Rev-erb-α modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy methods
Aim. Evidence-backed execution summary for Rev-erb-α modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy methods from Rev-erb-α modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Serum shock
reagent used in the protocol.
- Use
- We assessed circadian gene expression patterns in cells after synchronization by a 2-h horse serum shock on near confluent cells. After 2 h, the medium was changed to 2% serum-containing medium as above.
Rev-erb-α increases skeletal muscle mitochondrial biogenesis
reagent used in the protocol.
- Use
- As a functional reflection of impaired mitochondrial electron transport chain activity, ATP concentrations were severely reduced in Rev-erbα -deficient muscle ( ). Ampk, a 'fuel gauge' activated by liver kinase B1 (Lkb1 also known as serine threonine/kinase 11 or Stk11) when the AMP/ATP ratio increa...
Cell culture
reagent used in the protocol.
- Use
- We cultured C2C12 myoblasts in Dulbecco modified Eagle's medium (DMEM; 4.5 g l -1 D-Glucose; Gibco) supplemented with 10% fetal bovine serum and 1% gentamycin. Myogenic differentiation into myotubes was induced when cells reached 80% confluency by adding DMEM supplemented with 2% horse serum (HS) and 1%...
Retroviral production and infection
reagent used in the protocol.
- Use
- We cultured Phoenix Eco cells (Orbigen) in DMEM containing 10% fetal bovine serum, and 1% gentamycin at 37 °C under standard culture conditions. To generate cell lines constitutively over-expressing Rev-erb-α, we inserted the coding sequence of human Reverbα in the pBabe retrovirus plasmid (Addgene) u...
Determination of cellular respiration
reagent used in the protocol.
- Use
- C2C12 cells (1 × 10 6 cells ml -1 ) suspended in cell culture media (RPMI + 10% FCS) were placed into the chambers of the O2K oxygraph, operating at 37 °C. Routine respiration (R) was measured 20 min later. Then, 2 µg ml -1 oligomycin was injected into the chambers to obtain the oligomycin...
Oxygen consumption on permeabilized soleus fibers
reagent used in the protocol.
- Use
- After cervical dislocation, soleus or tibialis anterior muscles were excised and placed into a Petri dish containing ice-cold biopsy preservation solution (BIOPS) and permeabilized fibers were prepared as previously described. We placed fibers (3 to 6 mg wet weight) into the O2K oxygraph chambers (Oroboros Instrume...
Muscle mitochondria isolation
reagent used in the protocol.
- Use
- We cut muscles into small pieces and placed them into a Trypsin-EDTA solution for 15 min at 4 °C. Samples were rinsed with mitochondrial isolation buffer (in mM: sucrose 300, TES 5, EGTA 0.2, pH 7.2) and homogenized with a glass tissue grinder. After centrifugation at 800 g for 7 min, supernatant was collected...
ATP assay
reagent used in the protocol.
- Use
- We measured ATP concentrations using a commercially available kit (EnzyLight ™ ATP Assay Kit, Bioassay Systems, Hayward, CA, USA) following the manufacturer's instructions.
Rev-erb α-deficiency induces skeletal muscle autophagy
To gain further mechanistic insight, the distribution of Rev-erb-α binding sites in the vicinity of autophagy genes, assessed using chromatin immunoprecipitation (ChIP)-sequencing data, was examined and overlaid with epigenetic marks obtained in C2C12 myotubes, reasoning that intense transcriptional activity...
- Use
- To gain further mechanistic insight, the distribution of Rev-erb-α binding sites in the vicinity of autophagy genes, assessed using chromatin immunoprecipitation (ChIP)-sequencing data, was examined and overlaid with epigenetic marks obtained in C2C12 myotubes, reasoning that intense transcriptional activity...
Maximal exercise stress test and endurance capacity
Two days before the experiment, mice were acclimatized to a single lane treadmill by performing a 10 m min -1 run. The day of the exercise stress test, mice were placed into the treadmill enclosed in a metabolic chamber connected to an oxygen (O 2 ) sensor (Oxymax, Columbus Instruments, Columbus, OH). We measu...
