Impaired myogenesis in estrogen-related receptor γ (ERRγ)-deficient skeletal myocytes due to oxidative stress

Abstract

Specialized contractile function and increased mitochondrial number and oxidative capacity are hallmark features of myocyte differentiation. The estrogen-related receptors (ERRs) can regulate mitochondrial biogenesis or mitochondrial enzyme expression in skeletal muscle, suggesting that ERRs may have a role in promoting myogenesis. Therefore, we characterized myogenic programs in primary myocytes isolated from wild-type (M-ERRγWT) and muscle-specific ERRγ(-/-) (M-ERRγ(-/-)) mice. Myotube maturation and number were decreased throughout differentiation in M-ERRγ(-/-) primary myocytes, resulting in myotubes with reduced mitochondrial content and sarcomere assembly. Compared with M-ERRγWT myocytes at the same differentiation stage, the glucose oxidation rate was reduced by 30% in M-ERRγ(-/-) myotubes, while medium-chain fatty acid oxidation was increased by 34% in M-ERRγ(-/-) myoblasts and 36% in M-ERRγ(-/-) myotubes. Concomitant with increased reliance on mitochondrial β-oxidation, H(2)O(2) production was significantly increased by 40% in M-ERRγ(-/-) myoblasts and 70% in M-ERRγ(-/-) myotubes compared to M-ERRγWT myocytes. ROS activation of FoxO and NF-κB and their downstream targets, atrogin-1 and MuRF1, was observed in M-ERRγ(-/-) myocytes. The antioxidant N-acetyl cysteine rescued myotube formation and atrophy gene induction in M-ERRγ(-/-) myocytes. These results suggest that loss of ERRγ causes metabolic defects and oxidative stress that impair myotube formation through activation of skeletal muscle atrophy pathways.

Figure 1.

Differentiation is impaired in M-ERRγ^−/− primary myocytes. A) Bright-field images of M-ERRγWT and M-ERRγ^−/− primary myocytes were taken at the indicated days at ×100. Cells were analyzed in triplicate in several independent trials, and representative images are shown. B) Immunofluorescence analysis was performed for the structural targets actinin and TnI at MTd4 using primary antibodies against the indicated proteins and detected with Alexa Fluor 488 secondary antibody. Representative images from ≥50 cells analyzed are shown at ×600. C) Real-time PCR was carried out for myogenin and MyoD and normalized to 36B4 mRNA levels. Triplicate samples were analyzed, and results are expressed as means ± sd. D) Expression of the sarcomeric components TnI slow, skeletal actin, and MHC slow was analyzed by real-time PCR and normalized to 36B4. Samples were analyzed in triplicate, and data are expressed as means ± sd. *P < 0.05. E) Western blot analysis for MHC and TnI expression was performed in M-ERRγWT and M-ERRγ^−/− myocytes at the indicated differentiation days. β-Tubulin was included as a loading control. Scale bars = 100 μm (A); 10 μm (B).

Figure 2.

M-ERRγ^−/− primary myocytes exhibit mitochondrial defects. A) Immunofluorescence analysis for cytochrome c was assessed in M-ERRγWT and M-ERRγ^−/− myocytes at MTd4. Detection of primary antibodies was carried out using Alexa Fluor 568-conjugated secondary antibodies. At least 50 cells were analyzed, and representative images are shown at ×600. Scale bars = 10 μm. B) To assess mitochondrial number, DNA was isolated from M-ERRγWT and M-ERRγ^−/− myocytes at the indicated days. PCR was carried out for the mitochondria-encoded gene CoxI, and the nuclear encoded gene TFIID, and mitochondrial genome copy number was assessed from the ratio of CoxI to TFIID. Samples were analyzed in triplicate, and results are expressed as means ± se. *P < 0.05. C) Expression of ERRα, PGC-1α, and components of the ETC Ndufa8, Sdha, Cyt c, and Cox6c were analyzed by real-time PCR and normalized to 36B4 expression. Samples were analyzed in triplicate, and data are expressed as means ± sd. *P < 0.05.

Figure 3.

Oxidative metabolism is impaired in M-ERRγ^−/− MTs. A) Glucose oxidation rates were determined by ¹⁴CO₂ release after 1 h incubation with ¹⁴C-glucose. ³H-lauric acid (C12) and ³H-palmitic acid (C16) oxidation rates in M-ERRγWT and M-ERRγ^−/− myocytes were determined by ³H₂O release after a 2-h incubation with the indicated fatty acid. Experiments were performed in triplicate, and results are expressed as means ± se. *P < 0.05. B) Glucose uptake was determined by incubating M-ERRγWT and M-ERRγ^−/− MTd2 with 20 μM 2-NBDG for 20 min at 37°C. Fluorescence was measured on a Tecan Infinite M1000 plate reader (λ_ex 465 nm/λ_em 540 nm). Experiments were performed in triplicate, and results are expressed as means ± se. *P < 0.05. C) Uptake of BODIPY-labeled lauric acid or palmitic acid in M-ERRγWT and M-ERRγ^−/− MTd3 myocytes was assessed by fluorescence microscopy at ×600. At least 50 cells were analyzed from duplicate samples, and representative images are shown. Scale bars = 10 μm. D) Expression of metabolic transcripts in M-ERRγWT and M-ERRγ^−/− myocytes was analyzed by real-time PCR and normalized to 36B4 mRNA levels. Samples were analyzed in triplicate, and data are expressed as means ± se. *P < 0.05.

