Naked mole rats have distinctive cardiometabolic and genetic adaptations to their underground low-oxygen lifestyles (non-genetic data)

The naked mole-rat Heterocephalus glaber is a eusocial mammal exhibiting extreme longevity (37-year lifespan), extraordinary resistance to hypoxia and absence of cardiovascular disease. To identify the mechanisms behind these exceptional traits, metabolomics and RNAseq of cardiac tissue from naked mole-rats were compared to other African mole-rat genera. We identified metabolic and genetic adaptations unique to naked mole-rats including elevated glycogen, thus enabling glycolytic ATP generation during cardiac ischemia. Elevated normoxic expression of HIF-1α was observed while downstream hypoxia-responsive genes were down-regulated, suggesting adaptation to low-oxygen environments. Naked mole-rat hearts showed reduced succinate build-up during ischemia and negligible tissue damage following ischemia-reperfusion injury. These adaptive evolutionary traits reflect a unique hypoxic and eusocial lifestyle that collectively may contribute to their longevity and health span.

Methods
Langendorff Heart Perfusion
Beating hearts were excised from terminally anaesthetized naked mole rats (n=5) and C57/BL6 mice (n=5) (Charles River, UK) for Langendorff perfusions. After 20 minutes of equilibration, hearts were subject to 20 minutes of global normothermic ischemia. Hearts were snap frozen at the end of the protocol using a Wollenberger clamp pre-cooled in liquid N2. Ischemia/reperfusion (I/R) outcome in NMRs was compared to C57BL6 mouse heart due to their anatomical, physiological, and genetic similarity to humans. We were unable to repeat ex vivo cardiac perfusion protocols in all other 5 mole rat genera hearts as they are not kept in captive colonies in the UK. Myocardial infarct size quantification Myocardial infarct size was quantified. In brief, after 20 min of equilibration, Langendorff perfused hearts (n=3/ group) were subject to 20 of global normothermic ischemia and 2 hours of reperfusion. At the end of the protocol, hearts were perfused for 10 mins with 3% triphenyltetrazolium chloride (TTC) in KH Buffer followed by 10 min incubation in 3% TTC-KH. Tissue was sectioned (mouse heart gauge, Zivic instruments, USA) and infarct field was quantified using ImageJ Software.

NMR Spectroscopy 
1 H NMR Spectroscopy Powdered heart samples were subject to methanol / water / chloroform phase extraction. Frozen heart tissue was homogenised in 2 mL each of ice-cold methanol, chloroform, and Millipore water and vortexed. Samples were centrifuged for 1 hour at 3600 rpm at 4°C to separate aqueous, protein, and lipid layers. The upper aqueous phase was separated, 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 transferred to a fresh falcon tube containing 10 μL of universal pH indicator solution followed by vortexing and lyophilization. Dual-phase-extracted metabolites were reconstituted in 600 μL of 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 by titrating with 100 mM hydrochloric acid. 1H Nuclear magnetic resonance spectra were acquired using a vertical-bore, ultra-shielded Bruker 14.1. tesla (600 MHz) spectrometer with a bbo probe at 298K using the Bruker noesygppr1d pulse sequence. Acquisition parameters were 128 scans, 4 dummy scans and 20.8 ppm sweep width, acquisition time of 2.6s, pre-scan delay of 4s, 90° flip angle and experiment duration of 14.4 minutes per sample. TopSpin (version 4.0.5) software was used for data acquisition and for metabolite quantification. 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. Metabolite 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 and using Chenomx NMR Profiler Version 8.1 (Chenomx, Canada). Peak areas were normalized to the total metabolite peak area.

Western blotting 
Powdered heart samples were homogenised (100 µL of buffer per 10 mg of cardiac tissue) on ice in 100 mM Tris buffer (pH 7.4) supplemented with complete mini EDTA-free protease inhibitor (Roche) using a glass tissue grinder. The tissue homogenates were re-suspended in an equal volume of 2x reducing SDS sample buffer. Protein were resolved by SDS-PAGE (4 – 20 % Mini-PROTEAN TGX, Bio-Rad or Novex 4 – 20% Tris-Glycine, ThermoFisher Scientific) using Mini-Protean 3 system (Bio-Rad) or XCell4 SureLock Midi Cell and transferred using semi-dry system (Bio-Rad) to 22 μm PVDF or Nitrocellulose membranes (Bio-Rad). Following transfer, membranes were blocked for one hour with 5 % non-fat dried milk in TPBS at RT. After blocking, membranes were immunoprobed with primary antibodies dissolved in 5 % non-fat dried milk in TPBS overnight. Subsequently, membranes were washed three times with TPBS and incubated with IR-dye conjugated secondary antibodies in Intercept blocking buffer (LI-COR) or with horseradish peroxidase-coupled anti-rabbit IgG antibody in 5 % non-fat dried milk in TPBS for one hour at RT in dark. After the incubation, membranes were washed three times with TPBS and imaged using Odyssey DLx infrared imaging system (LI-COR) or ECL reagent (ThermoFisher Scientific) and imaging cabinet. Protein bands were analysed and quantified using Image Studio Lite (LI-COR) or ImageJ (NIH). List of vendors and dilution of antibodies used in this study: 

Anti HIF-1α Proteintech (#20960-1-AP); (Clone developed against protein sequence including amino acids 574 - 799 of the human HIF-1α protein; GenBank: BC012527.2) 1: 1000

Anti HIF-1α Abcam (#ab 179483); (Clone developed against recombinant fragment. This information is proprietary to Abcam and/or its suppliers) 1: 1000

Anti HIF-1α Novus lab (#NB100-479); (Clone developed against a fusion protein including amino acids 530 - 825 of the mouse HIF-1α protein; Uniprot #Q61221) 1: 1000

Anti β-Tubulin ThermoFisher Scientific (#62204) 1: 5000

Anti α-Actin Merck Millipore (#MAB1501) 1: 3000

Total OXPHOS Rodent WB Antibody Cocktail (#ab110413) 1: 1000

Statistical Analysis

Data are presented as mean ± SEM. Comparison between groups was performed by Student’s t-test (Gaussian data distribution), two-way analysis of variance (ANOVA) with Bonferroni’s correction for multiple comparison and one-way ANOVA using Bonferroni’s correction for multiple comparisons where applicable. Normality of data distribution was examined using Shapiro–Wilk’s normality test. Statistical analysis was performed using GraphPad Prism (v9) software. Unclassified principal component analysis (PCA) was performed using a PCA toolbox in Matlab. Data was autoscaled prior to PCA calculation using venetian blinds cross-validation and 5 cv groups. Classified discriminant analysis was performed using a classification toolbox in Matlab using linear discriminant analysis, bootstrap validation and 100 iterations. Hierarchical cluster analysis was performed in Matlab using the function clustergram. Differences were considered significant when P < 0.05.