10.5061/DRYAD.R2280GBB3
Aksentijevic, Dunja
0000-0002-8480-6727
King's College London
Karlstaedt, Anja
The University of Texas Health Science Center at Houston
Basalay, Marina
King's College London
O'Brien, Brett
King's College London
Sanchez-Tatay, David
King's College London
Eminaga, Seda
King's College London
Thakker, Alpesh
University of Birmingham
Tennant, Daniel
University of Birmingham
Fuller, William
University of Glasgow
Eykyn, Thomas
King's College London
Taegtmeyer, Heinrich
The University of Texas Health Science Center at Houston
Shattock, Michael
King's College London
Intracellular sodium elevation reprograms cardiac metabolism: Metabolomics data
Dryad
dataset
2020
British Heart Foundation
https://ror.org/02wdwnk04
RG/12/4/29426
2020-07-08T00:00:00Z
2020-07-08T00:00:00Z
en
51655 bytes
1
CC0 1.0 Universal (CC0 1.0) Public Domain Dedication
Intracellular Na elevation in the heart is a hallmark of pathologies where
both acute and chronic metabolic remodeling occurs. We assessed
whether acute (75μM ouabain 100nM blebbistatin) and chronic myocardial
Naiload (PLM3SA mouse) are causally linked to metabolic remodeling and
whether the hypertrophied failing heart shares a common Na-mediated
metabolic ‘fingerprint’. Control (PLMWT), transgenic (PLM3SA), ouabain
treated and hypertrophied Langendorff-perfused mouse hearts were studied
by 23Na, 31P, 13C NMR followed by 1H NMR metabolomic profiling. Elevated
Nai leads to common adaptive metabolic alterations preceding energetic
impairment: a switch from fatty acid to carbohydrate metabolism and
changes in steady-state metabolite concentrations (glycolytic,
anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca
exchanger by CGP37157 ameliorated the metabolic changes. In
silico modelling indicated altered metabolic fluxes (Krebs cycle, fatty
acid, carbohydrate, amino acid metabolism). Prevention of Nai overload or
inhibition of Na/Camitomay be a new approach to ameliorate metabolic
dysregulation in heart failure.
Frozen, weighed and pulverized hearts were subject to methanol/ water/
chloroform dual phase extraction adapted from Chung et al. (2017) The
upper aqueous phase was separated from the chloroform and protein
fractions. 20-30 mg chelex-100 was added to chelate paramagnetic ions,
vortexed and centrifuged at 3600 RPM for 1 minute at 4°C. The supernatant
was then added to a fresh Falcon tube containing 10 µL universal pH
indicator solution followed by vortexing and
lyophilisation. Dual-phase-extracted metabolites were reconstituted in 600
µL deuterium oxide (containing 8 g/L NaCl, 0.2 g/L KCl, 1.15 g/L Na2HPO4,
0.2 g/L KH2PO4 and 0.0075% w/v trimethylsilyl propanoic acid (TSP)) and
adjusted to pH ≈ 6.5 using 1 M hydrochloric acid and/or 1M sodium
hydroxide (<5 µL of each) prior to vortexing. The solution was
transferred to a 5 mm NMR tube (Norel Inc., USA) and then analysed using a
Bruker Avance III 400 MHz (9.4 T) wide-bore spectrometer (Bruker, Germany)
with a high-resolution broadband spectroscopy probe at 298 K. A NOESY 1D
pulse sequence was used with 128 scans, 2 dummy scans, total repetition
time 6.92 s, sweep width of 14 ppm and an acquisition duration of 15
minutes. Data were analysed using TopSpin software version 2.1 (Bruker,
Germany), FIDs were multiplied by a line broadening factor of 0.3 Hz and
Fourier-transformed, phase and automatic baseline-correction were applied.
