Mice –Transgenic model:
Specific skeletal muscle TRF2 KO mice (HSA-Cre+ / - ;Terf2flox/loxf) were generated as previously described 4. First, the previously characterized TRF2 floxed mice from the Jackson laboratory (strain B6; 129P2-Terf2tm1Tdl/J) 10 were backcrossed with C57BL/6 strain for 6 generations. Next, mice were crossed with the HSA-Cre +/- strain expressing the Cre-recombinase under the control of the Human Skeletal Actin (HSA) gene promoter specifically expressed in striated muscle fibers. The colony and protocols were controlled and approved by the Comité Institutionnel d‘Éthique Pour l‘Animal de Laboratoire (CIEPAL) and the Ministry of Education and Scientific Research under the number APAFIS#10444-2017052414468355v2 and APAFIS#31428-2021032913431387 v2. All experiments were performed at the ANIPHY core (ENS, Lyon), agreement n°C693880803 and at the PLATEAU DE BIOLOGIE EXPERIMENTALE (PBES) core (ENS-Lyon), agreement n°D691230303.
1, 3, 12 and 15 months old C57BL6/J mice were directly ordered to Charles River and maintain for 1 week adaptation within the IRCAN Experimental Core Facility (Nice) before organ collection.
Fiber size and nucleus position:
Mice were sacrificed, fresh muscle were dissected: gastrocnemius-plantaris-soleus (GPS) and tibialis anterior (TA) and then flash frozen in cold isopentane steam (dry-ice) in an OCT block (Optimal Cutting Temperature compound). Transverse frozen sections (10μm) of each muscle mice were prepared using a Leica cryostat and mounted onto SuperFrost Slides. Full tissues and slides were kept at -80°C for storage.
For staining, muscle slides were melted at room temperature for 30min and fixed for 10min with 4% paraformaldehyde. Next, slides were washed three times in PBS 1X, 3 min and permeabilized in 0.5% Triton X-100 in PBS for one hour and washed again in PBS 1X. After 1 hour blocking using a solution of M.O.M IgG diluent (Vector Laboratories, Burlingame, USA) at room temperature, slides were washed twice in PBS 1X for 5 min and incubated with M.O.M protein diluent solution for 5min. Next, slides were incubated with primary antibodies in M.O.M protein diluent (1:20) over night at 4°C. Each slide was treated simultaneously with 4 conditions separated using a Dako pen (Agilent); MyHC-I (Isoform BA-D5, DSHB, University of Iowa), MyHCIIa (Isoform SC-71, DSHB, University of Iowa) specific to type I and type IIa fibers, respectively, as well as combined MyHC-I + MyHC-IIa and no primary antibody. After three washes of 5 min each in PBS 1X, slides were incubated for 40min in M.O.M protein diluent containing the secondary antibody (Alexa 488 Donkey anti-mouse; 1:200) and washed three times 5min in PBS 1X. Last, slides were mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, USA). Images were taken using an Axio Imager 2 and a 20X lens (Zeiss).
Muscular fibers size was determine using Imaris software. More than 1000 fibers were analyzed per mice, and 3 to 5 mice per genotype and age.
For Peripherical or central nucleus position, 100 fibers were counted per mice (3 to 6 mice per genotype and age).
In vivo isometric force measurement.
Mice were initially anesthetized in an induction chamber using 4% isoflurane. The right hindlimb was shaved before an electrode cream was applied at the knee and heel regions to optimize electrical stimulation. Each anaesthetized mouse was placed supine in a strictly non-invasive ergometer (NIMPHEA_Research, AII Biomedical SAS, Grenoble, France) offering the possibility to electrically stimulate the plantar flexor mouse muscles and to record the resulting force production. Throughout a typical experiment, anesthesia was maintained by air inhalation through a facemask continuously supplied with 2.0-2.5% isoflurane. The cradle also includes an electrical heating blanket in order to maintain the animal at a physiological temperature during anesthesia. Electrical stimuli were delivered through two electrodes located below the knee and the Achilles tendon. The right foot was positioned and firmly immobilized through a rigid slipper on a pedal of an ergometer allowing for the measurement of the force produced by the plantar flexor muscles. The right knee was also firmly maintained using a rigid fixation in order to optimize isometric force recordings. Monophasic rectangular pulses of 0.2 ms were delivered using a constant-current stimulator (Digitimer DS7AH, Hertfordshire, UK; maximal voltage: 400 V). The force-frequency curves were determined by stepwise increasing stimulation frequency, with > 30 s between stimuli in order to avoid effects due to fatigue. After a 3 min recovery period, force was assessed during a fatigue protocol consisting of 180 stimulation trains delivered at 30 Hz (300 ms on, 700 ms off). The corresponding force was averaged every five contractions and was expressed in relation to maximal force Force signal was sampled at 1000 Hz using a Powerlab system and Labchart software (ADinstruments).
