Cardiac Myocyte-specific Knock-out of Calcium-independent Phospholipase A2γ (iPLA2γ) Decreases Oxidized Fatty Acids during Ischemia/Reperfusion and Reduces Infarct Size.

Calcium-independent phospholipase A2γ (iPLA2γ) is a mitochondrial enzyme that produces lipid second messengers that facilitate opening of the mitochondrial permeability transition pore (mPTP) and contribute to the production of oxidized fatty acids in myocardium. To specifically identify the roles of iPLA2γ in cardiac myocytes, we generated cardiac myocyte-specific iPLA2γ knock-out (CMiPLA2γKO) mice by removing the exon encoding the active site serine (Ser-477). Hearts of CMiPLA2γKO mice exhibited normal hemodynamic function, glycerophospholipid molecular species composition, and normal rates of mitochondrial respiration and ATP production. In contrast, CMiPLA2γKO mice demonstrated attenuated Ca(2+)-induced mPTP opening that could be rapidly restored by the addition of palmitate and substantially reduced production of oxidized polyunsaturated fatty acids (PUFAs). Furthermore, myocardial ischemia/reperfusion (I/R) in CMiPLA2γKO mice (30 min of ischemia followed by 30 min of reperfusion in vivo) dramatically decreased oxidized fatty acid production in the ischemic border zones. Moreover, CMiPLA2γKO mice subjected to 30 min of ischemia followed by 24 h of reperfusion in vivo developed substantially less cardiac necrosis in the area-at-risk in comparison with their WT littermates. Furthermore, we found that membrane depolarization in murine heart mitochondria was sensitized to Ca(2+) by the presence of oxidized PUFAs. Because mitochondrial membrane depolarization and calcium are known to activate iPLA2γ, these results are consistent with salvage of myocardium after I/R by iPLA2γ loss of function through decreasing mPTP opening, diminishing production of proinflammatory oxidized fatty acids, and attenuating the deleterious effects of abrupt increases in calcium ion on membrane potential during reperfusion.

Dysfunctional cardiac mitochondrial bioenergetic, lipidomic, and signaling in a murine model of Barth syndrome.

Barth syndrome is a complex metabolic disorder caused by mutations in the mitochondrial transacylase tafazzin. Recently, an inducible tafazzin shRNA knockdown mouse model was generated to deconvolute the complex bioenergetic phenotype of this disease. To investigate the underlying cause of hemodynamic dysfunction in Barth syndrome, we interrogated the cardiac structural and signaling lipidome of this mouse model as well as its myocardial bioenergetic phenotype. A decrease in the distribution of cardiolipin molecular species and robust increases in monolysocardiolipin and dilysocardiolipin were demonstrated. Additionally, the contents of choline and ethanolamine glycerophospholipid molecular species containing precursors for lipid signaling at the sn-2 position were altered. Lipidomic analyses revealed specific dysregulation of HETEs and prostanoids, as well as oxidized linoleic and docosahexaenoic metabolites. Bioenergetic interrogation uncovered differential substrate utilization as well as decreases in Complex III and V activities. Transgenic expression of cardiolipin synthase or iPLA2γ ablation in tafazzin-deficient mice did not rescue the observed phenotype. These results underscore the complex nature of alterations in cardiolipin metabolism mediated by tafazzin loss of function. Collectively, we identified specific lipidomic, bioenergetic, and signaling alterations in a murine model that parallel those of Barth syndrome thereby providing novel insights into the pathophysiology of this debilitating disease.

Activation of mitochondrial calcium-independent phospholipase A2γ (iPLA2γ) by divalent cations mediating arachidonate release and production of downstream eicosanoids.

Calcium-independent phospholipase A(2)γ (iPLA(2)γ) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)γ gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)γ in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)γ resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)γ mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)γ selective), but not its enantiomer, (S)-BEL (iPLA(2)ß selective) or pyrrolidine (cytosolic PLA(2)α selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)γ activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)γ. Intriguingly, Ca(2+)-induced iPLA(2)γ activation was completely inhibited by long-chain acyl-CoA (IC(50) ∼20 µm) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)γ is activated by divalent cations and inhibited by acyl-CoA modulating the generation of biologically active metabolites that regulate mitochondrial bioenergetic and signaling functions.