Figure 4.

ROS production is elevated in M-ERRγ^−/− primary myocytes. A) Production of hydrogen peroxide (H₂O₂) was assessed using Amplex Red. Primary myocytes (top) or isolated mitochondria (bottom) from M-ERRγWT and M-ERRγ^−/− myocytes were incubated with 50 μM Amplex Red and 0.1 U/ml horseradish peroxidase for 30 min at 37°C. Isolated mitochondria were incubated with no respiratory substrate (no subs), 5 mM succinate (Succ), or 5 mM succinate plus 1 mM ADP (succ+ADP). Fluorescence was measured on a Tecan Infinite M200 Pro plate reader (λ_ex 540 nm/λ_em 590 nm). Experiments were performed in triplicate, and results are expressed as means ± se. *P < 0.05. B) Expression of the antioxidant SOD2, catalase, Gpx1, Gpx3, and mitochondrial uncoupling proteins 2 and 3 (UCP2 and UCP3) was analyzed by real-time PCR in M-ERRγWT and M-ERRγ^−/− primary myocytes during differentiation. Results were normalized to 36B4 mRNA levels. Experiments were performed in triplicate, and results are expressed as means ± se. *P < 0.05.

Figure 5.

NF-κB is activated in M-ERRγ^−/− myocytes. A) Top panels: phosphorylation status of NF-κB was analyzed in M-ERRγWT and M-ERRγ^−/− myocytes by Western blot. β-Tubulin was included as a loading control. Bottom panel: quantitation of P-NF-κB vs. NF-κB levels from ≥2 independent lysates was performed using ImageJ software. B) Immunofluorescence analysis indicates that P-NF-κB is localized in the nucleus of M-ERRγ^−/− MBs. At least 50 cells were analyzed, and representative images are shown at ×600. Scale bars = 10 μm. C) Real-time PCR analysis of NF-κB target genes MuRF1, interleukin-6 (IL-6), and IκBα was performed and normalized to 36B4 expression levels. Experiments were performed in triplicate, and results are expressed as means ± se. *P < 0.05.

Figure 6.

NF-κB inhibitor QNZ partially rescues MT formation and reduces NF-κB target gene expression in M-ERRγ^−/− primary myocytes. A) Bright-field images of M-ERRγWT and M-ERRγ^−/− MTd2 myocytes treated with the indicated concentrations of QNZ (Santa Cruz Biotechnology) when switched into differentiation medium. At least 50 cells from duplicate samples were analyzed, and representative images are shown at ×100. Scale bars = 100 μm. B) Expression of MuRF1, IκBα, IL-6, and SOD2 in M-ERRγWT and M-ERRγ^−/− primary myocytes treated with the indicated concentrations of QNZ during differentiation was analyzed by real-time PCR and normalized to 36B4 mRNA levels. Experiments were performed in triplicate, and results are expressed as means ± se. *P < 0.05.

Figure 7.

FoxO1 and FoxO3a are activated in M-ERRγ^−/− mice. A) Expression of FoxO1, FoxO3a, and their target gene atrogin-1 in M-ERRγWT and M-ERRγ^−/− myocytes during differentiation was analyzed by real-time PCR and normalized to 36B4 expression levels. Experiments were performed in triplicate, and results are expressed as means ± se. *P < 0.05. B) Top panels: Western blot analysis of phospho-FoxO1, FoxO1, phospho-FoxO3a, and FoxO3a in M-ERRγWT and M-ERRγ^−/− myocytes during differentiation. Arrow indicates position of P-FoxO1 band. β-Tubulin was included as a loading control. Bottom panel: quantitation of phospho-FoxO1 vs. FoxO1 and phospho-FoxO3a vs. FoxO3a from ≥2 independent lysates was performed using ImageJ software.

Figure 8.

NAC treatment rescues MT formation and reduces atrogene expression in M-ERRγ^−/− myocytes. A) Bright-field images of M-ERRγWT and M-ERRγ^−/− MTd2 myocytes treated with the indicated concentrations of NAC at the time of plating. Cells were analyzed in triplicate in 3 independent trials, and representative images are shown at ×100. B) Immunofluorescence analysis for cytochrome c in M-ERRγWT and M-ERRγ^−/− MTd3 myocytes treated with 1 mM NAC when plated. At least 50 cells were analyzed, and representative images are shown at ×600. C) Real-time PCR analysis of atrogin-1 and MuRF1 expression in M-ERRγWT and M-ERRγ^−/− myocytes treated with 1 mM NAC when plated. Results were normalized to 36B4 mRNA expression levels. Experiments were performed in triplicate, and results are expressed as means ± se. Scale bars = 100 μm (A); 10 μm (B). *P < 0.05.