Approval for animal experiments
All procedures for animal care and experimentation were performed according to protocol HS-2847, which was approved by the University of Ottawa Animal Care Committee, and following guidelines provided by the Canadian Council on Animal Care (CCAC). In addition, these procedures followed the guidelines provided by ARRIVE (Animals in Research: Reporting In Vivo Experiments).
Animal breeding
The Institute Clinique de la Souris (ICS) generated a Mrpl54 constitutive knock-down mouse model on a C57BL/6N(Taconic) background (C57BL/6NTac-Mrpl54tm1b(EUCOMM)Wtsi/IcsOrl; colony name ICS-EPD1076_5_A09)48,49. Mrpl54+/- breeding pairs were kept in ventilated racks and their progeny were housed in groups of 2-5 mice until their transfer to the metabolic health and longevity studies. Litter sizes were small (mean 4-5 pups) and production dropped off after the fourth litter. To generate the cohorts needed, CD1 host mothers were used for three rounds of in vitro fertilization, in addition to traditional breeding. At age 21-days, animals were weaned, ear-tagged, and tissue sampled from the ear for genotyping.
Genotyping
DNA for genotyping was extracted from the ear tissue sample according to the Phire Animal Direct PCR protocol (F-170, Thermo Scientific). Genotyping proceeded according to the ICS protocol (Forward primer Ef 4877 5’-GACCCACATAAGCAGGGAAGGAGATG-3’, reverse primer L3r 4879 5’-CAATCTCCTGAGAATGTAGCCCACCAT-3’, Invitrogen). The Mrpl54 knock-out allele generated a 402 base-pair (bp) fragment, and the WT allele generated a 1095 bp fragment.
Animal husbandry
WT and Mrpl54+/- animals, separated by sex, were housed under a 12:12-hour light-dark cycle in a dedicated room set to 23°C. All mice were fed a standard chow diet (Envigo; Teklad Global 2018; 18.6% crude protein, 6.2% fat, 44.2% available carbohydrates). Female and male mice were initially housed 5-10 per cage in a large rat cage (at least 100cm2 per mouse) to reduce stress50. Male cages experienced aggression and fighting and the male mice in cages that experienced injuries or deaths due to male-on-male aggression were reassigned to individual-housing. Animals in the metabolic health study were handled or weighed at least once per week. Animals in the longevity study were weighed once per month until age-related weight loss was observed, after which mice were weighed weekly or daily as needed.
RNA isolation and Real-time qPCR
For total RNA isolation, a small piece of snap frozen mouse tissue was placed in 1ml TRIzol (Sigma-Aldrich or Invitrogen). Samples were homogenized with a 5 mm steel bead using a TissueLyser II (Qiagen) for 3 min at a frequency of 30 Hz. RNA was isolated according to manufacturer’s protocol. From 1 μg of extracted RNA, genomic DNA was eliminated with a gDNA Wipeout Buffer and subsequently reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). The diluted cDNA was prepared with SYBR Green (Roche), and the quantitative gene expression was measured using the LightCycler 480 Instrument (Roche). The primer pairs used were as follows: Mrpl54 forward 5’-AAAAAGCCAGTTGGCAAGGG-3’, reverse 5’-ATGTGTGGTGAGCTGAGTGG-3’; 36B4 (acidic ribosomal phosphoprotein gene) forward 5′-ACGGGTACAAACGAGTCCTG-3′, reverse 5′-GCCTTGACCTTTTCAGCAAG-3′; and B2m (beta2 microglobulin) forward 5′-GGCTCACACTGAATTCACCC-3, 5′-GTCTCGATCCCAGTAGACGG-3′. To normalize tissue sample expression (threshold cycle [Ct] values) to the expression of housekeeping genes, the delta-delta Ct method was used. The geometric means of 36B4 and B2m genes were used to normalize Mrpl54 tissue sample expression (delta Ct values) to the WT samples.
Metabolic Health Analyses
The metabolic health of male and female WT and Mrpl54+/- mice (n=7-17 in each group) were tested in three separate cohorts at 6-, 18-, and 24-months of age. The battery of tests started with EchoMRI body composition (EchoMRI-700 Analyzer), followed by indirect calorimetry using the comprehensive laboratory animal monitoring system (CLAMS, Columbus Instruments), oral glucose tolerance, intraperitoneal insulin tolerance, endurance treadmill, cold tolerance, heart rate and blood pressure measurement (at 18- and 24-months), and then necropsy (Supplemental Fig. S1). Mice were allowed to recover 1-2 weeks between tests.
