Animals

The different mouse models used in this study were generated as follows:

The skeletal muscle-specific Sesn1 transgenic mouse model (Sesn1SkM-Tg) was generated in C57BL/6 background and carry a transgene coding for human Sesn1 under the control of MCK promoter. The human Sesn1 cDNA sequence (from LV-Sesn119) was subcloned into the pBluescript-MCK plasmid (a kind gift from Markus Rüegg). A 4 kb PacI digestion fragment was excised, microdialyzed, and microinjected into the pronuclei of fertilized mouse eggs (C57BL/6J × C57BL/6J) at the Mouse Mutant Core Facility, Institute for Research in Biomedicine (Barcelona, Spain). Embryos were implanted into pseudo-pregnant foster females (ICR), and transgenic pups were identified. DNA samples from tail clips of subsequent litters were screened by PCR with primers spanning different sequences of MCK promoter and Sesn1 cDNA (Primer set 1 forward CTGCCCCCGGGTCACCACC reverse TCGATTCAGGTCATATAGCGGGT; Primer set 2 forward CAGGGCTTATACGTGCCTGGGACTC reverse TGTGGGTGGAAAACCATCACTAACG; Primer set 3 forward GCCCCCGGGTCACCACCAAG reverse GGCCAAGCGCATGGATCCTTTTA) that amplified a 1550, 600, and 76 bp fragments, respectively. The transgene was maintained on the C57BL/6J background throughout the study.

The skeletal muscle-specific Sesn2 transgenic mouse model (Sesn2SkM-Tg) was generated by crossing mice that express Sesn2 from a Tet-regulated promoter (provided by Dr. M. Karin, UCSD, San Diego, USA) with mice harboring the skeletal muscle-specific MCK-tTA construct (obtained from Dr. M. Ruegg, Biozentrum, Basel, Switzerland). Non-transgenic littermates (that were WT for Sesn1 and Sesn2 expressions) were used as controls for the two transgenic mouse lines, and named Sesn1WT and Sesn2WT mice, respectively. Sesn1 KO mice were provided by Dr. J.H. Lee (University of Michigan, USA) and analyzed in comparison with normal, control WT mice. Atg7SkM-KO mice were generated as previously described32, and FoxO1,3,4SkM-KO mice were obtained by crossing FoxO1/3/4-floxed mice (a kind gift from Dr. M. Sandri, University of Padova, Italy) with the Pax7Cre line (provided by Dr. M. Capecchi, University of Utah, USA). Non-transgenic littermates (that were WT for FoxO1,3,4 and for Atg7 expression) were used as controls for Atg7SkM-KO mice and for FoxO1,3,4SkM-KO mice, and named Atg7WT and FoxO1,3,4WT mice, respectively. Therefore, Sesn1WT, Sesn2WT, WT, Atg7WT, and FoxO1,3,4WT mice are all equally WT, non-transgenic mice, but each one is used as littermate control of each individual genetically modified mouse line.

Mice were housed in standard cages under 12 h light/dark cycles with ad libitum access to food and water. All experiments were performed on 4–5-month-old male mice. All animal experiments were approved by the Ethics Committee of the Barcelona Biomedical Research Park (PRBB) and performed according to Catalan and European legislation.

Induction of muscle atrophy

The immobilization protocol was performed unilaterally on anesthetized animals as formerly described39. Briefly, the right hindlimb was immobilized with rigid plastic sticks fixed with a medical adhesive bandage. This procedure prevented movement of the immobilized leg alone. Denervation was performed as previously described40. In brief, a 5 mm segment of the sciatic nerve was surgically removed down to the gluteus maximum from the right leg. Mice not subjected to atrophy-promoting conditions were used as controls (Basal). Muscles were removed at 3 or 10 days after atrophy induction and frozen in liquid nitrogen for subsequent analyses.

In vivo gene electrotransfer and AAV injection

Expression plasmids used for electrotransfer studies were purified using a Endofree plasmid kit (Qiagen) and dissolved in 0.9% NaCl. 45 min before electrotransfer, muscles were pretreated with hyaluronidase (10 U/muscle). Afterwards, naked plasmids (60 μg DNA) were injected into the tibialis anterior muscle and 10 pulses of 20 ms each were applied to each hindlimb at 175 V/cm and 1 Hz using an electroporator (ECM 830; BTX). Empty vector was used as control.

AAV for in vivo expression of Sesn1 and Sesn2 were generated and provided by the Virus Production Unit (UPV, UAB, Barcelona). AAVs were diluted in 0.9% NaCl at 0.25*1013 gc/ml and directly injected into muscles (40 µl/TA and 10 µl/EDL and soleus muscles). AAV-GFP was used as a control. Four days after transduction mice were subjected to the immobilization protocol.

