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

Creatine: a miserable life without it

“…In a parallel metabolomics analysis using 1 H-NMR spectroscopy, significant changes in a number of key metabolites were also detected that implied impairment of glycolysis, fatty acid oxidation and the TCA cycle, resulting in a substraterich but energy-poor heart. These results revealed hitherto unsuspected feedback mechanisms between creatine content and many key metabolites in the heart (Zervou et al 2016) that argue for a pleiotropic action of creatine, in addition to those effects already known (Wallimann et al 2011). Such sophisticated combined proteomic and metabolomics approaches are likely to add yet another dimension to the fields of CK and creatine research, as also exemplified by Stockebrand et al (2016).…”

“…The transgenic mouse lacking AGAT, the enzyme for the first step of endogenous creatine synthesis, is a true creatine-deficient mutant, provided that this animal is not receiving any alimentary creatine that can be adsorbed via the creatine transporter (CrT) by the intestine. This is in contrast to the GAMT knockout counterpart (Zervou et al 2016), which is able to synthesize guanidinoacetate (GAA). This GAA may then be phosphorylated to produce energy-rich P-GAA, which in turn can be used as a substrate by CK instead of PCr (Heerschap et al 2007).…”

“…Thus, the results described above do not justify the conclusions taken by Lygate et al (2013), specifically in light of the fact that reduced PCr concentrations and PCr/ATP ratios, as well as a reduced flux through the CK reaction, have been consistently seen in human myocardial infarction (Bottomley et al 2009). Zervou et al (2016) show in an elegant study that mice over-expressing the creatine transporter (CrT), and thus displaying elevated cardiac total creatine levels, were protected against ischemia/reperfusion injury via improved energy reserves. However, mice with very high cardiac creatine concentrations developed more or less severe cardiac hypertrophy and dysfunction.…”

“…Again, obviously, the CrT knockout phenotype is not lethal, but its efficiency of energy utilization is severely impaired, even though this may be mitigated by compensatory metabolic changes, as for example also seen in the AGAT and GAMT knockout mice (see Stockebrand et al 2016;Zervou et al 2016), as well as in the CK knockouts (for review see Wallimann et al 2011). It is to be expected that in the near future, a "true creatine knockout mouse" will be generated.…”

The creatine kinase system as a therapeutic target for myocardial ischaemia–reperfusion injury

“…One plausible explanation is that CK activity is insufficient to adequately phosphorylate the enlarged creatine pool, resulting in 2-fold higher ADP and reduced |Δ G ATP |, closely mimicking the impaired bioenergetics observed in heart failure models [ 44 – 46 ]. Further studies, utilising metabolomics and proteomics approaches, showed that excessive creatine has wide-ranging effects on cellular energy metabolism, including reduced glycolytic function [ 47 , 48 ].…”