EchoMRI and Indirect Calorimetry
Each mouse was loaded into an A100 antenna insert and placed into the EchoMRI (EchoMRI-700 Analyzer) to determine body composition (whole-body, lean, and fat mass). Immediately afterward, mice were placed in individual CLAMS (Columbus Instruments Oxymax System) plexiglass cages for 48-72 hours. The ambient temperature was set to thermoneutrality (28°C), and the light-dark cycle was 12:12. Mice had ad libitum access to water and powdered chow (Teklad 2018 Diet). At regular intervals (every 18-26 min), the following measurements were recorded: O2 volume (VO2), CO2 volume (VCO2), respiratory exchange ratio (RER), ambulatory and rearing motion, and the amount of food consumed in grams.
Oral Glucose Tolerance Test (OGTT)
Mice were fasted overnight for 16-hours prior to the OGTT. Whole-blood glucose levels were measured using the Accu-check Performa glucometer and accompanying strips (Roche Diagnostics, Canada). For the 6M cohort, mice were placed in a restraint tube during the blood sampling from the left lateral tail vein. For the 18M and 24M cohorts, mice remained unrestrained on the home-cage hopper during blood sampling. At time 0, freshly made 20% α-D-glucose (Sigma-Aldrich PN158968) was orally administered using a flexible straight metal gavage at a dose of 1.0g per kg of body mass. Blood glucose was measured at baseline (prior to oral glucose dose), and at 15-, 30-, 45-, 60-, 90-, 120-, 150-, and 180-min post-gavage.
Intraperitoneal Insulin Tolerance Test (iITT)
Two weeks prior to the iITT, intraperitoneal insulin titrations were conducted on a subset of mice to determine the correct dose to elicit an insulin response. Mice were fasted in the morning for 5-6 hours prior to the iITT. Whole-blood glucose levels were measured using the Accu-check Performa glucometer and accompanying strips (Roche Diagnostics, Canada). During blood sampling from the right lateral tail vein, mice remained unrestrained on their home-cage hopper. At time 0, mice received an intraperitoneal injection of insulin (NovoRapid Insulin aspart, 100U/mL, DIN 02245397). For the 6M cohort, females received an insulin dose of 1.5 U/kg body mass and males received 2.5 U/kg. For the 18M cohort, females received an insulin dose of 2 U/kg and males received 4.5 U/kg. For the 24M cohort, females received an insulin dose of 1 U/kg and males received 2 U/kg. Blood glucose was measured at baseline (prior to insulin injection), and at 15-, 30-, 45-, 60-, 75-, 90-, 105-, and 120-min post-injection. In the event the blood glucose level dropped below 2.0 mmol/L, emergency α-D-glucose (Sigma-Aldrich PN 158968) was orally administered at a dose of 1 g per kg of body mass.
Treadmill
Mice were acclimated to the treadmill (Exer-3/6, Columbus Instruments) over three consecutive days where they were placed on an unmoving belt for 5 min, then ran at a constant speed of 15 cm/s at an elevation of +5° with an electro-stimulus (0.1 mA, 1 Hz) applied to the resting grid. Those mice that achieved fewer than 5 electro-stimulations over 5 min by the third acclimation day were permitted to continue with the endurance test. The endurance test was conducted the day following the last day of acclimation using the following profile – accelerate to 15 cm/s in 30 sec and then run at 15 cm/s for 12 min, accelerating +3.0 cm/s over 30 sec every 12 min51. Mice ran until the exhaustion criterion was met (5 electro-stimulus contacts [0.1 mA, 1 Hz] within 5 sec) at which point the electro-stimulus was stopped, and the running time and maximum speed attained were recorded
Cold Tolerance
The baseline rectal temperature of each mouse was measured using a lubricated rectal thermometer at an insertion depth ~2 cm. Mice were placed in individual cages with access to fresh water and a plastic house, but without access to bedding or food, and then placed in a +4° C refrigerated room with all cages at the same height. Rectal temperature was measured every hour for up to 4-6 hours. If rectal temperature reached 25° C or below52, the mouse was removed from the cold room and placed in a 37° C incubator with access to food and water.
