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      Proteomic and metabolomic changes driven by elevating myocardial creatine suggest novel metabolic feedback mechanisms

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          Abstract

          Mice over-expressing the creatine transporter have elevated myocardial creatine levels [Cr] and are protected against ischaemia/reperfusion injury via improved energy reserve. However, mice with very high [Cr] develop cardiac hypertrophy and dysfunction. To investigate these contrasting effects, we applied a non-biased hypothesis-generating approach to quantify global protein and metabolite changes in the LV of mice stratified for [Cr] levels: wildtype, moderately elevated, and high [Cr] (65–85; 100–135; 160–250 nmol/mg protein, respectively). Male mice received an echocardiogram at 7 weeks of age with tissue harvested at 8 weeks. RV was used for [Cr] quantification by HPLC to select LV tissue for subsequent analysis. Two-dimensional difference in-gel electrophoresis identified differentially expressed proteins, which were manually picked and trypsin digested for nano-LC–MS/MS. Principal component analysis (PCA) showed efficient group separation (ANOVA P ≤ 0.05) and peptide sequences were identified by mouse database (UniProt 201203) using Mascot. A total of 27 unique proteins were found to be differentially expressed between normal and high [Cr], with proteins showing [Cr]-dependent differential expression, chosen for confirmation, e.g. α-crystallin B, a heat shock protein implicated in cardio-protection and myozenin-2, which could contribute to the hypertrophic phenotype. Nuclear magnetic resonance (¹H-NMR at 700 MHz) identified multiple strong correlations between [Cr] and key cardiac metabolites. For example, positive correlations with α-glucose ( r² = 0.45; P = 0.002), acetyl-carnitine ( r² = 0.50; P = 0.001), glutamine ( r² = 0.59; P = 0.0002); and negative correlations with taurine ( r² = 0.74; P < 0.0001), fumarate ( r² = 0.45; P = 0.003), aspartate ( r² = 0.59; P = 0.0002), alanine ( r² = 0.66; P < 0.0001) and phosphocholine ( r² = 0.60; P = 0.0002). These findings suggest wide-ranging and hitherto unexpected adaptations in substrate utilisation and energy metabolism with a general pattern of impaired energy generating pathways in mice with very high creatine levels.

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          The online version of this article (doi:10.1007/s00726-016-2236-x) contains supplementary material, which is available to authorized users.

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          A little sugar goes a long way: The cell biology of O-GlcNAc

          Unlike the complex glycans decorating the cell surface, the O-linked β-N-acetyl glucosamine (O-GlcNAc) modification is a simple intracellular Ser/Thr-linked monosaccharide that is important for disease-relevant signaling and enzyme regulation. O-GlcNAcylation requires uridine diphosphate–GlcNAc, a precursor responsive to nutrient status and other environmental cues. Alternative splicing of the genes encoding the O-GlcNAc cycling enzymes O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) yields isoforms targeted to discrete sites in the nucleus, cytoplasm, and mitochondria. OGT and OGA also partner with cellular effectors and act in tandem with other posttranslational modifications. The enzymes of O-GlcNAc cycling act preferentially on intrinsically disordered domains of target proteins impacting transcription, metabolism, apoptosis, organelle biogenesis, and transport.
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            Cardiac-specific overexpression of GLUT1 prevents the development of heart failure attributable to pressure overload in mice.

            Increased rates of glucose uptake and glycolysis have been repeatedly observed in cardiac hypertrophy and failure. Although these changes have been considered part of the fetal gene reactivation program, the functional significance of increased glucose utilization in hypertrophied and failing myocardium is poorly understood. We generated transgenic (TG) mice with cardiac-specific overexpression of insulin-independent glucose transporter GLUT1 to recapitulate the increases in basal glucose uptake rate observed in hypertrophied hearts. Isolated perfused TG hearts showed a greater rate of basal glucose uptake and glycolysis than hearts isolated from wild-type littermates, which persisted after pressure overload by ascending aortic constriction (AAC). The in vivo cardiac function in TG mice, assessed by echocardiography, was unaltered. When subjected to AAC, wild-type mice exhibited a progressive decline in left ventricular (LV) fractional shortening accompanied by ventricular dilation and decreased phosphocreatine to ATP ratio and reached a mortality rate of 40% at 8 weeks. In contrast, TG-AAC mice maintained LV function and phosphocreatine to ATP ratio and had <10% mortality. We found that increasing insulin-independent glucose uptake and glycolysis in adult hearts does not compromise cardiac function. Furthermore, we demonstrate that increasing glucose utilization in hypertrophied hearts protects against contractile dysfunction and LV dilation after chronic pressure overload.
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              Taurine depletion caused by knocking out the taurine transporter gene leads to cardiomyopathy with cardiac atrophy.

