DOI: 10.5061/dryad.w9ghx3fts
title: Naked mole rats have distinctive cardiometabolic and genetic adaptations to their underground low-oxygen lifestyles (non-genetic data)

description: 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.
description: 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 N<sub>2</sub>.
 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 &lt;
 0.05. 
description: # Naked mole rats have distinctive cardiometabolic and genetic adaptations
 to their underground low-oxygen lifestyles (non-genetic data) Experiments
 were carried out on heart tissue collected from naked mole rat
 (Heterocephalus glaber) and from seven other members of African mole rat
 genera, Cape mole-rat (Georychus capensis), Cape dune mole-rat (Bathyergus
 suillus), Common mole-rat (Cryptomys hottentotus hottentotus), Natal
 mole-rat (C. h. natalenesis), Mahali mole rat (C. h. mahali), Highveld
 mole-rat (C. h. pretoriae) and Damaraland mole-rats (Fukomys damarensis)
 representing differing burrow and soil types, degrees of sociality,
 lifespan and hypoxia tolerance. Tissue was also collected from C57/Bl6
 mouse heart. We carried out the study to understand what adaptation exist
 in the hearts of naked mole rats versus all the others so they are
 resistant to heart disease including heart attacks. ### Description of the
 Data and file structure Raw data is provided in an Excel table: 1H NMR
 metabolomic quantification of metabolites in normoxia and post-20-min
 ischemia, ex vivo heart function, infarct size quantification ( area of
 scar), western blotting for HIF1 alpha, components of mitochondrial
 electron transport chain. Tabs in Excel file are labelled with exactly
 what data is from what experiment. Experimental details are given in the
 metadata of this dataset. All rows and columns are labelled with sample
 legend and biochemical parameters that were assessed. Data was not derived
 from any other sources. Units: * 1H metabolomics data -normalised peak
 area arbitrary units * Ex vivo cardiac function: Left Ventricular
 Developed Pressure (LVDP) -mmHg, Heart Rate (HR)-beats per minute *
 Infarct size % of total cardiac area * Effluent lactate- ng/microlitre *
 Effluent succinate - normalised peak area (arbitrary units) * Plasma
 biochemistry: glucose (mM), insulin ( picogram/ml) triglycerides (mmol/L),
 lactate dehydrogenase (U/L) * Western blotting: normalised intensity
 (arbitrary units)