Heart Rate & Blood Pressure
Mice in the 18M and 24M cohorts were acclimated to the blood pressure and heart rate monitoring equipment (BP-2000-M-6 Series II Blood Pressure Analysis System, Visitech Systems) over a period of 3-5 days53. The procedure for acclimation and testing was the same – mice were individually placed in an opaque mouse holder on a warmed 36° C plate with the tail pulled through an inflatable tail cuff. Mice rested in position for 5 min, followed by 5 preliminary readings and then 10 actual readings. The program parameters were as follows: 2.5-sec pause between readings, 10-sec analysis pulse, system maximum set to 170 mmHg. Test day immediately followed the last day of successful acclimation, where blood pressure was recorded for at least 6 out of 10 readings. The outcomes recorded were diastolic blood pressure, systolic blood pressure, and heart rate.
EchoMRI & Necropsy
The day before the necropsy, body composition was determined using the EchoMRI-700 as described above. The mice were fasted overnight for 10-12 hours and then refed 1-2 hours before the necropsy. Mice were euthanized through injection with ketamine/xylazine cocktail (0.1 mg/kg body weight) followed by exsanguination through cardiac puncture. The following tissues and organs were collected, weighed, fast-frozen in liquid N2 and then stored at -80° C for further analysis: pancreas, spleen, diaphragm, heart, liver, kidneys, quadriceps, gastrocnemius, soleus, tibialis anterior, and brain54. For the 24-month cohort necropsy, the right kidney, quadriceps, and gastrocnemius/soleus were suspended in Optimal Cutting Temperature fixative (Fisher Health Care, Cat# 4585) and placed on dry ice (kidney) or in isopentane (Sigma, CAT# 1003301470) supercooled in liquid N2 (muscles), then stored at -80°C for further analysis.
Longevity Study and Growth
Male and female WT and Mrpl54+/- mice (n=80-112 per group) were initially group-housed 5-10 per cage (at least 100 cm2 per mouse). Male cages experienced aggression and fighting from age 3-months onward. In case of a fight-related injury or death, the males were reassigned to individual cages to improve animal welfare. Mice were weighed monthly, and median life expectancy (50% survival time point), 24-month life expectancy (all mice alive at 24 months were censored), and maximum lifespan (lifespan of top 10% of surviving animals) were calculated. Where a human-endpoint (HEP) was identified and time-permitting, mice were necropsied to identify tumour burden and to collect tissue and plasma for future analysis. HEPs included (a) >15% weight loss over 2 weeks, (b) skin lesions not responding to treatment, (c) head tilt/rolling or loss of balance, (d) ulcerated or bulging eye, (e) ulcerated mass, (f) respiratory distress, (g) inability to express bladder, or (h) a distended abdomen or growing internal mass50. Mice that were euthanized because of ulcerative dermatitis that did not respond to treatment (a C57BL/6 strain-specific condition55), or because of an unacceptable pain level from an ulcerated or bulging eye, were censored from the longevity analysis.
To assess growth over time, the body mass of WT and Mrpl54+/- male and female animals were weighed monthly. Animals destined for the 6-, 18-, and 24-month metabolic health analyses were included in the growth analysis, up until the month they entered the metabolic health study.
Respiration of Isolated Liver Mitochondria
7-week-old male WT and Mrpl54+/- mice were fasted overnight for 12 hours, killed by cervical dislocation, and their livers weighed then rapidly dissected directly into ice-cold isolation buffer (300 mM D-sucrose, 10 mM Tris-HCl, 1 mM EGTA-Tris Base; pH 7.2)56. The suspension was homogenized in a 15 mL Potter-Elvehjem homogenizer, and the supernatant centrifuged twice at 1000g for 10 min at 4° C (Sorvall RC-6 Plus Centrifuge, Thermo Scientific). The supernatant was centrifuged 8000g for 10 min at 4° C and then aspirated without disturbing the pellet. The pellet was gently resuspended in ice-cold suspension buffer (300 mM D-sucrose, 10 mM Tris-HCl, 0.05 mM EGTA-Tris Base; pH 7.2) and centrifuged at 8000g for 10 min at 4° C. The final pellet was gently re-suspended in 300 µL suspension buffer and kept on ice.
