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

Abstract

The nuclear receptor Rev-erb-α modulates hepatic lipid and glucose metabolism, adipogenesis and the inflammatory response in macrophages. We show here that Rev-erb-α is highly expressed in oxidative skeletal muscle and that its deficiency in muscle leads to reduced mitochondrial content and oxidative function, as well as upregulation of autophagy. These cellular effects resulted in both impaired mitochondrial biogenesis and increased clearance of this organelle, leading to compromised exercise capacity. On a molecular level, Rev-erb-α deficiency resulted in deactivation of the Lkb1-Ampk-Sirt1-Ppargc-1α signaling pathway. These effects were recapitulated in isolated fibers and in muscle cells after knockdown of the gene encoding Rev-erb-α, Nr1d1. In complementary experiments, Rev-erb-α overexpression in vitro increased the number of mitochondria and improved respiratory capacity, whereas muscle overexpression or pharmacological activation of Rev-erb-α in vivo increased exercise capacity. This study identifies Rev-erb-α as a pharmacological target that improves muscle oxidative function by modulating gene networks controlling mitochondrial number and function.

Figure 1. Rev-erbα-deficient mice display reduced voluntary activity and exercise performance

(a) Western blot analysis of mouse muscles; actin was used as control. Qua, quadriceps; Sol, soleus; Gas, gastrocnemius; EDL, extensor digitorum longus; Dia, diaphragm; TA, tibialis anterior (n = 7 per group). (b) Wheel voluntary activity of Rev-erbα^−/− mice compared to Rev-erbα^+/+ mice (n = 5 per genotype). (c) Basal (at rest) and maximal aerobic capacity (VO₂) during a progressive treadmill test to exhaustion in an enclosed single lane treadmill (n = 9–10 per genotype). (d) Endurance capacity of Rev-erbα^−/− mice compared to Rev-erbα^+/+ mice (n = 6 per genotype). (e–f) Running time (e) and distance (f) until exhaustion in endurance exercise (n = 6 per genotype). Results are expressed as means ± sem; *P < 0.05, ***P < 0.001 by unpaired t-test (a–c,e–f) and by log-rank Mantel cox (d).

Figure 2. Rev-erb-α modulates mitochondria content and function

(a) Mitochondrial DNA (mtDNA) content (n = 6 per genotype). (b) RT-qPCR analysis of mitochondrial respiratory chain subunit genes in soleus muscle (n = 7 per genotype). (c) Western blot analysis of mitochondrial complexes in soleus from Rev-erbα^−/− and Rev-erbα^+/+ mice (n = 4–5 per genotype). (d) Mitochondrial respiration in permeabilized soleus fiber from Rev-erbα^−/− and Rev-erbα^+/+ mice (Succ, succinate; Rot, rotenone) (n = 7 per genotype). (e) Mitochondrial respiration from isolated muscle mitochondria (n = 5 per genotype). (f) Mitochondrial fatty acid β-oxidation-dependent respiration in isolated permeabilized soleus fibers from Rev-erbα^−/− and Rev-erbα^+/+ mice (n = 6 per genotype). (g) Skeletal muscle (soleus) expression of genes encoding proteins involved in fatty acid oxidation (n = 7 per genotype). (h) Electron microscopy analysis of Rev-erbα^−/− and Rev-erbα^+/+ muscle. Black arrows: swollen, less dense mitochondria; white arrowheads: normal mitochondria. Representative pictures from n = 5 mice per genotype. Results are expressed as means ± sem; *P < 0.05 and **P < 0.01, by unpaired t-test.

Figure 3. Rev-erb-α modulates mitochondrial respiration

(a) Mitochondria content in Rev-erb-α-expressing (Rev-erbα) or control (pBabe) retrovirus infected C2C12 cells by flow cytometry analysis of green mitotracker (MT) staining (Left, representative histogram; Right, mean fluorescence intensity (MFI)) (n = 6 per condition). (b) Functional mitochondria content by flow cytometry analysis of JC–1 (Left) and JC1red/JC1green (i.e. high/low mitochondrial membrane potential) MFI ratio (Right) (n = 6 per condition). (c) Oxygen consumption in Rev-erbα and control pBabe cells (n = 5 per condition). (d) RT-qPCR analysis of fatty acid oxidation gene expression in differentiated C2C12 cells infected with Rev-erbα or control (pBabe) retrovirus (n = 6 per group). Results are expressed as means ± sem; *P < 0.05, *** P < 0.001 by unpaired t-test.