Genetic ablation of calcium-independent phospholipase A(2)γ (iPLA(2)γ) attenuates calcium-induced opening of the mitochondrial permeability transition pore and resultant cytochrome c release.

Herein, we demonstrate that calcium-independent phospholipase A(2)γ (iPLA(2)γ) is a critical mechanistic participant in the calcium-induced opening of the mitochondrial permeability transition pore (mPTP). Liver mitochondria from iPLA(2)γ(-/-) mice were markedly resistant to calcium-induced swelling in the presence or absence of phosphate in comparison with wild-type littermates. Furthermore, the iPLA(2)γ enantioselective inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one ((R)-BEL) was markedly more potent than (S)-BEL in inhibiting mPTP opening in mitochondria from wild-type liver in comparison with hepatic mitochondria from iPLA(2)γ(-/-) mice. Intriguingly, low micromolar concentrations of long chain fatty acyl-CoAs and the non-hydrolyzable thioether analog of palmitoyl-CoA markedly accelerated Ca(2+)-induced mPTP opening in liver mitochondria from wild-type mice. The addition of l-carnitine enabled the metabolic channeling of acyl-CoA through carnitine palmitoyltransferases (CPT-1/2) and attenuated the palmitoyl-CoA-mediated amplification of calcium-induced mPTP opening. In contrast, mitochondria from iPLA(2)γ(-/-) mice were insensitive to fatty acyl-CoA-mediated augmentation of calcium-induced mPTP opening. Moreover, mitochondria from iPLA(2)γ(-/-) mouse liver were resistant to Ca(2+)/t-butyl hydroperoxide-induced mPTP opening in comparison with wild-type littermates. In support of these findings, cytochrome c release from iPLA(2)γ(-/-) mitochondria was dramatically decreased in response to calcium in the presence or absence of either t-butyl hydroperoxide or phenylarsine oxide in comparison with wild-type littermates. Collectively, these results identify iPLA(2)γ as an important mechanistic component of the mPTP, define its downstream products as potent regulators of mPTP opening, and demonstrate the integrated roles of mitochondrial bioenergetics and lipidomic flux in modulating mPTP opening promoting the activation of necrotic and necroapoptotic pathways of cell death.

Myocardial regulation of lipidomic flux by cardiolipin synthase: setting the beat for bioenergetic efficiency.

Lipidomic regulation of mitochondrial cardiolipin content and molecular species composition is a prominent regulator of bioenergetic efficiency. However, the mechanisms controlling cardiolipin metabolism during health or disease progression have remained elusive. Herein, we demonstrate that cardiac myocyte-specific transgenic expression of cardiolipin synthase results in accelerated cardiolipin lipidomic flux that impacts multiple aspects of mitochondrial bioenergetics and signaling. During the postnatal period, cardiolipin synthase transgene expression results in marked changes in the temporal maturation of cardiolipin molecular species during development. In adult myocardium, cardiolipin synthase transgene expression leads to a marked increase in symmetric tetra-18:2 molecular species without a change in total cardiolipin content. Mechanistic analysis demonstrated that these alterations result from increased cardiolipin remodeling by sequential phospholipase and transacylase/acyltransferase activities in conjunction with a decrease in phosphatidylglycerol content. Moreover, cardiolipin synthase transgene expression results in alterations in signaling metabolites, including a marked increase in the cardioprotective eicosanoid 14,15-epoxyeicosatrienoic acid. Examination of mitochondrial bioenergetic function by high resolution respirometry demonstrated that cardiolipin synthase transgene expression resulted in improved mitochondrial bioenergetic efficiency as evidenced by enhanced electron transport chain coupling using multiple substrates as well as by salutary changes in Complex III and IV activities. Furthermore, transgenic expression of cardiolipin synthase attenuated maladaptive cardiolipin remodeling and bioenergetic inefficiency in myocardium rendered diabetic by streptozotocin treatment. Collectively, these results demonstrate the unanticipated role of cardiolipin synthase in maintaining physiologic membrane structure and function even under metabolic stress, thereby identifying cardiolipin synthase as a novel therapeutic target to attenuate mitochondrial dysfunction in diabetic myocardium.