- Use
- Two days before the experiment, mice were acclimatized to a single lane treadmill by performing a 10 m min -1 run. The day of the exercise stress test, mice were placed into the treadmill enclosed in a metabolic chamber connected to an oxygen (O 2 ) sensor (Oxymax, Columbus Instruments, Columbus, OH). We measu...
Oxygen consumption on permeabilized soleus fibers
After cervical dislocation, soleus or tibialis anterior muscles were excised and placed into a Petri dish containing ice-cold biopsy preservation solution (BIOPS) and permeabilized fibers were prepared as previously described. We placed fibers (3 to 6 mg wet weight) into the O2K oxygraph chambers (Oroboros Instrume...
- Use
- After cervical dislocation, soleus or tibialis anterior muscles were excised and placed into a Petri dish containing ice-cold biopsy preservation solution (BIOPS) and permeabilized fibers were prepared as previously described. We placed fibers (3 to 6 mg wet weight) into the O2K oxygraph chambers (Oroboros Instrume...
Mitotracker-FACS quantification
Rev-erb-α over-expressing, sh Rev-erbα cells and their respective control cells were washed with PBS, trypsinized and incubated at 37 °C for 20 min with 100 nM MitoTracker Green FM and Red FM dyes (Molecular Probes). Mitotracker Green probe preferentially accumulates in mitochondria, allowing estimati...
- Use
- Rev-erb-α over-expressing, sh Rev-erbα cells and their respective control cells were washed with PBS, trypsinized and incubated at 37 °C for 20 min with 100 nM MitoTracker Green FM and Red FM dyes (Molecular Probes). Mitotracker Green probe preferentially accumulates in mitochondria, allowing estimati...
Quantitative RT-QPCR
We isolated total RNA from tissues by guanidinium thiocyanate/phenol/chloroform extraction and from C2C12 cells using the Trizol reagent (Invitrogen). Isolated RNA was reverse transcribed into cDNA using commercially available reagents (Superscript II kit; Applied Bioscience). We performed quantitative PCR (qPCR) wi...
- Use
- We isolated total RNA from tissues by guanidinium thiocyanate/phenol/chloroform extraction and from C2C12 cells using the Trizol reagent (Invitrogen). Isolated RNA was reverse transcribed into cDNA using commercially available reagents (Superscript II kit; Applied Bioscience). We performed quantitative PCR (qPCR) wi...
Electron microscopy analysis of muscle sections
We examined ultrastructural muscle morphology using transmission electron microscopy. To this end, muscle tissue blocks were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Post-fixation was performed in 1% OsO 4 in 0.1 M cacodylate buffer (pH 7.4) supplemented with 1.5% K 4 [Fe(CN) 6 |. Subsequentl...
- Use
- We examined ultrastructural muscle morphology using transmission electron microscopy. To this end, muscle tissue blocks were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Post-fixation was performed in 1% OsO 4 in 0.1 M cacodylate buffer (pH 7.4) supplemented with 1.5% K 4 [Fe(CN) 6 |. Subsequentl...
Immunofluorescence assay
Frozen cross sections of muscle were fixed with acetone and incubated with antibodies directed to CD31 (1:100, BD Biosciences, BD557355), Pax7 (1:20, Developmental Studies Hybridoma Bank, PAX7), fiber types (1:25, Developmental Studies Hybridoma Bank, A4.840), and laminin (1:50, Sigma L9393). After incubation with t...
- Use
- Frozen cross sections of muscle were fixed with acetone and incubated with antibodies directed to CD31 (1:100, BD Biosciences, BD557355), Pax7 (1:20, Developmental Studies Hybridoma Bank, PAX7), fiber types (1:25, Developmental Studies Hybridoma Bank, A4.840), and laminin (1:50, Sigma L9393). After incubation with t...
Flow cytometry Map1lc3a analysis
Rev-erb-α over-expressing and control cells were seeded in 6 well-plates and serum-starved in presence or in absence of Bafilomycin (50 nM) or NH 4 Cl (25 mM) overnight at 37 °C. Before staining with a Map1lc3a antibody (1:50, MBL International M152-3 clone 4E12), we fixed the cells with 4% paraformaldehyd...
