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 Na_iload (PLM^3SA mouse) are causally linked to metabolic remodeling and whether the hypertrophied failing heart shares a common Na-mediated metabolic ‘fingerprint’. Control (PLM^WT), transgenic (PLM^3SA), ouabain treated and hypertrophied Langendorff-perfused mouse hearts were studied by ²³Na, ³¹P, ¹³C NMR followed by ¹H NMR metabolomic profiling. Elevated Na_i 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 Na_i overload or inhibition of Na/Ca_mitomay 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 tcaCALC^tm software (v2.07).