Oxidized fatty acid analysis by charge-switch derivatization, selected reaction monitoring, and accurate mass quantitation.

A highly sensitive, specific, and robust method for the analysis of oxidized metabolites of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) was developed using charge-switch derivatization, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) with selected reaction monitoring (SRM) and quantitation by high mass accuracy analysis of product ions, thereby minimizing interferences from contaminating ions. Charge-switch derivatization of LA, AA, and DHA metabolites with N-(4-aminomethylphenyl)-pyridinium resulted in a 10- to 30-fold increase in ionization efficiency. Improved quantitation was accompanied by decreased false positive interferences through accurate mass measurements of diagnostic product ions during SRM transitions by ratiometric comparisons with stable isotope internal standards. The limits of quantitation were between 0.05 and 6.0pg, with a dynamic range of 3 to 4 orders of magnitude (correlation coefficient r(2)>0.99). This approach was used to quantitate the levels of representative fatty acid metabolites from wild-type (WT) and iPLA2γ(-/-) mouse liver identifying the role of iPLA2γ in hepatic lipid second messenger production. Collectively, these results demonstrate the utility of high mass accuracy product ion analysis in conjunction with charge-switch derivatization for the highly specific quantitation of diminutive amounts of LA, AA, and DHA metabolites in biologic systems.

Activation of group VI phospholipase A2 isoforms in cardiac endothelial cells.

The endothelium comprises a cellular barrier between the circulation and tissues. We have previously shown that activation of protease-activated receptor 1 (PAR-1) and PAR-2 on the surface of human coronary artery endothelial cells by tryptase or thrombin increases group VIA phospholipase A(2) (iPLA(2)ß) activity and results in production of multiple phospholipid-derived inflammatory metabolites. We isolated cardiac endothelial cells from hearts of iPLA(2)ß-knockout (iPLA(2)ß-KO) and wild-type (WT) mice and measured arachidonic acid (AA), prostaglandin I(2) (PGI(2)), and platelet-activating factor (PAF) production in response to PAR stimulation. Thrombin (0.1 IU/ml) or tryptase (20 ng/ml) stimulation of WT endothelial cells rapidly increased AA and PGI(2) release and increased PAF production. Selective inhibition of iPLA(2)ß with (S)-bromoenol lactone (5 µM, 10 min) completely inhibited thrombin- and tryptase-stimulated responses. Thrombin or tryptase stimulation of iPLA(2)ß-KO endothelial cells did not result in significant PAF production and inhibited AA and PGI(2) release. Stimulation of cardiac endothelial cells from group VIB (iPLA(2)γ)-KO mice increased PAF production to levels similar to those of WT cells but significantly attenuated PGI(2) release. These results indicate that cardiac endothelial cell PAF production is dependent on iPLA(2)ß activation and that both iPLA(2)ß and iPLA(2)γ may be involved in PGI(2) release.

Genetic ablation of calcium-independent phospholipase A2gamma prevents obesity and insulin resistance during high fat feeding by mitochondrial uncoupling and increased adipocyte fatty acid oxidation.