- Use
- Rev-erb-α over-expressing and control cells were seeded in 6 well-plates and serum-starved in presence or in absence of Bafilomycin (50 nM) or NH 4 Cl (25 mM) overnight at 37 °C. Before staining with a Map1lc3a antibody (1:50, MBL International M152-3 clone 4E12), we fixed the cells with 4% paraformaldehyd...
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Open quote workflowStep-by-step procedure
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Training
We examined Rev-erb-α expression in control (untrained) and exercised mice. Mice were trained for 8 weeks, 5 days per week, 1 to 2 h per day starting at a speed of 8 m min -1 up to 16 m min -1 the last 4 weeks.
Cell culture
We cultured C2C12 myoblasts in Dulbecco modified Eagle's medium (DMEM; 4.5 g l -1 D-Glucose; Gibco) supplemented with 10% fetal bovine serum and 1% gentamycin. Myogenic differentiation into myotubes was induced when cells reached 80% confluency by adding DMEM supplemented with 2% horse serum (HS) and 1% gentamycin at 37 °C in a humidified incubator under 5% CO 2 during 2 to 5 days. When indicated, we added SR9009 or SR9011 (5 µM) to the medium one day prior to differentiation and for the length of the differentiation period (8 days).
ONLINE METHODS
Rev-erb-α over-expression: we cloned the coding sequence of Rev-erb-α in front of a MCK promoter allowing muscle-specific expression and introduced in a 1/2 AAV vector (Sirion Biotech, Germany). We injected 2.1 × 10 10 infectious particles intra-muscularly in the tibialis anterior muscle using the tibialis anterior muscle of the contra-lateral limb as control. Pharmacological activation: SR9009 was administered i.p. at 100 mpk for 30 days as previously described.
Maximal exercise stress test and endurance capacity
Two days before the experiment, mice were acclimatized to a single lane treadmill by performing a 10 m min -1 run. The day of the exercise stress test, mice were placed into the treadmill enclosed in a metabolic chamber connected to an oxygen (O 2 ) sensor (Oxymax, Columbus Instruments, Columbus, OH). We measured basal oxygen consumption (VO 2b ) after a 30 min resting period; then, mice were encouraged to run on the treadmill at 10 m min -1 and 0% incline. Every 3 min, the treadmill speed was incremented by 4 m min -1 until the mice reached exhaustion in order to determine their maximal VO 2 (VO 2 max). The speed at which VO 2 max was obtained was considered as the maximal running speed (Vmax). One week later, we tested the mice for their endurance capacity. After a 6 min run at 30% of their maximal running speed, the treadmill speed was set at 70% of the VO 2 max....
Daily wheel running activity
To record daily running wheel behavior, we placed the animals into individual cages containing running wheels for 3 weeks (Campden, Phymep, Paris). Wheel revolution was recorded daily during the last two weeks and averaged.
Retroviral production and infection
We cultured Phoenix Eco cells (Orbigen) in DMEM containing 10% fetal bovine serum, and 1% gentamycin at 37 °C under standard culture conditions. To generate cell lines constitutively over-expressing Rev-erb-α, we inserted the coding sequence of human Reverbα in the pBabe retrovirus plasmid (Addgene) using the BamHI-SalII sites to generate pBabe-Rev-erb-α (Rev-erbα). We used the empty vector (pBabe) as control. To down-regulate Rev-erbα expression, we inserted the sh Rev-erbα sequence in psilencer 5.1-U6 Retro (Ambion) using the BamH1 and HindIII sites. Phoenix cells (100,000 per cm 2 ) were transfected with the pBabe plasmid constructs (20 µg) using the cationic lipid RPR 120535B as previously described. C2C12 cells were infected with the supernatant from pBabe or pBabe-Rev-erbα and shControl (shCTL) or sh Rev-erbα, and puromycin-res...
Determination of cellular respiration
C2C12 cells (1 × 10 6 cells ml -1 ) suspended in cell culture media (RPMI + 10% FCS) were placed into the chambers of the O2K oxygraph, operating at 37 °C. Routine respiration (R) was measured 20 min later. Then, 2 µg ml -1 oligomycin was injected into the chambers to obtain the oligomycin-inhibited leak rate of respiration (4o, Leak, 'L'), ie. uncoupled respiration. Finally, pulses of FCCP (1 µM) were added into the chambers until maximal oxygen consumption was reached (3u, OXPHOS, 'P'). It represents the maximal respiratory capacity. The respiratory control ratio (RCR), i.e. the coupled oxidative phosphorylation, is expressed as 3u/4o ratio.
