Concerted regulation of skeletal muscle metabolism and contractile properties by the orphan nuclear receptor Nr2f6

Abstract

Background: The maintenance of skeletal muscle plasticity upon changes in the environment, nutrient supply, and exercise depends on regulatory mechanisms that couple structural and metabolic adaptations. The mechanisms that interconnect both processes at the transcriptional level remain underexplored. Nr2f6, a nuclear receptor, regulates metabolism and cell differentiation in peripheral tissues. However, its role in the skeletal muscle is still elusive. Here, we aimed to investigate the effects of Nr2f6 modulation on muscle biology in vivo and in vitro.

Methods: Global RNA-seq was performed in Nr2f6 knockdown C2C12 myocytes (N = 4-5). Molecular and metabolic assays and proliferation experiments were performed using stable Nr2f6 knockdown and Nr2f6 overexpression C2C12 cell lines (N = 3-6). Nr2f6 content was evaluated in lipid overload models in vitro and in vivo (N = 3-6). In vivo experiments included Nr2f6 overexpression in mouse tibialis anterior muscle, followed by gene array transcriptomics and molecular assays (N = 4), ex vivo contractility experiments (N = 5), and histological analysis (N = 7). The conservation of Nr2f6 depletion effects was confirmed in primary skeletal muscle cells of humans and mice.

Results: Nr2f6 knockdown upregulated genes associated with muscle differentiation, metabolism, and contraction, while cell cycle-related genes were downregulated. In human skeletal muscle cells, Nr2f6 knockdown significantly increased the expression of myosin heavy chain genes (two-fold to three-fold) and siRNA-mediated depletion of Nr2f6 increased maximal C2C12 myocyte's lipid oxidative capacity by 75% and protected against lipid-induced cell death. Nr2f6 content decreased by 40% in lipid-overloaded myotubes and by 50% in the skeletal muscle of mice fed a high-fat diet. Nr2f6 overexpression in mice resulted in an atrophic and hypoplastic state, characterized by a significant reduction in muscle mass (15%) and myofibre content (18%), followed by an impairment (50%) in force production. These functional phenotypes were accompanied by the establishment of an inflammation-like molecular signature and a decrease in the expression of genes involved in muscle contractility and oxidative metabolism, which was associated with the repression of the uncoupling protein 3 (20%) and PGC-1α (30%) promoters activity following Nr2f6 overexpression in vitro. Additionally, Nr2f6 regulated core components of the cell division machinery, effectively decoupling muscle cell proliferation from differentiation.

Conclusions: Our findings reveal a novel role for Nr2f6 as a molecular transducer that plays a crucial role in maintaining the balance between skeletal muscle contractile function and oxidative capacity. These results have significant implications for the development of potential therapeutic strategies for metabolic diseases and myopathies.

Keywords: Metabolism; Muscle atrophy; Nr2f6; Skeletal muscle; Transcription.

Figure 1

Nr2f6 knockdown derepresses the expression of genes involved in metabolism and myogenesis. (A) Network of ontology terms enriched in the differentially expressed genes in the transcriptomics of transient Nr2f6 knockdown in C2C12 cells. Groups of similar terms were manually curated and encircled as indicated. Upregulated elements in red and downregulated in blue (N = 4–5). (B, E) Gene ontology enrichment of downregulated and upregulated genes. (C) Panel of myogenic differentiation markers differentially regulated by Nr2f6 knockdown with myogenic regulatory factors (MRFs) and myosin isoforms and their respective fibre expression patterns.^S69,S70 (D) Gene expression measured by RT‐qPCR of markers of myogenic differentiation in primary human skeletal muscle myotubes (HSMC) transfected with control non‐target RNAi (siScr) or siNr2f6 (N = 5–6). Circles represent individual donors. *P < 0.05 using ratio paired two‐tailed Student's t‐test.

Figure 2

Nr2f6 depletion increases fatty acid oxidation and protects cells against lipid‐induced stress. (A) Fatty acid‐dependent oxygen consumption (FAO) assay in control siScr and siNr2f6 C2C12 myocytes using palmitate (palm.) as substrate. Data displayed as mean ± SD. (B) Calculated respiratory parameters of the FAO assay are displayed as a line on the mean and minimum to max bars (N = 3). *P < 0.05 using unpaired two‐tailed Student's t‐test. (C) Mitochondrial and total superoxide production following palmitate treatment in shGFP and shNr2f6 stable C2C12 cells (N = 3–4). (D) Relative gene expression using RT‐qPCR in stable Nr2f6 knockdown (shNr2f6) C2C12 cells and control shGFP stable cells (N = 4–8). (E) Control shGFP or Nr2f6 knockdown cells were exposed to 500 μM palmitate (palm.), or vehicle (vehi.) for 20 h, and relative cell death was measured by propidium iodide staining (N = 5). (F) Relative Nr2f6 protein content in the gastrocnemius of mice fed with a control chow diet or high‐fat diet (HFD) for 16 weeks. Inlet: Representative western blot image (N = 6). Boxplot with whiskers spanning minimum to maximal and box edges 25th–75th percentile, the line at the median and + at the mean. *P < 0.05 using unpaired two‐tailed Student's t‐test.