Phospholipases are critical enzyme mediators participating in many aspects of cellular function through modulating the generation of lipid 2nd messengers, membrane physical properties, and cellular bioenergetics. Here, we demonstrate that mice null for calcium-independent phospholipase A(2)γ (iPLA(2)γ(-/-)) are completely resistant to high fat diet-induced weight gain, adipocyte hypertrophy, hyperinsulinemia, and insulin resistance, which occur in iPLA(2)γ(+/+) mice after high fat feeding. Notably, iPLA(2)γ(-/-) mice were lean, demonstrated abdominal lipodystrophy, and remained insulin-sensitive despite having a marked impairment in glucose-stimulated insulin secretion after high fat feeding. Respirometry of adipocyte explants from iPLA(2)γ(-/-) mice identified increased rates of oxidation of multiple different substrates in comparison with adipocyte explants from wild-type littermates. Shotgun lipidomics of adipose tissue from wild-type mice demonstrated the anticipated 2-fold increase in triglyceride content after high fat feeding. In sharp contrast, the adipocyte triglyceride content was identical in iPLA(2)γ(-/-) mice fed either a standard diet or a high fat diet. Respirometry of skeletal muscle mitochondria from iPLA(2)γ(-/-) mice demonstrated marked decreases in state 3 respiration using multiple substrates whose metabolism was uncoupled from ATP production. Shotgun lipidomics of skeletal muscle revealed a decreased content of cardiolipin with an altered molecular species composition thereby identifying the mechanism underlying mitochondrial uncoupling in the iPLA(2)γ(-/-) mouse. Collectively, these results identify iPLA(2)γ as an obligatory upstream enzyme that is necessary for efficient electron transport chain coupling and energy production through its participation in the alterations of cellular bioenergetics that promote the development of the metabolic syndrome.

Genetic ablation of calcium-independent phospholipase A2{gamma} leads to alterations in hippocampal cardiolipin content and molecular species distribution, mitochondrial degeneration, autophagy, and cognitive dysfunction.

Genetic ablation of calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) results in profound alterations in hippocampal phospholipid metabolism and mitochondrial phospholipid homeostasis resulting in enlarged and degenerating mitochondria leading to autophagy and cognitive dysfunction. Shotgun lipidomics demonstrated multiple alterations in hippocampal lipid metabolism in iPLA(2)gamma(-/-) mice including: 1) a markedly elevated hippocampal cardiolipin content with an altered molecular species composition characterized by a shift to shorter chain length molecular species; 2) alterations in both choline and ethanolamine glycerophospholipids, including a decreased plasmenylethanolamine content; 3) increased oxidized phosphatidylethanolamine molecular species; and 4) an increased content of ceramides. Electron microscopic examination demonstrated the presence of enlarged heteromorphic lamellar structures undergoing degeneration accompanied by the presence of ubiquitin positive spheroid inclusion bodies. Purification of these enlarged heteromorphic lamellar structures by buoyant density centrifugation and subsequent SDS-PAGE and proteomics identified them as degenerating mitochondria. Collectively, these results identify the obligatory role of iPLA(2)gamma in neuronal mitochondrial lipid metabolism and membrane structure demonstrating that iPLA(2)gamma loss of function results in a mitochondrial neurodegenerative disorder characterized by degenerating mitochondria, autophagy, and cognitive dysfunction.

Genetic ablation of calcium-independent phospholipase A(2)beta causes hypercontractility and markedly attenuates endothelium-dependent relaxation to acetylcholine.

Activation of phospholipases leads to the release of arachidonic acid and lysophospholipids that play prominent roles in regulating vasomotor tone. To identify the role of calcium-independent phospholipase A(2)beta (iPLA(2)beta) in vasomotor function, we measured vascular responses to phenylephrine (PE) and ACh in mesenteric arterioles from wild-type (WT; iPLA(2)beta(+/+)) mice and those lacking the beta-isoform (iPLA(2)beta(-/-)) both ex vivo and in vivo. Vessels isolated from iPLA(2)beta(-/-) mice demonstrated increased constriction to PE, despite lower basal smooth muscle calcium levels, and decreased vasodilation to ACh compared with iPLA(2)beta(+/+) mice. PE constriction resulted in initial intracellular calcium release with subsequent steady-state constriction that depended on extracellular calcium influx. Endothelial denudation had no effect on vessel tone or PE-induced constriction although the dilation to ACh was significantly reduced in iPLA(2)beta(+/+) vessels. In contrast, vessels from iPLA(2)beta(-/-) constricted by 54% after denudation, indicating smooth muscle hypercontractility. In vivo, blood pressure, resting vessel diameter, and constriction of mesenteric vessels to PE were not different in iPLA(2)beta(-/-) vessels compared with WT mouse vessels. However, relaxation after ACh administration in situ was attenuated, indicating an endothelial inability to induce dilation in response to ACh. In cultured endothelial cells, inhibition of iPLA(2)beta with (S)-(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL) decreased endothelial nitric oxide synthase phosphorylation and reduced endothelial agonist-induced intracellular calcium release as well as extracellular calcium influx. We conclude that iPLA(2)beta is an important mediator of vascular relaxation and intracellular calcium homeostasis in both smooth muscle and endothelial cells and that ablation of iPLA(2)beta causes agonist-induced smooth muscle hypercontractility and reduced agonist-induced endothelial dilation.