Chemical shifts were normalised by setting the TSP signal to 0 ppm. Peaks
of interest were initially integrated automatically using a pre-written
integration region text file and then manually adjusted where
required. Assignment of metabolites to their respective peaks was carried
out based on previously obtained in-house data, confirmed by chemical
shift, NMR spectra of standards acquired under the same conditions
and confirmed using Chenomx NMR Profiler Version 8.1 (Chenomx,
Canada). Peak areas were normalized to the TSP peaks and metabolite
concentrations quantified per gram tissue wet weight. Intracellular
concentration of NADH, ATP+ADP, phosphocreatine, creatine, lactate,
succinate, fumarate, carnitine, phosphocholine, choline, acetyl carnitine,
acetate, aspartate, glutamine, glycine, alanine was analysed. The fold
change with respect to the control group was then calculated for each
metabolite. LC-MS/MS Lyophilised aqueous metabolite extracts were
reconstituted in 350µL ultrapure water (Millipore Corporation, USA). A
series of mixed standards were prepared in ultrapure water containing
0.0025-50 µM of each metabolite. The LC-MS/MS method was adapted from
Bylund et al (2007). An Agilent 1100 HPLC system (Agilent Technologies,
USA) consisting of an autosampler, a binary pump, a degasser unit and a
column oven coupled to an Applied Biosystems Sciex API 3000 mass
spectrometer with Turbo Ionspray interface (MDS Sciex, Canada).
Chromatograpic separation was achieved using a Supelcogel C610-H column
(300 mm x 7.7 mm) with a Supelcogel H guard column (50 mm x 4.6 mm)
(Supelco, USA) with an isocratic flow (0.4 mL/min) of mobile phase
consisting of 0.01% v/v formic acid and methanol (90:10) and an injection
volume of 100µL. The HPLC eluate was split (4:1) just before the Turbo
Ionspray interface resulting in a flow of 0.1 mL/min into the mass
spectrometer. The mass spectrometer acquisition parameters were exactly as
described in Bylund et al (2007). In order to eliminate peak to peak
interference, two separate acquisitions were performed for each sample and
standard. Acquisition 1 included α-ketoglutarate (145>101 m/z),
citrate (191>87 m/z), isocitrate (191>155 m/z), fumarate
(115>71 m/z) and lactate (89>43 m/z) whilst Acquisition 2
included pyruvate (87>43 m/z), malate (133>115 m/z) and
succinate (117>73 m/z). Data was acquired using Analyst software
(version 1.4.2) and metabolite concentrations in the samples were
interpolated using calibration curves of each metabolite. GC-MS/MS Polar
metabolites were extracted from the frozen pulverized cardiac tissue
(50mg) using the modified Folch method involving methanol water and
chloroform with some modifications. Namely, a 200 µl of ice-cold distilled
water with 1 mcg Norvalin as internal standard was added to the samples
and 1 hour sonication was performed in cold conditions. This was followed
by addition of 500 µl HPLC grade methanol (ice cold) to each samples with
1 hour sonication in ice cold conditions. Subsequently, the methanol:
water extract was transferred using glass Pasteur pipette to a new
labelled high grade Eppendorf tube and 500 µl chloroform was added to each
tube, vortexed for 1 minute followed by 15 minutes shaking on the shaker
at high speed. Subsequently, the Eppendorf tubes were centrifuged at 13000
rpm, 4 C for 15 minutes and the top polar layer was aspirated to a clean
Eppendorf tubes. The polar extract was dried using a speedvac and stored
in -80 freezer for subsequent derivatisation. Derivatization method All
derivatization steps were carried out in a fume hood. In order to
derivatize proteinogenic amino acids, organic acids and glycolytic
intermediates for GC-MS analysis, the dried extract was incubated at 95 °C
in open tubes in order to remove any residual moisture in the samples. The
dried extract was solubilized in 40 μl of 2 % methoxyamine HCL in pyridine
(Sigma-Aldrich, Dorset,UK) followed by 60 minutes incubation at 60°C and
subsequently 60 μl N-tertbutyldimethylsilyl-N-methyltrifluoroacetamide
(MTBSTFA) with 1% (w/v) tertbutyldimethyl-chlorosilane (TBDMSCI)
(Sigma-Aldrich, Dorset, UK) derivatization reagent was added. The
suspension was incubated for an hour at 60 °C in a well-sealed tube to
prevent evaporation. Finally the samples were centrifuged at 13000 rpm for
5 minutes and the clear supernatant was transferred to a chromatography
vial with a glass insert (Thermo Fisher, Scientific, Chromacol,