Pharmacological treatments

Mice were injected with rapamycin (4 mg/kg body weight) (which induces autophagy) or vehicle (DMSO) intraperitoneally (i.p.) every other day for 2 weeks. Colchicine (which inhibits autophagy) was injected i.p. (0.4 mg/kg*day) 2 days before sacrifice. The proteasome inhibitor Bortezomib (0.1 mg/kg) or vehicle (DMSO) was injected i.p. every other day for 2 weeks. Mice were treated with 3 mM spermidine in drinking water for 2 weeks.

Muscle force measurement

Ex vivo force measurements of EDL and soleus muscles was assessed as previously described41. Briefly, mice were sacrificed, and muscles were immediately excised and placed into a dish containing oxygenated Krebs–Henseleit solution. Muscles were mounted vertically in a temperature controlled (30 °C) chamber and immersed in the Krebs–Ringer bicarbonate buffer solution, with 10 mM glucose, also continuously oxygenated. One end of the muscle was linked to a fixed clamp, while the other end was connected to the lever-arm of an Aurora Scientific Instruments 300B actuator/transducer system, using a nylon thread. The optimum muscle length (Lo) was determined from micromanipulations of muscle length to produce the maximum isometric twitch force. Maximum isometric-specific tetanic force was determined from the plateau of the curve of the relationship between specific isometric force with a stimulation frequency ranging from 1 to 200 Hz. Force was normalized per muscle area (determined by dividing the muscle mass by the product of longitude and the density of muscle (1.06 mg/mm3)) to calculate the specific force (mN/mm2).

Muscle histology and immunohistochemistry

Muscles were embedded in OCT solution (TissueTek), frozen in isopentane cooled with liquid nitrogen and stored at −80 °C until analysis. 10 μm muscle cryosections were collected and stained for hematoxylin/eosin (H/E) or Sirius red (Sigma-Aldrich).

For immunohistochemistry assays, muscle cryosections were examined by standard immunohistochemical procedures for the expression of myosin heavy chain (MHC) isoforms. The primary monoclonal antibodies employed were anti-myosin I (A4.840), anti-myosin IIA (A4.74), and anti-myosin IIB (BF-F3) (Developmental Studies Hybridoma Bank).

Digital images were acquired using the Leica DMR600B microscope equipped with a DFC300FX camera. Fiber type distribution, CSA, and percentage of muscle area positive for Sirius red staining were quantified using Image J software, as previously reported42,43.

For myonuclei quantification, muscle sections were immunostained for dystrophin (1/400), the secondary antibody was coupled to Alexa-488 and nuclei were stained with DAPI (Invitrogen). Images were acquired using a Leica TCS SP5 confocal scanning microscope system.

Fluorescence microscopy analysis of muscle sections

TA muscles were removed, fixed in PFA 2% for 4 h at 4 °C and incubated with 15% sucrose overnight at 4 °C. Then, muscles were embedded in OCT solution (TissueTek), immediately frozen in liquid nitrogen-cooled isopentane and stored at −80 °C. 10 μm cryosections of TA muscle (which has been transfected with mRFP-GFP-LC344) were analyzed using a Leica TCS SP5 confocal scanning microscope system. Colocalization of RFP-LC3 and GFP-LC3 puncta was determined on the maximum projection of 10-z sections. Note: Measuring autophagy flux through this method is based on the concept of lysosomal quenching of GFP. GFP is a stably folded protein and relatively resistant to lysosomal proteases. However, the low pH inside the lysosome quenches the fluorescent signal of GFP, which makes it difficult to trace the delivery of GFP–LC3 to lysosomes. In contrast, RFP exhibits more stable fluorescence in acidic compartments, and mRFP–LC3 can be readily detected in autolysosomes. By exploiting the difference in the nature of these two fluorescent proteins (that is, lysosomal quenching of GFP fluorescence versus lysosomal stability of RFP fluorescence), autophagic flux can be morphologically traced with an mRFP–GFP–LC3 tandem construct. With this tandem construct, autophagosomes and autolysosomes are labeled with yellow (mRFP and GFP) and red (mRFP only) signals, respectively.

RNA isolation, reverse transcription, and quantitative PCR

Total RNA from TA muscles was isolated with QIAzol Lysis Reagent (Qiagen) and quantified with Nanodrop. M-MLV Reverse Transcriptase (Promega) was used to synthesize cDNAs from 1 µg total RNA following the manufacturer’s instructions. RT-qPCR reactions were performed with SYBR Green in 384-well plates using the Roche LC-480 cycler (Roche Applied Science). All data were normalized to L7 expression. Primer sequences are listed in Table 1.

