Nuclear receptor/microRNA circuitry links muscle fiber type to energy metabolism
Zhenji Gan, John Rumsey, Bethany C. Hazen, Ling Lai, Teresa C. Leone, Rick B. Vega, Hui Xie, Kevin E. Conley, Johan Auwerx, Steven R. Smith, Eric N. Olson, Anastasia Kralli, Daniel P. Kelly
Zhenji Gan, John Rumsey, Bethany C. Hazen, Ling Lai, Teresa C. Leone, Rick B. Vega, Hui Xie, Kevin E. Conley, Johan Auwerx, Steven R. Smith, Eric N. Olson, Anastasia Kralli, Daniel P. Kelly
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Research Article Muscle biology

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.

Authors

Zhenji Gan, John Rumsey, Bethany C. Hazen, Ling Lai, Teresa C. Leone, Rick B. Vega, Hui Xie, Kevin E. Conley, Johan Auwerx, Steven R. Smith, Eric N. Olson, Anastasia Kralli, Daniel P. Kelly

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Figure 4

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

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PPARβ/δ and ERRγ function cooperatively to control miR-208b and miR-499 ...
(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.