Oxygen consumption on permeabilized soleus fibers
After cervical dislocation, soleus or tibialis anterior muscles were excised and placed into a Petri dish containing ice-cold biopsy preservation solution (BIOPS) and permeabilized fibers were prepared as previously described. We placed fibers (3 to 6 mg wet weight) into the O2K oxygraph chambers (Oroboros Instruments, Innsbruck, Austria). In the first chamber, we added glutamate (10 mM) and malate (2 mM) to obtain state 2 respiration. Then, we injected 2.5 mM ADP into the chamber to measure ADP-coupled oxygen consumption (state 3). Further addition of succinate (10 mM) allowed estimation of the entire OXPHOS capacity. Finally, complex I and III were inhibited with rotenone (0.5 µM) and antimycin A (2.5 µM), respectively. In the second chamber, we added palmitoyl-carnitine (5 or 20 µM) and malate (2 mM). After stabilization, coupled respiration was obtained with 2.5 mM...
Measurement outputs
What raw and processed outputs should exist?
We assessed circadian gene expression patterns in cells after synchronization by a 2-h horse serum shock on near confluent cells. After 2 h, the medium was changed to 2% serum-...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
We examined Rev-erb-α expression in control (untrained) and exercised mice. Mice were trained for 8 weeks, 5 days per week, 1 to 2 h per day starting at a speed of 8 m min...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Mitochondrial DNA content was ~40% lower in skeletal muscle from Rev-erbα -/- mice compared to wild-type littermates ( ), suggesting a regulatory role of Rev-er...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Although gene expression analysis of fiber type markers suggested a switch toward a more glycolytic profile (tropomyosin 3, a marker of oxidative type 1 fiber, myosin heavy chai...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
Although gene expression analysis of fiber type markers suggested a switch toward a more glycolytic profile (tropomyosin 3, a marker of oxidative type 1 fiber, myosin heavy chain IIa and IIx, markers of mostly oxidative fibers, are lower in Rev-erbα-deficient soleus and q...
from paperScoring or quantification
Quantify the primary readouts for this experiment: We assessed circadian gene expression patterns in cells after synchronization by a 2-h horse serum shock on near confluent cells. After 2 h, the medium was changed to 2% serum-...; We examined Rev-erb-α expression in control (untrained) and exercised mice. Mice were trained for 8 weeks, 5 days per week, 1 to 2 h per day starting at a speed of 8 m min...; Mitochondrial DNA content was ~40% lower in skeletal muscle from Rev-erbα -/- mice compared to wild-type littermates ( ), suggesting a regulatory role of Rev-er...; Although gene expression analysis of fiber type markers suggested a switch toward a more glycolytic profile (tropomyosin 3, a marker of oxidative type 1 fiber, myosin heavy chai....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Although gene expression analysis of fiber type markers suggested a switch toward a more glycolytic profile (tropomyosin 3, a marker of oxidative type 1 fiber, myosin heavy chai...; Next, we determined the effect of pharmacological activation of Rev-erb-α, by treating mice with the synthetic ligand SR9009, on exercise capacity. Notably, in an enduranc...; Values are means ± sem of the indicated number of measurements. Statistical significance was determined using two-tailed unpaired Student's t test with a significance...
from paperReporting output
Report representative outputs alongside summary comparisons for We assessed circadian gene expression patterns in cells after synchronization by a 2-h horse serum shock on near confluent cells. After 2 h, the medium was changed to 2% serum-..., We examined Rev-erb-α expression in control (untrained) and exercised mice. Mice were trained for 8 weeks, 5 days per week, 1 to 2 h per day starting at a speed of 8 m min..., Mitochondrial DNA content was ~40% lower in skeletal muscle from Rev-erbα -/- mice compared to wild-type littermates ( ), suggesting a regulatory role of Rev-er..., Although gene expression analysis of fiber type markers suggested a switch toward a more glycolytic profile (tropomyosin 3, a marker of oxidative type 1 fiber, myosin heavy chai....
