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

Sevasti Zervou; Xiaoke Yin; Adam A. Nabeebaccus; Brett O'Brien; Rebecca L. Cross; Debra J. McAndrew; Andrew Atkinson; Thomas Eykyn; Manuel Mayr; Stefan Neubauer; Craig A. Lygate

doi:10.1007/s00726-016-2236-x

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

Sevasti Zervou, Xiaoke Yin, Adam A. Nabeebaccus, Brett O'Brien, Rebecca L. Cross, Debra J. McAndrew, Andrew Atkinson, Thomas Eykyn, Manuel Mayr, Stefan Neubauer, Craig A. Lygate^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

17 Citations (Scopus)

192 Downloads (Pure)

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.

Original language	English
Pages (from-to)	1-13
Number of pages	13
Journal	AMINO ACIDS
Early online date	3 May 2016
DOIs	https://doi.org/10.1007/s00726-016-2236-x
Publication status	E-pub ahead of print - 3 May 2016

Keywords

Cardiac energetics
Creatine kinase
Creatine transporter
Metabolism
Transgenic mice

Access to Document

10.1007/s00726-016-2236-xLicence: CC BY

Proteomicandmetabolomicchanges_ZervouAccepted11April2016_GOLDFPVFinal published version, 1.8 MBLicence: CC BY

Cite this

@article{79d9e1cdc8bf4d3fbe11b746c5b17923,

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.",

keywords = "Cardiac energetics, Creatine kinase, Creatine transporter, Metabolism, Transgenic mice",

author = "Sevasti Zervou and Xiaoke Yin and {A. Nabeebaccus}, Adam and Brett O'Brien and {L. Cross}, Rebecca and McAndrew, {Debra J.} and Andrew Atkinson and Thomas Eykyn and Manuel Mayr and Stefan Neubauer and Lygate, {Craig A.}",

year = "2016",

month = may,

day = "3",

doi = "10.1007/s00726-016-2236-x",

language = "English",

pages = "1--13",

journal = "AMINO ACIDS",

issn = "0939-4451",

publisher = "Springer Wien",

}

TY - JOUR

T1 - Proteomic and metabolomic changes driven by elevating myocardial creatine suggest novel metabolic feedback mechanisms

AU - Zervou, Sevasti

AU - Yin, Xiaoke

AU - A. Nabeebaccus, Adam

AU - O'Brien, Brett

AU - L. Cross, Rebecca

AU - McAndrew, Debra J.

AU - Atkinson, Andrew

AU - Eykyn, Thomas

AU - Mayr, Manuel

AU - Neubauer, Stefan

AU - Lygate, Craig A.

PY - 2016/5/3

Y1 - 2016/5/3

N2 - 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.

AB - 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.

KW - Cardiac energetics

KW - Creatine kinase

KW - Creatine transporter

KW - Metabolism

KW - Transgenic mice

UR - http://www.scopus.com/inward/record.url?scp=84965062085&partnerID=8YFLogxK

U2 - 10.1007/s00726-016-2236-x

DO - 10.1007/s00726-016-2236-x

M3 - Article

AN - SCOPUS:84965062085

SN - 0939-4451

SP - 1

EP - 13

JO - AMINO ACIDS

JF - AMINO ACIDS

ER -

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

Abstract

Keywords

Access to Document

Other files and links

Fingerprint

Cite this