Calcium-independent phospholipases in the heart: mediators of cellular signaling, bioenergetics, and ischemia-induced electrophysiologic dysfunction.

Myocardial function is intimately dependent on the precise spatiotemporal regulation of membrane-bound proteins and ion channels. Phospholipases play critical roles in the maintenance of membrane structure and function, thereby fundamentally integrating dynamic alterations in myocardial performance with membrane composition and dynamics. The major phospholipases in myocardium belong to a family of proteins known as calcium-independent phospholipases (iPLA2s). In addition to their role in maintaining normal membrane structure and function, iPLA2 catalytic activity results in the generation of a variety of lipid second messengers that facilitate cellular signaling. Through its multiple effects on cardiac myocyte bioenergetics, cellular signaling, and membrane function, the iPLA2 family of enzymes is of primary importance in modulating the pathologic sequelae of myocardial ischemia, diabetic cardiomyopathy, and remodeling during hemodynamic stress. This review will provide a brief overview of myocardial iPLA2s and their significance in cardiac pathology and physiology.

Shotgun lipidomics reveals the temporally dependent, highly diversified cardiolipin profile in the mammalian brain: temporally coordinated postnatal diversification of cardiolipin molecular species with neuronal remodeling.

Large-scale neuronal remodeling through apoptosis occurs shortly after birth in all known mammalian species. Apoptosis, in large part, depends upon critical interactions between mitochondrial membranes and cytochrome c. Herein, we examined the hypothesis that the large-scale reorganization of neuronal circuitry after birth is accompanied by profound alterations in cardiolipin (CL) content and molecular species distribution. During embryonic development, over 100 CL molecular species were identified and quantitated in murine neuronal tissues. The embryonic CL profile was notable for the presence of abundant amounts of relatively short aliphatic chains (e.g., palmitoleic and oleic acids). In sharp contrast, after birth, the CL profile contained a remarkably complex repertoire of CL molecular species, in which the signaling fatty acids (i.e., arachidonic and docosahexaenoic acids) were markedly increased. These results identify the rapid remodeling of CL in the perinatal period with resultant alterations in the physical properties of the mitochondrial membrane. The complex distribution of aliphatic chains in the neuronal CL pool is separate and distinct from that in other organs (e.g., heart, liver, etc.), where CL molecular species contain predominantly only one major type of aliphatic chain (e.g., linoleic acid). Analyses of mRNA levels by real-time quantitative polymerase chain reactions suggested that the alterations in CL content were due to the combined effects of both attenuation of de novo CL biosynthesis and decreased remodeling of CL. Collectively, these results provide a new perspective on the complexity of CL in neuronal signaling, mitochondrial bioenergetics, and apoptosis.

Functional lipidomics: the roles of specialized lipids and lipid-protein interactions in modulating neuronal function.

Lipids fulfill multiple specialized roles in neuronal function. In brain, the conduction of electrical impulses, synaptic function, and complex signaling pathways depend on the temporally and spatially coordinated interactions of specialized lipids (e.g., arachidonic acid and plasmalogens), proteins (e.g., ion channels, phospholipases and cyclooxygenases) and integrative lipid-protein interactions. Recent technical advances in mass spectrometry have allowed unparalled insight into the roles of lipids in neuronal function. Through shotgun lipidomics and multidimensional mass spectrometry, in conjunction with the identification of new classes of phospholipases (e.g., calcium dependent and calcium independent intracellular phospholipases), new roles for lipids in cerebral function have been accrued. This review summarizes the advances in our understanding of the types of lipids and phospholipases in the brain and the role of functional lipidomics in increasing our chemical understanding of complex neuronal processes.

