Nuclear receptor/microRNA circuitry links muscle fiber type to energy metabolism

Abstract

The mechanisms involved in the coordinate regulation of the metabolic and structural programs controlling muscle fitness and endurance are unknown. Recently, the nuclear receptor PPARβ/δ was shown to activate muscle endurance programs in transgenic mice. In contrast, muscle-specific transgenic overexpression of the related nuclear receptor, PPARα, results in reduced capacity for endurance exercise. We took advantage of the divergent actions of PPARβ/δ and PPARα to explore the downstream regulatory circuitry that orchestrates the programs linking muscle fiber type with energy metabolism. Our results indicate that, in addition to the well-established role in transcriptional control of muscle metabolic genes, PPARβ/δ and PPARα participate in programs that exert opposing actions upon the type I fiber program through a distinct muscle microRNA (miRNA) network, dependent on the actions of another nuclear receptor, estrogen-related receptor γ (ERRγ). Gain-of-function and loss-of-function strategies in mice, together with assessment of muscle biopsies from humans, demonstrated that type I muscle fiber proportion is increased via the stimulatory actions of ERRγ on the expression of miR-499 and miR-208b. This nuclear receptor/miRNA regulatory circuit shows promise for the identification of therapeutic targets aimed at maintaining muscle fitness in a variety of chronic disease states, such as obesity, skeletal myopathies, and heart failure.

Trial registration: ClinicalTrials.gov NCT00401791.

Figure 1. PPARβ/δ and PPARα regulate opposing muscle fiber-type programs.

(A) Expression of the slow-twitch myosin Myh7 and representative slow/fast-twitch troponin genes (qRT-PCR) in soleus muscle from indicated genotypes (n = 5–13 mice per group). (B) Cross-section of soleus muscle from 3- to 4-month-old male MCK-PPARβ/δ and MCK-PPARα mice (n = 5 mice per group) stained for myosin I ATPase activity as well as MHC fiber typing by immunofluorescence (IF) of soleus of indicated genotypes (MHC1 [green], MHC2a [blue], MHC2b [red], and MHC2x [unstained]). Scale bar: 500 μm. (C) Quantification of immunofluorescence data shown in B expressed as mean percentage total muscle fibers. (D) Muscle fiber typing in GC of WT and PPARα-null (PPARα KO) mice (n = 5 mice per group) as described in B. Scale bar: 500 μm. (E) Quantification of contractile protein gene expression in GC from indicated genotypes (n = 6–9 mice per group). (F) qRT-PCR results and Western blot analysis of Myh expression in skeletal myotubes harvested from WT mice subjected to adenovirus-based overexpression of PPARβ/δ shRNA (KD) compared with control shRNA (con) (n = 3). *P < 0.05 vs. corresponding controls. All values represent mean ± SEM and are shown as arbitrary units (AU) normalized to corresponding controls.

Figure 2. PPARβ/δ and PPARα control distinct miRNA subnetworks in skeletal muscle.

(A and B) qRT-PCR analysis of Myh7/miR-208b and Myh7b/miR-499 levels in GC muscles of indicated genotypes (n = 5–8 mice per group). Schematics at the top indicate the location of the miR-208b and miR-499 genes within the Myh7 and Myh7b genes. Exons are denoted in the boxes. (C and D) Mean expression levels (qRT-PCR) in myotubes harvested from GC of WT mice or indicated genotypes subjected to adenovirus-based overexpression (OE) of PPARβ/δ compared with GFP control or shRNA-mediated KD of PPARβ/δ compared with control shRNA (con) (n = 3). *P < 0.05 vs. corresponding controls. All values represent mean ± SEM and are shown as arbitrary units normalized to corresponding controls.

Figure 3. miR-208b and miR-499 mediate opposing effects of PPARβ/δ and PPARα on muscle fiber-type determination.

(A) qRT-PCR analysis of Myh7 and Myh4 transcripts in myotubes harvested from muscles of MCK-PPARβ/δ and NTG mice subjected to inhibition of miR-208b and/or miR-499 (n = 4). (–), negative control anti-miR. (B) Muscle fiber typing, as described in the legend for Figure 1B, of soleus muscle from indicated genotypes (n = 5 mice per group). Scale bar: 500 μm. (C and D) Gene expression (qRT-PCR) in soleus muscle from indicated genotypes (n = 5–8 mice per group). (E) Mean running time and distance for indicated genotypes during endurance exercise on a motorized treadmill (n = 5–7 mice per group). *P < 0.05 vs. NTG. One-way ANOVA was used for statistics. All values represent mean ± SEM and are shown as arbitrary units normalized to corresponding controls.