Hertfordshire, UK) and proceeded immediately to GC-MS analysis. GC-MS/MS
analysis For analysis of the derivatized samples an Agilent 7890B Series
GC/MSD gas chromatograph with a polydimethylsiloxane GC column coupled,
with a mass spectrometer (GC-MS) (Agilent Technologies UK Limited,
Stockport, UK) was used. Prior to sample analysis the GC-MS was tuned to a
full width at half maximum (FWHM) peak width of 0.60 a.m.u. in the mass
range of 50 to 650 mass to charge ratio (m/z) using PFTBA tuning
solution. 1 μl of sample was injected into the GC-MS in splitless mode
with helium carrier gas at a rate of 1.0 ml min-1. The inlet liner
containing glass wool was set to a temperature of 270 °C. Oven temperature
was set at 100 °C for 1 minute before ramping to 280 °C at a rate of 5 °C
min-1. Temperature was further ramped to 320 °C at a rate of 10 °C
min-1held at 320 °C for 5 minutes. Compound detection was carried out in
full scan mode in the mass range 50 to 650 m/z, with 2-4 scans sec-1, a
source temperature of 250 °C, a transfer line temperature of 280 °C and a
solvent delay time of 6.5 minutes. The injector needle was cleaned with
acetonitrile three times before measurement commencement and three times
following every measurement thereafter. The raw GC-MS data was converted
to common data format (CDF) using the acquisition software and further
processing of the isotope data including isotope correction and mass
isotopomer analysis /batch quantification was performed on metabolite
detector software. To determine absolute concentration, a 7 point
calibration series covering the mass range of 0 µM – 8.46 µM was prepared
in triplicates with 100 µl of 8.5 µM of internal standard added to each
sample of the calibration series and were extracted as the method outlined
above. The dried extract were then derivatised followed by GCMS analysis.
For absolute quantification, the ratio of peak area of each concentration
to the peak area of internal standard was calculated and plotted against
the ratio of the concentration of analyte with respect to the
concentration of internal standard to generate the equation and estimate
the linear dynamic range. Subsequently, the raw peak area for each analyte
of interest was calculated using the metabolite detector software followed
by normalizing the response to the internal standard peak area. Tissue
extraction for 13C NMR Frozen hearts were weighed and ground to a fine
powder under liquid nitrogen and extracted at 4°C with 6% perchloric acid
(PCA) in a ration 5:1. The suspension was centrifuged at 4000 RPM, 4°C for
10min and a known volume of supernatant decanted and neutralised with 6M
KOH to pH7.0 at 4°C. The mixture was centrifuged and the supernatant
lyophilised at -40°C. Lyophilised tissue extracts were reconstituted in
0.6ml of 50mM deuterated phosphate (KH2PO4) buffer pH 7.0 lyophilised and
resuspended in D2O. A small amount of chelating resin (Chelex-100) was
added to samples to remove any paramagnetic ions and filtered through a
0.22 mm syringe filter into 3mm NMR tube. High resolution 13C NMR of
tissue extracts High-resolution 1H-decoupled 13C NMR spectra were
acquired under automation at 298K on a Bruker Avance III 700 (16.4 T) NMR
spectrometer (Bruker Biospin, Coventry, UK) equipped with a 5 mm TCI
helium-cooled cryoprobe and a refrigerated SampleJet sample changer. The
temperature was allowed to stabilise for 3 min after insertion into the
magnet. Tuning, matching and shimming was performed automatically for each
sample and the 1H pulse length was calibrated on each sample and was
typically around 8 µs. 1D 1H-decoupled 13C spectra (zgpg60) were acquired
with 8192 transients, a spectral width of 200 ppm, 64K data points, a
mixing time of 10 ms, relaxation delay of 1 s and repetition time of 2s.
1H-decoupling was achieved using a WALTZ65 sequence during the relaxation
delay and acquisition. Spectra were processed in the manufacturer’s
software (Topspin 3.2.6). Free induction decays were multiplied with an
exponential function (line broadening of 0.25 Hz), Fourier transformed,
phase correction was performed manually and automatic baseline correction
was applied. Representative spectra are shown in Figure 1B. The relative
contributions of exogenous 13C substrates (palmitate vs glucose) to
oxidative phosphorylation were determined from 13C glutamate isotopomer
labelling patterns (Figure 1B) using tcaCALCtm software (v2.07).