Harold F. Sims

A FETAL FATTY-ACID OXIDATION DISORDER AS A CAUSE OF LIVER DISEASE IN PREGNANT WOMEN

Background Acute fatty liver of pregnancy and the HELLP syndrome (hemolysis, elevated liver-enzyme levels, and a low platelet count) are serious hepatic disorders that may occur during pregnancy in...

Molecular Heterogeneity in Very-Long-Chain Acyl-CoA Dehydrogenase Deficiency Causing Pediatric Cardiomyopathy and Sudden Death

BACKGROUND Genetic defects are being increasingly recognized in the etiology of primary cardiomyopathy (CM). Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first step in the beta-oxidation spiral of fatty acid metabolism, the crucial pathway for cardiac energy production. METHODS AND RESULTS We studied 37 patients with CM, nonketotic hypoglycemia and hepatic dysfunction, skeletal myopathy, or sudden death in infancy with hepatic steatosis, features suggestive of fatty acid oxidation disorders. Single-stranded conformational variance was used to screen genomic DNA. DNA sequencing and mutational analysis revealed 21 different mutations on the VLCAD gene in 18 patients. Of the mutations, 80% were associated with CM. Severe CM in infancy was recognized in most patients (67%) at presentation. Hepatic dysfunction was common (33%). RNA blot analysis and VLCAD enzyme assays showed a severe reduction in VLCAD mRNA in patients with frame-shift or splice-site mutations and absent or severe reduction in enzyme activity in all. CONCLUSIONS Infantile CM is the most common clinical phenotype of VLCAD deficiency. Mutations in the human VLCAD gene are heterogeneous. Although mortality at presentation is high, both the metabolic disorder and cardiomyopathy are reversible.

Genetic Ablation of Calcium-independent Phospholipase A2γ 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 A2 γ (iPLA2γ), which possesses dual mitochondrial and peroxisomal subcellular localization signals. To identify the roles of iPLA2γ in cellular bioenergetics, we generated mice null for the iPLA2γ gene by eliminating the active site of the enzyme through homologous recombination. Mice null for iPLA2γ 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 iPLA2γ is essential for maintaining efficient bioenergetic mitochondrial function through tailoring mitochondrial membrane lipid metabolism and composition.

Very-Long-Chain Acyl-Coenzyme A Dehydrogenase Deficiency in Mice

Abstract— Fatty acid oxidation (FAO) defects are inborn errors of metabolism clinically associated with cardiomyopathy and sudden infant death syndrome (SIDS). FAO disorders often present in infancy with myocardial dysfunction and arrhythmias after exposure to stresses such as fasting, exercise, or intercurrent viral illness. It is uncertain whether the heart, in the absence of stress, is normal. We generated very-long-chain acyl-coenzyme A dehydrogenase (VLCAD)-deficient mice by homologous recombination to define the onset and molecular mechanism of myocardial disease. We found that VLCAD-deficient hearts have microvesicular lipid accumulation, marked mitochondrial proliferation, and demonstrated facilitated induction of polymorphic ventricular tachycardia, without antecedent stress. The expression of acyl-CoA synthase (ACS1), adipophilin, activator protein 2, cytochrome c, and the peroxisome proliferator activated receptor &ggr; coactivator-1 were increased immediately after birth, preceding overt histological lipidosis, whereas ACS1 expression was markedly downregulated in the adult heart. We conclude that mice with VLCAD deficiency have altered expression of a variety of genes in the fatty acid metabolic pathway from birth, reflecting metabolic feedback circuits, with progression to ultrastructural and physiological correlates of the associated human disease in the absence of stress.

Inherited long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency and a fetal-maternal interaction cause maternal liver disease and other pregnancy complications

Fetal-maternal interactions are critical determinants of maternal health during pregnancy and perinatal outcome. This review explores the causative relationship of a fetal disorder of mitochondrial fatty acid oxidation, long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, and the serious maternal liver diseases of pregnancy-preeclampsia, the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelet counts), and acute fatty liver of pregnancy. Features of the metabolic adaptation necessitated during the fetal-neonatal transition; common phenotypes of pediatric fatty acid oxidation disorders, including neonatal hypoketotic, hypoglycemia and hepatic crisis; and clinical abnormalities of HELLP and acute fatty liver of pregnancy are presented. Evidence that a common mutation in the alpha-subunit (LCHAD) of trifunctional protein, E474Q, is always one of the mutant alleles in fetal isolated LCHAD deficiency associated with these disorders of pregnancy that cause high maternal, fetal, and newborn morbidity and mortality is reviewed. Recommendations for molecular testing for LCHAD deficiency in families with life-threatening maternal liver disease are given.