inferred from protocolStructured statistical methods
Although gene expression analysis of fiber type markers suggested a switch toward a more glycolytic profile (tropomyosin 3, a marker of oxidative type 1 fiber, myosin heavy chai...; Next, we determined the effect of pharmacological activation of Rev-erb-α, by treating mice with the synthetic ligand SR9009, on exercise capacity. Notably, in an enduranc...; Values are means ± sem of the indicated number of measurements. Statistical significance was determined using two-tailed unpaired Student's t test with a significance...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
We examined Rev-erb-α expression in control (untrained) and exercised mice. Mice were trained for 8 weeks, 5 days per week, 1 to 2 h per day starting at a speed of 8 m min -1 up to 16 m min -1 the last 4 weeks.
We cultured C2C12 myoblasts in Dulbecco modified Eagle's medium (DMEM; 4.5 g l -1 D-Glucose; Gibco) supplemented with 10% fetal bovine serum and 1% gentamycin. Myogenic differentiation into myotubes was induced when cells reached 80% confluency by adding DMEM supplemented with 2% horse serum (HS) and 1% gentamycin at 37 °C in a humidified incubator under 5% CO 2 during 2 to 5 days. When indicated, we added SR9009 or SR9011 (5 µM) to the medium one day prior to differentiation and for the length of the differentiation period (8 days).
Rev-erb-α over-expression: we cloned the coding sequence of Rev-erb-α in front of a MCK promoter allowing muscle-specific expression and introduced in a 1/2 AAV vector (Sirion Biotech, Germany). We injected 2.1 × 10 10 infectious particles intra-muscularly in the tibialis anterior muscle using the tibialis anterior muscle of the contra-lateral limb as control. Pharmacological activation: SR9009 was administered i.p. at 100 mpk for 30 days as previously described.
Two days before the experiment, mice were acclimatized to a single lane treadmill by performing a 10 m min -1 run. The day of the exercise stress test, mice were placed into the treadmill enclosed in a metabolic chamber connected to an oxygen (O 2 ) sensor (Oxymax, Columbus Instruments, Columbus, OH). We measured basal oxygen consumption (VO 2b ) after a 30 min resting period; then, mice were encouraged to run on the treadmill at 10 m min -1 and 0% incline. Every 3 min, the treadmill speed was incremented by 4 m min -1 until the mice reached exhaustion in order to determine their maximal VO 2 (VO 2 max). The speed at which VO 2 max was obtained was considered as the maximal running speed (Vmax). One week later, we tested the mice for their endurance capacity. After a 6 min run at 30% of their maximal running speed, the treadmill speed was set at 70% of the VO 2 max. The experiment was stopped once mice stayed for 5 continuous seconds on the electrical grid. Time to exhaustion and total running distance were determined.
To record daily running wheel behavior, we placed the animals into individual cages containing running wheels for 3 weeks (Campden, Phymep, Paris). Wheel revolution was recorded daily during the last two weeks and averaged.
We cultured Phoenix Eco cells (Orbigen) in DMEM containing 10% fetal bovine serum, and 1% gentamycin at 37 °C under standard culture conditions. To generate cell lines constitutively over-expressing Rev-erb-α, we inserted the coding sequence of human Reverbα in the pBabe retrovirus plasmid (Addgene) using the BamHI-SalII sites to generate pBabe-Rev-erb-α (Rev-erbα). We used the empty vector (pBabe) as control. To down-regulate Rev-erbα expression, we inserted the sh Rev-erbα sequence in psilencer 5.1-U6 Retro (Ambion) using the BamH1 and HindIII sites. Phoenix cells (100,000 per cm 2 ) were transfected with the pBabe plasmid constructs (20 µg) using the cationic lipid RPR 120535B as previously described. C2C12 cells were infected with the supernatant from pBabe or pBabe-Rev-erbα and shControl (shCTL) or sh Rev-erbα, and puromycin-resistant infected cells were used within 15 days after infection. We verified Rev-erbα over-expression by RT-qPCR on Rev-erb-α C2C12 cells compared to pBabe control cells ( ). We verified Rev-erb-α nuclear localization by western blot analysis ( ), and we verified its repressive transcr...