Figure 3

Nr2f6 inhibits PGC1‐α and UCP3 gene expression. (A) Relative gene expression using RT‐qPCR in stable Nr2f6‐myc overexpression myotubes (N = 4–5). (B) Densitometry and representative images of PGC‐1α western blot in stable Nr2f6‐myc overexpression myotubes (N = 5). (C) Relative gene expression by RT‐qPCR in stable Nr2f6 knockdown myotubes (N = 3–5). (D) PGC‐1α 2kbp luciferase reporter assay in HEK293 cells overexpressing HA‐tagged Nr2f6 (Nr2f6‐HA) or control empty vector (EV). (E) Luciferase activity of UCP3 promoter transactivation assay in cells transfected with Nr2f6‐HA or siNr2f6 (N = 3). (F) Relative gene expression by RT‐qPCR in human primary skeletal myotubes (HSMC) transfected with siNr2f6 or siScr (N = 6). Boxplot with whiskers spanning minimum to maximal and box edges 25th–75th percentile, the line at the median and + at the mean. *P < 0.05 using ratio paired two‐tailed Student's t‐test for the experiments with human cells and unpaired for the other comparisons.

Figure 4

The transcriptional landscape of Nr2f6 overexpression exhibits a decrease in metabolism and an increase in inflammatory markers (A, B) Gene ontology enrichment of downregulated and upregulated genes. (C, D, E) Validation of selected markers modulated in the microarray by RT‐qPCR (N = 4). Insert on (C): Representative western blot for validation of Nr2f6 overexpression in the tibialis anterior samples in empty vector control (EV) and Nr2f6 electroporated (Nr2f6‐myc) muscles. Circles represent individual samples. *P < 0.05 using ratio paired two‐tailed Student's t‐test. The numbers above some bars indicate the P‐value. CAT, catalase; CD, cluster differentiation; CPT1B, carnitine palmitoyltransferase 1B; F4‐80, EGF‐like module‐containing mucin‐like hormone receptor‐like 1; mLIF, monocyte locomotion inhibitory factor; MYH1 and MYH2, myosin heavy chain 1–2; MYOD1, myogenic differentiation 1; MYOG, myogenin; NDUFA1, NADH:Ubiquinone oxidoreductase subunit A1; PDK4, pyruvate dehydrogenase kinase 4; PPARGC1A, PPARG coactivator‐1 α; SDHB, succinate dehydrogenase b; SOD1 and SOD2, superoxidase dismutase 1–2; TGFB, transforming growth factor beta; UCP3, uncoupling protein 3; UQCRC1, ubiquinol‐cytochrome c reductase core protein 1; VEGF, vascular endothelial growth factor.

Figure 5

Overexpression of Nr2f6 induces muscle atrophy and impairs muscle force production. (A) Weight of tibialis anterior muscles (TA) electroporated with empty vector (EV) or Nr2f6‐myc coding plasmid. Top: Representative photo of electroporated muscles (N = 12). (B) Representative images of myosin heavy chain staining in the electroporated TAs for fibre type determination. In green, MHC IIA; in red, MHC IIB; unstained fibres as IIX. No substantial number of MHCI fibres were stained, therefore the corresponding channel was omitted (N = 7). (C, D) Total and type‐segmented fibre counts (N = 7). (E) Ex vivo contraction maximal force production in FDB muscles electroporated with control empty vector (EV) or Nr2f6‐coding plasmid (N = 5). Data are displayed as individual animals and bars at the mean. *P < 0.05 using ratio paired two‐tailed Student's t‐test.

Figure 6

Nr2f6 increases myoblast proliferation rates. (A) Interaction network of genes consistently regulated by Nr2f6 overexpression and knockdown and with detected Nr2f6 binding motif at the promoter region. In blue: Genes downregulated; in red: Genes upregulated. The number of connections of each gene increases clockwise. (B) Representative images of the western blot of control (EV) or Nr2f6‐myc electroporated tibialis anterior muscles. Densitometric quantitation of the indicated protein bands is provided in Figure S5B (N = 4). (C, E) Proliferation curves of stable C2C12 cell lines and the calculated doubling time. (D, F) RT‐qPCR of cell cycle arrest markers in Nr2f6 knockdown and overexpression stable cell lines, respectively (N = 4–6). Boxplot with whiskers spanning minimum to maximal and box edges 25th–75th percentile, the line at the median and + at the mean. *P < 0.05 using unpaired Student's t‐test. The numbers above some bars and next to antibodies indicate the P‐value when >0.05.