Maternal Acute Fatty Liver of Pregnancy Associated with Fetal Trifunctional Protein Deficiency: Molecular Characterization of a Novel Maternal Mutant Allele

Acute fatty liver of pregnancy (AFLP) is a devastating late gestational complication with many similarities to the inherited disorders of mitochondrial fatty acid oxidation. We report the molecular defects in a woman with AFLP and her infant who subsequently was diagnosed with trifunctional protein (TFP) deficiency. We used single-stranded conformation variance and DNA sequence analyses of the human TFP α-subunit gene, which encodes the long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity, to demonstrate a C to T mutation (C1678T) in exon 16 present on one allele in the mother and the affected infant. This creates a premature termination codon (R524Stop) in the LCHAD domain. Using reverse transcriptase-PCR amplification of theα-subunit mRNA from cultured fibroblasts, we demonstrated that transcripts containing this R524Stop mutation are present at very low levels, presumably because of rapid mRNA degradation. The affected infant also had the common E474Q mutation (nucleotide G1528C) on the second allele. Thus, he is a compound heterozygote. The father and two normal siblings are heterozygous for this E474Q mutation. This initial delineation of the R524Stop mutation provides evidence of the heterogeneity of genetic defects responsible for TFP deficiency and AFLP.

Genetic Ablation of Calcium-independent Phospholipase A2γ Leads to Alterations in Hippocampal Cardiolipin Content and Molecular Species Distribution, Mitochondrial Degeneration, Autophagy, and Cognitive Dysfunction

Genetic ablation of calcium-independent phospholipase A2γ (iPLA2γ) 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 iPLA2γ−/− 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 iPLA2γ in neuronal mitochondrial lipid metabolism and membrane structure demonstrating that iPLA2γ loss of function results in a mitochondrial neurodegenerative disorder characterized by degenerating mitochondria, autophagy, and cognitive dysfunction.

Deletion of the n-terminus of murine map2 by gene targeting disrupts hippocampal ca1 neuron architecture and alters contextual memory

Microtubule-associated protein-2 (MAP2) is a brain specific A-kinase anchoring protein that targets the cyclic AMP-dependent protein kinase holoenzyme (PKA) to microtubules. Phosphorylation of MAP2 by different protein kinases is crucial for neuronal growth. The N-terminus of MAP2 contains the binding site for regulatory subunit II of cAMP-dependent protein kinase (PKA-RIIbeta). Using homologous recombination, we created a mutant line of mice (delta1-158) that express truncated MAP2 lacking the N-terminal peptide and the PKA binding site. Deletion of the PKA binding site from the MAP2 gene resulted in decreased efficiency of MAP2 phosphorylation. Biochemical and immunohistochemical studies demonstrate major changes in the morphology of hippocampal neurons in delta1-158 mice. Behavioral tests indicate that delta1-158 mice were impaired (exhibited less conditioned freezing) relative to Wild-Type (WT) controls during a test of contextual, but not during auditory cue, fear conditioning when tested at 8 weeks or 8 months of age. The delta1-158 mice displayed a heightened sensitivity to shock at 8 weeks, but not at 8 months of age. We conclude that PKA binding to MAP2 and MAP2 phosphorylation is essential for the selective development of contextual memory.

Genetic Ablation of Calcium-independent Phospholipase A2γ 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 A2γ (iPLA2γ−/−) are completely resistant to high fat diet-induced weight gain, adipocyte hypertrophy, hyperinsulinemia, and insulin resistance, which occur in iPLA2γ+/+ mice after high fat feeding. Notably, iPLA2γ−/− 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 iPLA2γ−/− 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 iPLA2γ−/− mice fed either a standard diet or a high fat diet. Respirometry of skeletal muscle mitochondria from iPLA2γ−/− 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 iPLA2γ−/− mouse. Collectively, these results identify iPLA2γ 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.

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.