross-linking of 12-[(4-azidosalicyl)amino]dodecanoyl coenzyme A to the enzyme inhibited enzyme activity. Enzyme activity was optimum at pH 7.0, and the enzyme did not utilize other lysophospholipids as substrate. The purified enzyme was heat-labile and exhibited an isoelectric point of pH 5.4. To determine the enzymes kinetic mechanism the effect of varying concentrations of linoleoyl coenzyme A and monolysocardiolipin on initial velocity were determined. Double-reciprocal plots revealed parallel lines consistent with a ping pong kinetic mechanism. When the enzyme was incubated in the absence of monolysocardiolipin, coenzyme A was produced from linoleoyl coenzyme A at a rate consistent with the formation of an enzyme-linoleate intermediate. The true K(m) value for linoleoyl coenzyme A and true K(m) value for monolysocardiolipin were 100 and 44 microM, respectively. The calculated V(max) was 6802 pmol/min per mg of protein. A polyclonal antibody, raised in rabbits to the purified protein, cross-reacted with the protein in crude pig liver mitochondrial fractions. In liver mitochondria prepared from thyroxine-treated rats, the level of the protein was elevated compared with euthyroid controls indicating that expression of monolysocardiolipin acyltransferase is regulated by thyroid hormone. The study represents the first purification and characterization of a monolysocardiolipin acyltransferase activity from any organism.
Journal of Biological Chemistry 05/2003; 278(15):12716-21. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We examined the effect of etomoxir treatment on de novo cardiolipin (CL) biosynthesis in H9c2 cardiac myoblast cells. Etomoxir treatment did not affect the activities of the CL biosynthetic and remodeling enzymes but caused a reduction in [1-14C]palmitic acid or [1-14C]oleic acid incorporation into CL. The mechanism was a decrease in fatty acid flux through the de novo pathway of CL biosynthesis via a redirection of lipid synthesis toward 1,2-diacyl-sn-glycerol utilizing reactions mediated by a 35% increase (P < 0.05) in membrane phosphatidate phosphohydrolase activity. In contrast, etomoxir treatment increased [1,3-3H]glycerol incorporation into CL. The mechanism was a 33% increase (P < 0.05) in glycerol kinase activity, which produced an increased glycerol flux through the de novo pathway of CL biosynthesis. Etomoxir treatment inhibited 1,2-diacyl-sn-glycerol acyltransferase activity by 81% (P < 0.05), thereby channeling both glycerol and fatty acid away from 1,2,3-triacyl-sn-glycerol utilization toward phosphatidylcholine and phosphatidylethanolamine biosynthesis. In contrast, etomoxir inhibited myo-[3H]inositol incorporation into phosphatidylinositol and the mechanism was an inhibition in inositol uptake. Etomoxir did not affect [3H]serine uptake but resulted in an increased formation of phosphatidylethanolamine derived from phosphatidylserine. The results indicate that etomoxir treatment has diverse effects on de novo glycerolipid biosynthesis from various metabolic precursors. In addition, etomoxir mediates a distinct and differential metabolic channeling of glycerol and fatty acid precursors into CL.
The Journal of Lipid Research 02/2003; 44(2):415-23. · 4.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Monolysocardiolipin acyltransferase (MLCL AT) catalyzes the acylation of monolysocardiolipin to cardiolipin in mammalian tissues. We previously reported that cardiac cardiolipin levels, MLCL AT and cardiolipin synthase activities were all elevated in rats made hyperthyroid by thyroxine treatment. In this study, we examined if cardiac mitochondrial MLCL AT activity was dependent upon the biosynthesis and level of cardiolipin in the heart. Rat heart mitochondrial MLCL AT activity was determined under conditions in which the levels of cardiac cardiolipin and cardiolipin synthase activity were either reduced or unaltered using four different disease models in the rat. In addition, these parameters were examined in a murine model of cardiac cell differentiation.
In rats made hypothyroid by treatment with 6-n-propyl-2-thiouracil in the drinking water for 34 days, cardiac cardiolipin content was decreased 29% (p < 0.025) and this was associated with a 32% decrease (p < 0.025) in cardiolipin synthase and a 35% reduction (p < 0.025) in MLCL AT activities. Streptozotocin-induced diabetes or hyperinsulinemia in rats did not affect cardiac cardiolipin content nor MLCL AT and cardiolipin synthase activities. Finally, cardiolipin content, MLCL AT and cardiolipin synthase activities were unaltered during murine P19 teratocarcinoma cell differentiation into cardiac myocytes. In all models, phospholipase A2 activities were unaltered compared with controls.
We propose a general model in which the expression of MLCL AT activity is regulated in concert with the biosynthesis and level of cardiolipin in the heart.