              The sulfur-containing beta-amino acid, taurine, is the most abundant free amino acid in cardiac and skeletal muscle. Although its physiological function has not been established, it is thought to play an important role in ion movement, calcium handling, osmoregulation and cytoprotection. To begin examining the physiological function of taurine, we generated taurine transporter- (TauT-) knockout mice (TauTKO), which exhibited a deficiency in myocardial and skeletal muscle taurine content compared with their wild-type littermates. The TauTKO heart underwent ventricular remodeling, characterized by reductions in ventricular wall thickness and cardiac atrophy accompanied with the smaller cardiomyocytes. Associated with the structural changes in the heart was a reduction in cardiac output and increased expression of heart cardiac failure (fetal) marker genes, such as ANP, BNP and beta-MHC. Moreover, ultrastructural damage to the myofilaments and mitochondria was observed. Further, the skeletal muscle of the TauTKO mice also exhibited decreased cell volume, structural defects and a reduction of exercise endurance capacity. Importantly, the expression of Hsp70, ATA2 and S100A4, which are upregulated by osmotic stress, was elevated in both heart and skeletal muscle of the TauTKO mice. Taurine depletion causes cardiomyocyte atrophy, mitochondrial and myofiber damage and cardiac dysfunction, effects likely related to the actions of taurine. Our data suggest that multiple actions of taurine, including osmoregulation, regulation of mitochondrial protein expression and inhibition of apoptosis, collectively ensure proper maintenance of cardiac and skeletal muscular structure and function.
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                Author and article information

                Contributors
                szervou@well.ox.ac.uk
                xiaoke.yin@kcl.ac.uk
                adam.a.nabeebacus@kcl.ac.uk
                brett.obrien@kcl.ac.uk
                rebeccalilliancross@gmail.com
                debra.mcandrew@well.ox.ac.uk
                andrew.atkinson@kcl.ac.uk
                thomas.eykyn@kcl.ac.uk
                manuel.mayr@kcl.ac.uk
                stefan.neubauer@cardiov.ox.ac.uk
                +44 1865 287603 , clygate@well.ox.ac.uk
                Journal
                Amino Acids
                Amino Acids
                Amino Acids
                Springer Vienna (Vienna )
                0939-4451
                1438-2199
                3 May 2016
                3 May 2016
                2016
                : 48
                : 1969-1981
                Affiliations
                [1 ]Division of Cardiovascular Medicine, Radcliffe Department of Medicine, and the BHF Centre of Research Excellence, University of Oxford, Oxford, UK
                [2 ]King’s British Heart Foundation Centre, King’s College London, London, UK
                [3 ]Division of Imaging Sciences and Biomedical Engineering, King’s College London, London, UK
                [4 ]Randall Division of Cell and Molecular Biophysics, and the BHF Centre of Research Excellence, Centre for Biomolecular Spectroscopy, King’s College London, London, UK
                Author notes

                Handling Editor: T. Wallimann and R. Harris.

                Article
                2236
                10.1007/s00726-016-2236-x
                4974297
                27143170
                8d3a87fd-a040-4b3b-a6de-a073db72e76e
                © The Author(s) 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

                History
                : 20 January 2016
                : 11 April 2016
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100000274, British Heart Foundation;
                Award ID: RG/13/8/30266
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100004440, Wellcome Trust;
                Award ID: RG/13/8/30266
                Funded by: BHF Centre of Research Excellence, Kings College London
                Award ID: RE/08/003
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100000327, Heart Research UK;
                Funded by: Department of Health via the National Institute for Health Research (NIHR)
                Funded by: FundRef http://dx.doi.org/10.13039/501100000289, Cancer Research UK;
                Funded by: FundRef http://dx.doi.org/10.13039/501100000266, Engineering and Physical Sciences Research Council;
                Funded by: Department of Health (England)
                Award ID: C1060/A10334
                Award Recipient :
                Funded by: Medical Research Council (UK)
                Categories
                Original Article
                Custom metadata
                © Springer-Verlag Wien 2016

                Genetics
                cardiac energetics,metabolism,creatine kinase,creatine transporter,transgenic mice
                Genetics
                cardiac energetics, metabolism, creatine kinase, creatine transporter, transgenic mice

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