The respiratory function of isolated liver mitochondria was measured using a Clark-type electrode (Oxygraph System, Hansatech Instruments Ltd.). The chambers were calibrated at 23° C with continuous stirring with a PTFE-coated magnetic follower bar. The mitochondrial suspension was quantified (Bio-Rad DC Protein Assay, BMG Labtech POLARstar Omega plate reader) and then suspended in miR05 respiration buffer (110 mM D-sucrose, 60 mM lactobionic acid, 20 mM taurine, 20 mM HEPES, 10 mM KH2PO4, 3 mM MgCl2, 0.5 mM EGTA, 1 g/L fatty-acid free BSA; pH 7.1 at 23° C)57 at 0.5 mg protein/mL. Following a baseline recording, O2 consumption rate was measured following sequential additions of: 1) glutamate + malate (G/M, 5:2.5 mM) to give Complex I (CI)-driven State 2 respiration, 2) ADP (2 mM) to give CI-driven State 3 respiration, 3) Amytal (2 mM) to inhibit CI, 4) succinate (10 mM) to give Complex II (CII)-driven State 3 respiration, 5) carbonyl cyanide m-chlorophenylhydrazone (CCCP) titrations to decouple OXPHOS until maximum respiration was reached (0.01-0.05 µM); 6) antimycin-A (8 µM) to inhibit Complex III (CIII); 7) N,N,N',N'-Tetramethyl-p-phenylenediamine dihydrochloride (TMPD) + ascorbate (5:0.3 mM) to test Complex IV (CIV) respiration; and finally 8) KCN (0.6 mM) to inhibit CIV.
Mass Spectrometry (MS)-based Proteomics
Crude mitochondria were isolated from the livers of WT (n=3) and Mrpl54+/- (n=3) male mice, as described above, and then purified using 30% Percoll gradient ultracentrifugation at 95,000g for 30 min at +4° C (Beckman sv41 rotor). Purified mitochondria were treated 1:200 with a protease inhibitor cocktail (Thermo Fisher Scientific HALT PI) then the mitochondrial proteins were left to precipitate overnight in precipitation buffer (50% acetone, 49.9% ethanol, 0.1% acetic acid). Proteins were suspended in 50 mM ammonium bicarbonate in 8 M urea and then quantified (Bio-Rad DC Protein Assay, BMG Labtech POLARstar Omega plate reader). To reduce disulphide bonds, 140 µg of mitochondrial protein was incubated with 1:100 of 1 M dithiothreitol (Sigma-Aldrich, SKU 43815), followed by 1:50 dark incubation in 1 M iodoacetamide (Sigma-Aldrich, SKU I1149). The urea concentration was reduced to <2 M and the protein sample was digested overnight at 37° C with 1:100 Trypsin Gold (Promega V5280). The following day, the digested proteins were passed through a Sep-Pak Vac 1 cc tC18 cartridge (Waters, WAT054960) to rid the sample of salts and contaminants. The sample was dried in a Savant DNA120 SpeedVac Concentrator (Thermo Electron Corporation) at room temperature and analysed using a nano-LC-MS/MS with an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) coupled to an ultraperformance LC system (Ultimate 3000 RSLC; Thermo Fisher Scientific). Data analysis was performed with Proteome Discoverer (version 1.3), and searches were performed with Mascot and Sequest against a mouse database (UniProt). Data were further processed, inspected, and visualized with Scaffold 4 (Proteome Software).