RNA sequencing
Mouse skeletal muscles were snap frozen in liquid nitrogen and preserved at -80°C. Total RNA was extracted using Tris Reagent (T9424 - Merck), according to manufacturer’s instructions. In brief, muscles were incubated in Tris Reagent in ceramic beads tubes (KT03961-1-003.2 Precellys) and dissociated with a Precellys tissue homogenizer. Add 200 µL chloroform and centrifuge 15 min at 12000g (4°C). Aqueous phase was transferred, RNA pellet was precipitated with 500 µL isopropanol and centrifuge again. Next, RNA pellet was washed twice in ethanol 75%. RNA quantification was determined by Nanodrop 1000 spectrophotometer (Thermo Scientific) and RNA quality with an Agilent 2100 Bioanalyser. Paired-end sequencing (read length: 2x 150 bp) was performed by NovoGene using an Illumina NovaSeq 6000 sequencer.
qPCR
After total RNA extraction, Reverse Transcription (RT) was performed with 500 ng RNA using High-Capacity RNA-to-cDNA Kit (Thermo Scientific). Each qPCR contained 5X diluted cDNA, 0.2 mM primers, and SYBR Green Master Mix (Roche, 4913914 001). Quantitative RT-PCR (qRT-PCR) was performed in triplicates using FastStart universal SYBR Green master Mix (Roche). Melting curves were analyzed (SYBR green) to exclude non-specific amplification products. We confirmed amplicon size at least once on agarose gels. Crossing-threshold (Ct) values were normalized to 36B4 housekeeping gene.
Telomere damage Induced Foci (TIFs):
Gastrocnemius-plantaris-soleus slides were prepared as mentioned above. After fixation, slides were permeabilized with 0.5% Triton X-100 for 20 min and dehydrated in increasing concentrations of ethanol for 3 min (50%, 70% and 100%). Hybridization of PNA telomeric probe was performed for at least 2 h at RT after denaturation (5 min) in 70% formamide, 10 mM Tris pH 7.2 and 1% blocking solution (Roche) at 80°C. Then, slides were washed in a 70% formamide, 10 mM Tris pH 7.2 solution for 30 min, followed by washes with 150 mM NaCl and 50 mM Tris pH 7.5 for 15 min. Next, slides were incubated 1h with blocking buffer (3% BSA and 0,3% Triton X-100) and incubated overnight at 4°C with 53BP1 antibody, rabbit polyclonal (NB 100-305, 1:200, Novus Biologicals). Slides were then washed three times with 1X PBS and incubated for 1 h with the corresponding secondary antibody. Finally, slides were prepared with a DAPI containing mounting solution (Vectashield, Vector Laboratories).
Pictures were taken using a DeltaVision Elite system (GE). An average of 70 stacks and 40 nuclei were taken per conditions. Images were then analyzed using FIJI (ImageJ). Total number of 53BP1 foci was determine and telomere-53BP1 colocalizations (TIFs) were determined by doing a stack-by-stack screening looking for at least one colocalizing pixel (yellow).
Statistical analysis
GraphPad Prism 7 software was used to generate graphs and to perform statistical analysis. p-values were obtained using either Mann-Whitney U test or the Kruskal-Wallis test. Differences were considered statistically significant when p < 0.05, as it follows: *p < 0.01, **p < 0.001, ***p < 0.0001. Absence of statistical annotation indicates non-significance (ns).