Table 1 Primers used for qPCR. Full size table

Transcriptomic analysis

For RNAseq analysis, total RNA from TA muscles was extracted using a protocol combining QIAzol Lysis Reagent and RNAeasy minikit columns (Qiagen) following the manufacturer’s instructions. RNAseq services were provided by the CNIC Genomics Unit, including quality control tests of total RNA using Agilent Bioanalyzer and Nanodrop spectrophotometry. cDNA library preparation and amplification were performed from 200 ng total RNA using NEBNext Ultra RNA Library Prep Kit for Illumina. RNAseq analysis was performed with 3–4 samples per condition, using Illumina Hiseq 2500. Sequencing reads were pre-processed by means of a pipeline that used FastQC, to asses read quality, and Cutadapt 1.7.1 to trim sequencing reads, eliminating Illumina adaptor remains, and to discard reads that were shorter than 30 bp. The resulting reads were mapped against the mouse transcriptome (GRCm38, release 76; aug2014 archive) and quantified using RSEM v1.2.20. Data were then processed with a differential expression analysis pipeline that used Bioconductor package LIMMA for normalization and differential expression testing. For differential expression analysis we filtered for genes that showed at least Log2FC 0.25 (≥+0.25 for upregulation; and ≤−0.25 for downregulation) and an adj. p value < 0.05.

FoxO promoter-reporter assay

TA muscles from Sesn1WT and Sesn1SkM-Tg mice were electrotransferred with a reporter plasmid with three copies of forkhead responsive element linked to the luciferase reporter gene (FHRE-luciferase; Addgene). After 3 days of immobilization, muscles were collected and luciferase activity was measured in muscle homogenates by using Dual-Luciferase Reporter Assay Kit (Promega Corporation, USA). Values were normalized to non-immobilized muscles.

Muscle protein extraction and Western blotting

Total homogenates from skeletal muscle were obtained in IP buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP-40, 5 mM EGTA, 5 mM EDTA, 20 mM NaF, 25 mM β-glycerophosphate, 0.1 mM sodium vanadate, 1 mM PMSF) supplemented with protease and phosphatase inhibitors (Complete Mini, Roche Diagnostic Corporation; phosphatase inhibitor cocktail, Sigma). Protein concentration was measured using the Bradford method (Protein Assay, Bio-Rad). 40 µg of protein were resolved by SDS–PAGE and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% milk in TBS-T for 1 h and incubated with primary antibodies overnight at 4 °C in 5% BSA in TBS-T (Tubulin 1:4000 and others 1:1000). Proteins were detected by the ECL method and quantified by scanning densitometry. The antibodies used are listed in Table 2. Uncropped versions of all blots shown in figures are supplied in the Source Data File.

Table 2 Antibodies. Full size table

Proteasome activity in muscle

Proteasome activity in total homogenates from TA muscles was determined by evaluating the cleavage of specific fluorogenic substrates. Muscles were homogenized in lysis buffer (50 mM Tris–HCl pH 7.5, 250 mM Sucrose, 5 mM MgCl 2 , 0.5 mM EDTA, 2 mM ATP, and 1 mM DTT) and centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was collected and protein concentration determined by the method of Bradford. For chymotrypsin-like activity, aliquots of 20 μg protein were incubated for 60 min at 37 °C in the presence of 100 μM of the fluorogenic substrate succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc LLVY-AMC). Each assay was conducted in the absence and presence of the specific proteasomal inhibitor MG132 (Sigma-Aldrich) at 20 μM. Fluorescence was read with a spectrofluorometer (390 nm excitation/460 nm emission; Tecan Infinite M200). The activity was expressed as units of fluorescence per microgram of protein, as a percentage of the control group. All samples were assayed in triplicate using at least four animals.

Bioinformatic analysis

Hierarchical clustering of expression values (after filtering expression values below one in all the samples) was carried out with Morpheus (https://software.broadinstitute.org/morpheus/) using one minus Pearson correlation with a complete linkage. GSEA of Sestrin-regulated genes was performed using GSEA web interface with the Molecular Signatures Database “hallmarks” and “transcription factor binding targets” genesets, to reveal pathways and cis-regulatory motifs which can function as potential transcription factor-binding sites, respectively45. The Java implementation code from the Broad Institute was used for direct GSEA comparisons of the raw data from our RNA-seq experiments or with GEO: GSE5395 data set with the “AKT-MTOR signaling” gene set (generated by combining the “MTORC1 Signaling” and the “PI3K-AKT- MTOR signaling” Molecular Signatures Database hallmarks), the growth and atrophy regulators gene set (assembled by combining the following Gene Ontology categories: positive and negative regulations of insulin-like growth factor receptor signaling pathway, positive and negative regulations of TORC1 signaling and positive and negative regulations of muscle atrophy) and the atrogenes gene set (that was manually curated from selected bibliography1,2,3,4,6,7,22,24). Bubble plots were generated using ggplot2 library in R or Seaborn in Python. Venn diagrams were generated using The BEG Ugent tool (http://bioinformatics.psb.ugent.be/webtools/Venn/).

Statistical analysis

For mouse experiments, no specific blinding method was used, but mice in each sample group were selected randomly. The sample size (n) of each experimental group is described in each corresponding figure legend.

GraphPad Prism software was used for all statistical analyses. Quantitative data displayed as histograms are expressed as means ± standard error of the mean (represented as error bars). Results from each group were averaged and used to calculate descriptive statistics.

Unpaired t-test (independent samples, two-sided) was used for pairwise comparisons among groups at each time point, unless indicated in figure legends. Statistical significance was set at a p < 0.05.