title: Proteomic and metabolomic changes driven by elevating myocardial creatine suggest novel metabolic feedback mechanisms.
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.
authors.affiliation: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, and the BHF Centre of Research Excellence, University of Oxford, Oxford, UK.
authors.forename: Sevasti
authors.initials: S
authors.lastname: Zervou
authors.affiliation: King's British Heart Foundation Centre, King's College London, London, UK.
authors.forename: Xiaoke
authors.initials: X
authors.lastname: Yin
authors.affiliation: King's British Heart Foundation Centre, King's College London, London, UK.
authors.forename: Adam A
authors.initials: AA
authors.lastname: Nabeebaccus
authors.affiliation: Division of Imaging Sciences and Biomedical Engineering, King's College London, London, UK.
authors.forename: Brett A
authors.initials: BA
authors.lastname: O'Brien
authors.affiliation: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, and the BHF Centre of Research Excellence, University of Oxford, Oxford, UK.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Cross
authors.affiliation: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, and the BHF Centre of Research Excellence, University of Oxford, Oxford, UK.
authors.forename: Debra J
authors.initials: DJ
authors.lastname: McAndrew
authors.affiliation: Randall Division of Cell and Molecular Biophysics, and the BHF Centre of Research Excellence, Centre for Biomolecular Spectroscopy, King's College London, London, UK.
authors.forename: R Andrew
authors.initials: RA
authors.lastname: Atkinson
authors.affiliation: Division of Imaging Sciences and Biomedical Engineering, King's College London, London, UK.
authors.forename: Thomas R
authors.initials: TR
authors.lastname: Eykyn
authors.affiliation: King's British Heart Foundation Centre, King's College London, London, UK.
authors.forename: Manuel
authors.initials: M
authors.lastname: Mayr
authors.affiliation: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, and the BHF Centre of Research Excellence, University of Oxford, Oxford, UK.
authors.forename: Stefan
authors.initials: S
authors.lastname: Neubauer
authors.affiliation: Division of Cardiovascular Medicine, Radcliffe Department of Medicine, and the BHF Centre of Research Excellence, University of Oxford, Oxford, UK. clygate@well.ox.ac.uk.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Lygate
mesh_terms: D000818:Animals; D003401:Creatine / Q000378:metabolism*; D004734:Energy Metabolism; D005786:Gene Expression Regulation; D008297:Male; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000235:genetics; D055432:Metabolomics; D051379:Mice; D008822:Mice, Transgenic; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics; D009200:Myocardial Contraction; D015428:Myocardial Reperfusion Injury / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D009206:Myocardium; D040901:Proteomics; D011817:Rabbits
chemical_list: D026901:Membrane Transport Proteins; D009124:Muscle Proteins; C080679:creatine transporter; D003401:Creatine
keywords: Cardiac energetics; Creatine kinase; Creatine transporter; Metabolism; Transgenic mice