C2C12 cells (1 × 10 6 cells ml -1 ) suspended in cell culture media (RPMI + 10% FCS) were placed into the chambers of the O2K oxygraph, operating at 37 °C. Routine respiration (R) was measured 20 min later. Then, 2 µg ml -1 oligomycin was injected into the chambers to obtain the oligomycin-inhibited leak rate of respiration (4o, Leak, 'L'), ie. uncoupled respiration. Finally, pulses of FCCP (1 µM) were added into the chambers until maximal oxygen consumption was reached (3u, OXPHOS, 'P'). It represents the maximal respiratory capacity. The respiratory control ratio (RCR), i.e. the coupled oxidative phosphorylation, is expressed as 3u/4o ratio.
After cervical dislocation, soleus or tibialis anterior muscles were excised and placed into a Petri dish containing ice-cold biopsy preservation solution (BIOPS) and permeabilized fibers were prepared as previously described. We placed fibers (3 to 6 mg wet weight) into the O2K oxygraph chambers (Oroboros Instruments, Innsbruck, Austria). In the first chamber, we added glutamate (10 mM) and malate (2 mM) to obtain state 2 respiration. Then, we injected 2.5 mM ADP into the chamber to measure ADP-coupled oxygen consumption (state 3). Further addition of succinate (10 mM) allowed estimation of the entire OXPHOS capacity. Finally, complex I and III were inhibited with rotenone (0.5 µM) and antimycin A (2.5 µM), respectively. In the second chamber, we added palmitoyl-carnitine (5 or 20 µM) and malate (2 mM). After stabilization, coupled respiration was obtained with 2.5 mM ADP. Addition of exogenous cytochrome c (10 µM) was done in all experiments in order to test external membrane integrity. Experiments were performed at 25 °C.
Machine-readable layer
[
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"name": "Rev-erb-α modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy methods",
"description": "Evidence-backed execution summary for Rev-erb-α modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy methods from Rev-erb-α modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy.",
"totalTime": "PT50450M",
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{
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"position": 1,
"name": "Training",
"text": "We examined Rev-erb-α expression in control (untrained) and exercised mice. Mice were trained for 8 weeks, 5 days per week, 1 to 2 h per day starting at a speed of 8 m min -1 up to 16 m min -1 the last 4 weeks."
},
{
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"text": "We cultured C2C12 myoblasts in Dulbecco modified Eagle's medium (DMEM; 4.5 g l -1 D-Glucose; Gibco) supplemented with 10% fetal bovine serum and 1% gentamycin. Myogenic differentiation into myotubes was induced when cells reached 80% confluency by adding DMEM supplemented with 2% horse serum (HS) and 1% gentamycin at 37 °C in a humidified incubator under 5% CO 2 during 2 to 5 days. When indicated, we added SR9009 or SR9011 (5 µM) to the medium one day prior to differentiation and for the length of the differentiation period (8 days)."
},
{
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"name": "ONLINE METHODS",
"text": "Rev-erb-α over-expression: we cloned the coding sequence of Rev-erb-α in front of a MCK promoter allowing muscle-specific expression and introduced in a 1/2 AAV vector (Sirion Biotech, Germany). We injected 2.1 × 10 10 infectious particles intra-muscularly in the tibialis anterior muscle using the tibialis anterior muscle of the contra-lateral limb as control. Pharmacological activation: SR9009 was administered i.p. at 100 mpk for 30 days as previously described."
},
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"text": "Two days before the experiment, mice were acclimatized to a single lane treadmill by performing a 10 m min -1 run. The day of the exercise stress test, mice were placed into the treadmill enclosed in a metabolic chamber connected to an oxygen (O 2 ) sensor (Oxymax, Columbus Instruments, Columbus, OH). We measured basal oxygen consumption (VO 2b ) after a 30 min resting period; then, mice were encouraged to run on the treadmill at 10 m min -1 and 0% incline. Every 3 min, the treadmill speed was incremented by 4 m min -1 until the mice reached exhaustion in order to determine their maximal VO 2 (VO 2 max). The speed at which VO 2 max was obtained was considered as the maximal running speed (Vmax). One week later, we tested the mice for their endurance capacity. After a 6 min run at 30% of their maximal running speed, the treadmill speed was set at 70% of the VO 2 max...."