Figure 4. PPARβ/δ and ERRγ function cooperatively to control miR-208b and miR-499 expression.

(A) The putative conserved ERR binding site within the Myh7 and Myh7b promoter regions. (B) ERR mRNA (GC, bottom) and ERRγ protein (GC and soleus, top) expression in muscle from indicated genotypes (n = 6–15 mice per group). (C) Myh transcript levels in myotubes harvested from muscles of MCK-PPARβ/δ and NTG mice subjected to ERRγ siRNA or scrambled control (Con) (n = 4). (D) Site-directed mutagenesis was used to abolish the ERR response element (top). The mMyh7b.Luc.1K (WT) or ERRmut.mMyh7b.Luc.1K promoter-reporters were used in cotransfection studies in C2C12 myotubes in the presence or absence of ERRβ or ERRγ (n = 3) (bottom). (E) Results of SYBR green–based quantification of ERRγ ChIP assays performed on WT primary mouse myotubes (n = 3). The graphs show enrichment relative (%) to input, while the schematics show PCR primer set location and the location of the putative conserved ERR binding sites relative to the Myh7 and Myh7b gene transcription start site (+1): –2882ERR-RE and –7ERR-RE (described in A) and a previously identified +20077ERR-RE (23) were analyzed. PCR primer sets located in the 2.0-kb (–2.0k) region upstream of the Myh7b promoter transcription start site were used as a negative control. –10.2 k, –9.4 k, –2882, +20077, and –7 represent locations in the promoter regions corresponding to the transcription start site = +1. *P < 0.05 vs. corresponding controls; ^‡P < 0.05 vs. control siRNA or vector alone. One-way ANOVA was used for statistics in C and D. All values represent mean ± SEM and are shown as arbitrary units normalized to corresponding controls.

Figure 5. ERRβ/ERRγ are required for type I muscle fiber formation.

(A) miR-208b and miR-499 levels in GC muscles of indicated genotypes (n = 6 mice per group). (B and C) Gene expression (qRT-PCR) in GC muscles from indicated genotypes (n = 6–10 mice per group). (D) Muscle fiber typing, as described in the legend for Figure 1B, of GC of WT and skeletal muscle–specific ERRγ/ERRβ double-deficient mice (n = 5–7 mice per group). Scale bar: 500 μm. (E) Mean distance (meters) traveled by 14-week-old female ERRβ/γ dmKO mice and control littermates during endurance (run-to-exhaustion) exercise on a motorized treadmill (n = 14 mice per group). *P < 0.05 vs. corresponding controls. All values represent mean ± SEM and are shown as arbitrary units normalized to corresponding controls.

Figure 6. The ERRγ/miRNA circuit is induced by CLFS in skeletal myotubes in culture.

(A) MHC fiber typing by immunofluorescence in CLFS or unstimulated myotubes (MHC1 [green], MHC2b [red]). Scale bar: 200 μm. Quantification of the immunofluorescence data shown on the left expressed as mean percentage total myotubes (n = 5). (B) qRT-PCR analysis of Myh7/miR-208b, Myh7b/miR-499, and Esrrg levels in CLFS and unstimulated mouse myotubes subjected to ERRγ siRNA or scrambled control (Con) (n = 3). *P < 0.05 vs. corresponding controls. One-way ANOVA was used for statistics in B. All values represent mean ± SEM and are shown as arbitrary units normalized to corresponding controls.

Figure 7. ERRγ/miRNA axis is associated with muscle contractile and metabolic properties in humans.

Samples from 5 to 8 “active” and 15 to 17 healthy sedentary controls were used for this analysis. (A) mRNA expression levels of MYH7B, MYH7, ESRRG, miR-499, and miR-208B were determined by qRT-PCR. Data represent the mean ± SEM. Significant differences were analyzed using 2-sample t test. *P < 0.05 vs. sedentary controls. (B) Correlation between ESRRG gene expression and that of muscle contractile genes, miR-499, and miR-208B. Spearman correlation analysis was used to determine the correlation. (C) Correlation between ESRRG and miR-499 expression and the type I fiber percentage, ATP_max, and VO_2max. Pearson correlation analysis was used to determine the correlation.

Figure 8. Model of nuclear receptor/miRNA circuit in the coordinate control of energy metabolism and muscle fiber type.

The schematic depicts a proposed model for a nuclear receptor/miRNA circuit that controls muscle type I fiber-type program. ERRγ (and likely ERRβ) activates miR-208b and miR-499, triggering a type I fiber program, while PPARα suppresses the program. The effect of exercise could stimulate this program via the transcriptional coactivator PGC-1α and possibly via ERRγ/β.