Figure 4. Rev-erb-α modulates mitochondrial biogenesis by interfering with Ampk-Sirt1-Ppargc1-α signaling

(a–g) Soleus Ppargc1-α protein relative amounts (a), mRNA concentrations of Tfam and Nuclear respiratory factor 1 (Nrf1) (b), cellular ATP concentrations (c), Stk11 mRNA concentrations (d), western blot analysis of Ampk phosphorylation (e), Nampt and Sirt1 mRNA concentrations (f), NAD+ and NADH cellular concentrations, (g) acetylated Ppargc1-α protein relative amounts in Rev-erbα^−/− and Rev-erbα^+/+ soleus muscle (n = 7 per genotype). (h) Ppargc1α, Tfam, Sirt1 and Nampt gene expression in Rev-erb-α (Rev-erbα) vs. control (pBabe) retrovirus infected C2C12 cells (n = 6 per group). (i) OXPHOS (3U) mitochondrial respiration and RCR in absence or presence of the Ampk phosphorylation inhibitor C compound (CC) in C2C12 cells infected with Rev-erb-α or control (pBabe) retrovirus. (n = 6 per condition) (j) Mitochondria content in Rev-erb-α-expressing (Rev-erbα) or control (pBabe) retrovirus infected C2C12 cells transfected with a siRNA to reduce Ampkα1 and Ampkα2 expression or a scramble control siRNA: green Mitotracker mean fluorescence intensity (MFI) (n = 3 per condition). Results are expressed as means ± sem *P < 0.05, **P < 0.01, and ***P < 0.001, ^§§P < 0.01by unpaired t-test.

Figure 5. Rev-erb-α modulates skeletal muscle autophagy

(a) RT-qPCR analysis of autophagy gene expression in soleus from Rev-erbα^−/− and Reverbα^+/+ littermates. (n = 7 per genotype). (b) Western blot analysis of soleus muscle Park2 and Bnip3 proteins in Rev-erbα^−/− mice compared to Rev-erbα^+/+ littermates. (c) Western blot analysis of Map1lc3a–I and Map1lc3a–II protein in soleus from Rev-erbα^−/− and Rev-erbα^+/+ mice (n = 3–4 per genotype). (d) Flow cytometry delta (treated – vehicle) mean fluorescence intensity (MFI) of Map1lc3a–II protein levels in C2C12 cells infected with Rev-erbα (Reverbα) vs control (pBabe) retrovirus and treated with lysosome inhibitors (50 nM bafilomycin or 25 mM NH₄Cl) or their respective vehicle after 16 h serum deprivation (n = 6 per condition). (e) Mitochondria content in shRev-erbα and control (shCTL) cells treated or not with 5 mM 3-methyl adenine (3MA) or 50 μM chloroquine (CQ) or 100 nM bafilomycin or 25 mM NH₄Cl: specific green mitotracker (MT) flow cytometry MFI (n = 6 per condition). (f) Rev-erb-α binding to regulatory regions of the indicated autophagy genes measured by ChIP-qPCR (n = 3 independent experiments). (g–h) ChIP-qPCR examining the Rev-erb-α binding regions from (e) for changes in H3K27 and H3K9 acetylation (n = 3 independent experiments). (f–h) ChiP experiments were conducted on Rev-erb-α over-expressing (Rev-erbα) and control (pBabe) C2C12 cells. Results are expressed as means ± sem *P < 0.05, **P < 0.01 and ***P < 0.001, by unpaired t-test.

Figure 6. Rev-erb-α over-expression or pharmacological activity improves mitochondrial function and exercise capacity

(a) Running distance and time until exhaustion in endurance exercise in mice treated with SR9009 (100 mpk for 30 days) (n = 6 per group). (b) Mitochondria content in C2C12 cells treated with SR9009 (5 μM), SR9011 (5 μM) or vehicle: representative bargraph of flow cytometry green mitotracker (upper panel) and red mitotracker (bottom panel) staining (n = 4 per condition). (c) Mitochondrial respiration in permeabilized fiber isolated from tibialis anterior muscle from mice intra-muscularly injected with a Rev-erb-α expressing AAV vector (n = 4 per group). Results are expressed as means ± sem; *P < 0.05, **P < 0.01 by unpaired t-test.