Smooth muscle cell arachidonic acid release, migration, and proliferation are markedly attenuated in mice null for calcium-independent phospholipase A2beta.

Pharmacologic evidence suggests that the lipid products generated by one or more calcium-independent phospholipases A(2) (iPLA(2)s) participate in the regulation of vascular tone through smooth muscle cell (SMC) Ca(2+) signaling and the release of arachidonic acid. However, the recent identification of new members of the iPLA(2) family, each inhibitable by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one, has rendered definitive identification of the specific enzyme(s) mediating these processes difficult. Accordingly, we used iPLA(2)beta(-/-) mice to demonstrate that iPLA(2)beta is responsible for the majority of thapsigargin and ionophore (A23187)-induced arachidonic acid release from SMCs. Both thapsigargin and A23187 stimulated robust [(3)H]arachidonate (AA) release from wild-type aortic SMCs that was dramatically attenuated in iPLA(2)beta(-/-) mice (>80% reduction at 5 min; p < 0.01). Moreover, iPLA(2)beta(-/-) mice displayed defects in SMC Ca(2+) homeostasis and decreased SMC migration and proliferation in a model of vascular injury. Ca(2+)-store depletion resulted in the rapid entry of external Ca(2+) into wild-type aortic SMCs that was significantly slower in iPLA(2)beta-null cells (p < 0.01). Furthermore, SMCs from iPLA(2)beta-null mesenteric arterial explants demonstrated decreased proliferation and migration. The defects in migration and proliferation in iPLA(2)beta-null SMCs were restored by 2 mum AA. Remarkably, the cyclooxygenase-2-specific inhibitor, NS-398, prevented AA-induced rescue of SMC migration and proliferation in iPLA(2)beta(-/-) mice. Moreover, PGE(2) alone rescued proliferation and migration in iPLA(2)beta(-/-) mice. We conclude that iPLA(2)beta is an important mediator of AA release and prostaglandin E(2) production in SMCs, modulating vascular tone, cellular signaling, proliferation, and migration.

Disrupted membrane homeostasis and accumulation of ubiquitinated proteins in a mouse model of infantile neuroaxonal dystrophy caused by PLA2G6 mutations.

Mutations in the PLA2G6 gene, which encodes group VIA calcium-independent phospholipase A2 (iPLA(2)beta), were recently identified in patients with infantile neuroaxonal dystrophy (INAD) and neurodegeneration with brain iron accumulation. A pathological hallmark of these childhood neurodegenerative diseases is the presence of distinctive spheroids in distal axons that contain accumulated membranes. We used iPLA(2)beta-KO mice generated by homologous recombination to investigate neurodegenerative consequences of PLA2G6 mutations. iPLA(2)beta-KO mice developed age-dependent neurological impairment that was evident in rotarod, balance, and climbing tests by 13 months of age. The primary abnormality underlying this neurological impairment was the formation of spheroids containing tubulovesicular membranes remarkably similar to human INAD. Spheroids were strongly labeled with anti-ubiquitin antibodies. Accumulation of ubiquitinated protein in spheroids was evident in some brain regions as early as 4 months of age, and the onset of motor impairment correlated with a dramatic increase in ubiquitin-positive spheroids throughout the neuropil in nearly all brain regions. Furthermore accumulating ubiquitinated proteins were observed primarily in insoluble fractions of brain tissue, implicating protein aggregation in this pathogenic process. These results indicate that loss of iPLA(2)beta causes age-dependent impairment of axonal membrane homeostasis and protein degradation pathways, leading to age-dependent neurological impairment. iPLA(2)beta-KO mice will be useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration with brain iron accumulation.

Genetic ablation of calcium-independent phospholipase A2gamma leads to alterations in mitochondrial lipid metabolism and function resulting in a deficient mitochondrial bioenergetic phenotype.