Generation of Primary Myoblasts
Hind limb skeletal muscles were excised from euthanized 7-week-old male WT (n=3) and Mrpl54+/- (n=3) mice. Under aseptic conditions, the muscles were rinsed in phosphate buffered solution (PBS, Wisent Inc., Cat# 311-010-CL), the fat excised, and the remaining muscle tissue placed in collagenase B-dispase solution, 1.5 mL per 0.5 g tissue (4 mg/mL dispase II [Sigma D4693], 10 mg/mL collagenase B [Roche 11088831001] in Hams-F10 [Multicell, Wisent Inc. Cat# 318 050-CL]). The tissue was mulched with a razor blade, incubated at 37° C for 30 min, and then homogenized through trituration until smooth. The homogenate was passed through a 100 micron cell strainer with PBS washes. The muscle cells were centrifuged at 300g for 5 min and the cell pellet resuspended in Ham’s Complete growth medium (4% bovine calf serum [cytiva, Cat# SH30077.03], 1% penicillin and streptomycin [gibco by Life Technologies, Cat# 15140122], 2.5 ng/µL human basic fibroblast growth factor [bFGF, Stemcell, Cat# 78003]). To rid the culture of fibroblasts, the cells were allowed to rest on a non-collagen-coated plate for two hours at 37° C before plating onto a 10 cm collagen-coated plate. To enrich myoblasts, 80% confluent plates were pre-plated on non-collagen-coated plates for one hour before plating on a collagen-coated plate.
Western Blotting
Protein was extracted by suspending isolated WT and Mrpl54+/- liver mitochondria or primary myoblast cells (10 cm plates at 80% confluence) in radioimmunoprecipitation (RIPA) lysis buffer (50 mM Tris HCl, 150 mM NaCl, 1% v/v Triton X-100, 0.5% w/v sodium deoxycholate, 0.1% w/v SDS; pH 8.0) augmented with protease and phosphatase inhibitors (1:100 HALT PI cocktail, Thermofisher Sci Cat# 78440; or Roche cOmplete ULTRA [Cat# 05892970001] plus phosStop [Cat# 4906845001] tablets), then flash frozen in liquid N2. The solution was centrifuged 16,000g for 10 min at 4°C and the supernatant containing the mitochondrial or cell lysate proteins quantified (Bio-Rad DC Protein Assay, BMG Labtech POLARstar Omega plate reader).
Protein samples were diluted to 1 µg/µL in Laemmli Buffer (Bio-Rad Cat# 1610737) augmented with 10% 𝛽-mercaptoethanol (Fisher Bioreagents, BP176-100) and run on a 10% SDS-PAGE using the TGX Stain-free FastCast Acrylamide Kit (Bio-Rad Cat# 1610183) and the Mini-PROTEAN system (Bio-Rad Cat# 1658006). SDS-PAGE was run at a constant 90 volts across the gel for 30 min and then 120 volts for 50-60 min in a 4° C cold room. The StainFree gels were activated once for 45 sec using a ChemiDoc Touch Imaging System (Bio-Rad) and then the proteins transferred to TransBlot Turbo Mini size 0.2 µm nitrocellulose membrane (Bio-Rad) using the Trans-Blot TurboTransfer System (Bio-Rad Cat# 1704150EDU).
The nitrocellulose membrane was imaged for total protein and then blocked for one hour in blocking buffer (5% w/v bovine serum albumin [Sigma, SKU A7906] in TBS-T buffer [50 mM Tris-HCl, pH 7.6; 150 mM NaCl; 0.1% Tween]), rocking gently at room temperature. Membranes were then incubated with primary antibody diluted in TBS-T buffer (1:1000 Total OXPHOS Rodent WB Antibody Cocktail [abcam Cat# ab110413] or 1:1000 HSP60 XP Rabbit mAB [Cell Signaling Technology CAT#12165]) overnight at 4°C with gentle rocking. After three washes with TBS-T, the membrane was incubated for one hour with the matching IgG, horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000 Cell Signaling Technology Anti-rodent Cat# 7076, or Anti-rabbit Cat# 7074P2), rocking at room temperature. After three washes in TBS-T, the membrane was visualized using enhanced chemiluminescent detection (BioRad Clarity Cat# 1705061) on the ChemiDoc Touch Imaging System (Bio-Rad).
Statistical Analysis
All data are presented as mean ± SEM, unless otherwise stated. Differences between two groups were assessed using two-tailed t-tests. Analysis of covariance (ANCOVA) was used to eliminate unwanted variance on the dependent variable. To compare the interaction between time and treatment, a two-way analysis of variance (ANOVA) tests was performed. Survival curves were analysed using the Kaplan-Meier method. Hazard ratios were calculated using the Log rank test. GraphPad Prism 9.4.1 (GraphPad Software, Inc.) was used for all statistical analyses, and p<0.05 was considered significant.