},
{
"@type": "HowToStep",
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"text": "To record daily running wheel behavior, we placed the animals into individual cages containing running wheels for 3 weeks (Campden, Phymep, Paris). Wheel revolution was recorded daily during the last two weeks and averaged."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Retroviral production and infection",
"text": "We cultured Phoenix Eco cells (Orbigen) in DMEM containing 10% fetal bovine serum, and 1% gentamycin at 37 °C under standard culture conditions. To generate cell lines constitutively over-expressing Rev-erb-α, we inserted the coding sequence of human Reverbα in the pBabe retrovirus plasmid (Addgene) using the BamHI-SalII sites to generate pBabe-Rev-erb-α (Rev-erbα). We used the empty vector (pBabe) as control. To down-regulate Rev-erbα expression, we inserted the sh Rev-erbα sequence in psilencer 5.1-U6 Retro (Ambion) using the BamH1 and HindIII sites. Phoenix cells (100,000 per cm 2 ) were transfected with the pBabe plasmid constructs (20 µg) using the cationic lipid RPR 120535B as previously described. C2C12 cells were infected with the supernatant from pBabe or pBabe-Rev-erbα and shControl (shCTL) or sh Rev-erbα, and puromycin-res..."
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"name": "Determination of cellular respiration",
"text": "C2C12 cells (1 × 10 6 cells ml -1 ) suspended in cell culture media (RPMI + 10% FCS) were placed into the chambers of the O2K oxygraph, operating at 37 °C. Routine respiration (R) was measured 20 min later. Then, 2 µg ml -1 oligomycin was injected into the chambers to obtain the oligomycin-inhibited leak rate of respiration (4o, Leak, 'L'), ie. uncoupled respiration. Finally, pulses of FCCP (1 µM) were added into the chambers until maximal oxygen consumption was reached (3u, OXPHOS, 'P'). It represents the maximal respiratory capacity. The respiratory control ratio (RCR), i.e. the coupled oxidative phosphorylation, is expressed as 3u/4o ratio."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Oxygen consumption on permeabilized soleus fibers",
"text": "After cervical dislocation, soleus or tibialis anterior muscles were excised and placed into a Petri dish containing ice-cold biopsy preservation solution (BIOPS) and permeabilized fibers were prepared as previously described. We placed fibers (3 to 6 mg wet weight) into the O2K oxygraph chambers (Oroboros Instruments, Innsbruck, Austria). In the first chamber, we added glutamate (10 mM) and malate (2 mM) to obtain state 2 respiration. Then, we injected 2.5 mM ADP into the chamber to measure ADP-coupled oxygen consumption (state 3). Further addition of succinate (10 mM) allowed estimation of the entire OXPHOS capacity. Finally, complex I and III were inhibited with rotenone (0.5 µM) and antimycin A (2.5 µM), respectively. In the second chamber, we added palmitoyl-carnitine (5 or 20 µM) and malate (2 mM). After stabilization, coupled respiration was obtained with 2.5 mM..."
}
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{
"@type": "HowToTool",
"name": "Rev-erb α-deficiency induces skeletal muscle autophagy"
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{
"@type": "HowToTool",
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},
{
"@type": "HowToTool",
"name": "Oxygen consumption on permeabilized soleus fibers"
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{
"@type": "HowToTool",
"name": "Mitotracker-FACS quantification"
},
{
"@type": "HowToTool",
"name": "Quantitative RT-QPCR"
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"@type": "HowToTool",
"name": "Electron microscopy analysis of muscle sections"
},
{
"@type": "HowToTool",
"name": "Immunofluorescence assay"
},
{
"@type": "HowToTool",
"name": "Flow cytometry Map1lc3a analysis"
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"supply": [
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"name": "Serum shock"
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"name": "Rev-erb-α increases skeletal muscle mitochondrial biogenesis"
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