Previously, we identified a novel calcium-independent phospholipase, designated calcium-independent phospholipase A(2) gamma (iPLA(2)gamma), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA(2)gamma in cellular bioenergetics, we generated mice null for the iPLA(2)gamma gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA(2)gamma display multiple bioenergetic dysfunctional phenotypes, including 1) growth retardation, 2) cold intolerance, 3) reduced exercise endurance, 4) greatly increased mortality from cardiac stress after transverse aortic constriction, 5) abnormal mitochondrial function with a 65% decrease in ascorbate-induced Complex IV-mediated oxygen consumption, and 6) a reduction in myocardial cardiolipin content accompanied by an altered cardiolipin molecular species composition. We conclude that iPLA(2)gamma is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.

Dramatic accumulation of triglycerides and precipitation of cardiac hemodynamic dysfunction during brief caloric restriction in transgenic myocardium expressing human calcium-independent phospholipase A2gamma.

Previously, we identified calcium-independent phospholipase A2gamma (iPLA2gamma) with multiple translation initiation sites and dual mitochondrial and peroxisomal localization motifs. To determine the role of iPLA2gamma in integrating lipid and energy metabolism, we generated transgenic mice containing the alpha-myosin heavy chain promoter (alphaMHC) placed proximally to the human iPLA2gamma coding sequence that resulted in cardiac myocyte-restricted expression of iPLA2gamma (TGiPLA2gamma). TGiPLA2gamma mice possessed multiple phenotypes including: 1) a dramatic approximately 35% reduction in myocardial phospholipid mass in both the fed and mildly fasted states; 2) a marked accumulation of triglycerides during brief caloric restriction that represented 50% of total myocardial lipid mass; and 3) acute fasting-induced hemodynamic dysfunction. Biochemical characterization of the TGiPLA2gamma protein expressed in cardiac myocytes demonstrated over 25 distinct isoforms by two-dimensional SDS-PAGE Western analysis. Immunohistochemistry identified iPLA2gamma in the peroxisomal and mitochondrial compartments in both wild type and transgenic myocardium. Electron microscopy revealed the presence of loosely packed and disorganized mitochondrial cristae in TGiPLA2gamma mice that were accompanied by defects in mitochondrial function. Moreover, markedly elevated levels of 1-hydroxyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-hydroxyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine were prominent in the TGiPLA2gamma myocardium identifying the production of signaling metabolites by this enzyme in vivo. Collectively, these results identified the participation of iPLA2gamma in the remarkable lipid plasticity of myocardium, its role in generating signaling metabolites, and its prominent effects in modulating energy storage and utilization in myocardium in different metabolic contexts.

Highly selective hydrolysis of fatty acyl-CoAs by calcium-independent phospholipase A2beta. Enzyme autoacylation and acyl-CoA-mediated reversal of calmodulin inhibition of phospholipase A2 activity.

Calcium-independent phospholipase A2beta (iPLA2beta) participates in numerous diverse cellular processes, such as arachidonic acid release, insulin secretion, calcium signaling, and apoptosis. Herein, we demonstrate the highly selective iPLA2beta-catalyzed hydrolysis of saturated long-chain fatty acyl-CoAs (palmitoyl-CoA approximately myristoyl-CoA >> stearoyl-CoA >> oleoyl-CoA approximately = arachidonoyl-CoA) present either as monomers in solution or guests in host membrane bilayers. Site-directed mutagenesis of the iPLA2beta catalytic serine (S465A) completely abolished acyl-CoA thioesterase activity, demonstrating that Ser-465 catalyzes both phospholipid and acyl-CoA hydrolysis. Remarkably, incubation of iPLA2beta with oleoyl-CoA, but not other long-chain acyl-CoAs, resulted in robust stoichiometric covalent acylation of the enzyme. Moreover, S465A mutagenesis or pretreatment of wild-type iPLA2beta with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one unexpectedly increased acylation of the enzyme, indicating the presence of a second reactive nucleophilic residue that participates in the formation of the fatty acyl-iPLA2beta adduct. Radiolabeling of intact Sf9 cells expressing iPLA2beta with [3H]oleic acid demonstrated oleoylation of the membrane-associated enzyme. Partial trypsinolysis of oleoylated iPLA2beta and matrix-assisted laser desorption ionization mass spectrometry analysis localized the acylation site to a hydrophobic 25-kDa fragment (residues approximately 400-600) spanning the active site to the calmodulin binding domain. Intriguingly, calmodulin-Ca2+ blocked acylation of iPLA2beta by oleoyl-CoA. Remarkably, the addition of low micromolar concentrations (5 microM) of oleoyl-CoA resulted in reversal of calmodulin-mediated inhibition of iPLA2 beta phospholipase A2 activity. These results collectively identify the molecular species-specific acyl-CoA thioesterase activity of iPLA2beta, demonstrate the presence of a second active site that mediates iPLA2beta autoacylation, and identify long-chain acyl-CoAs as potential candidates mediating calcium influx factor activity.

