Rev-erb-α modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy methods
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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...
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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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