Accumulation of long-chain acylcarnitine and 3-hydroxy acylcarnitine molecular species in diabetic myocardium: identification of alterations in mitochondrial fatty acid processing in diabetic myocardium by shotgun lipidomics.

Diabetic cardiomyopathy is the result of maladaptive changes in energy homeostasis. However, the biochemical mechanisms underlying dysfunctional lipid metabolism in diabetic myocardium are incompletely understood. Herein, we exploit shotgun lipidomics to demonstrate a 4-fold increase in acylcarnitines in diabetic myocardium, which was reversible upon insulin treatment. Analysis of acylcarnitine molecular species in myocardium unexpectedly identified acylcarnitine molecular species containing a mass shift of 16 amu in comparison to the anticipated molecular species. Synthesis of 3-hydroxy acylcarnitine identified the natural products as the 3-hydroxylated acylcarnitines through comparisons of diagnostic fragmentation patterns of synthetic and naturally occurring constituents using tandem mass spectrometry. Diabetes induced an increase of both calcium-independent phospholipase A(2) (iPLA(2)) mRNA and iPLA(2) activity in rat myocardium. Cardiac ischemia in myocardium genetically engineered to overexpress iPLA(2) dramatically increased the amount of acylcarnitine present in myocardium. Moreover, mechanism-based inactivation of iPLA(2) in either wild-type or transgenic myocardium ablated a substantial portion of the acylcarnitine increase. Collectively, these results identify discrete insulin remediable abnormalities in mitochondrial fatty acid processing in diabetic myocardium and identify iPLA(2) as an important enzymatic contributor to the pool of fatty acids that can be used for acylcarnitine synthesis and energy production in myocardium.

The human Kv1.1 channel is palmitoylated, modulating voltage sensing: Identification of a palmitoylation consensus sequence.

Voltage-dependent K(+) channels rely on precise dynamic protein interactions with surrounding plasma membrane lipids to facilitate complex processes such as voltage sensing and channel gating. Many transmembrane-spanning proteins use palmitoylation to facilitate dynamic membrane interactions. Herein, we demonstrate that the human Kv1.1 ion channel is palmitoylated in the cytosolic portion of the S(2)-S(3) linker domain at residue C243. Through heterologous expression of the human Kv1.1 protein in Sf9 cells, covalent radiolabeling with [(3)H]palmitate, chemical stability studies of the [(3)H]-palmitoylated protein, and site-directed mutagenesis, C243 was identified as the predominant site of palmitoylation. The functional sequelae of palmitoylation were examined by analysis of whole cell currents from WT and mutant channels, which identified a 20-mV leftward shift in the current-voltage relationship when palmitoylation at C243 (but not with other cysteine deletions) is prevented by site-directed mutagenesis, implicating a role for palmitoylated C243 in modulating voltage sensing through protein-membrane interactions. Database searches identified an amino acid palmitoylation consensus motif (ACP/RSKT) that is present in multiple other members of the Shaker subfamily of K(+) channels and in several other unrelated regulatory proteins (e.g., CD36, nitric oxide synthase type 2, and the mannose-6 phosphate receptor) that are known to be palmitoylated by thioester linkages at the predicted consensus site cysteine residue. Collectively, these results (i) identify palmitoylation as a mechanism for K(+) channel interactions with plasma membrane lipids contributing to electric field-induced conformational alterations, and ii) define an amino acid consensus sequence for protein palmitoylation.