Type I muscle fibers are equipped with a high-capacity mitochondrial system and a distinct set of contractile protein isoforms poised for endurance. The circuitry involved in the coordinate control of these seemingly distinct but highly interrelated processes is unknown. Recently, a clue was provided when the nuclear receptors PPARβ/δ and ERRγ, transcriptional regulators of cellular energy metabolism (28
), were independently shown to drive an increase in slow oxidative fiber composition and vascularity in mice (14
). Herein, we show that PPARβ/δ and ERRγ function to activate transcription of the Myh7
genes, increasing the levels of miR-208b and miR-499 and, thereby, triggering a cascade of muscle slow-twitch contractile protein gene expression (19
). The related nuclear receptor PPARα suppresses the activating effects of PPARβ/δ/ERRγ on Myh7/miR-208b
Muscle endurance is determined by multiple factors, including capacity for mitochondrial fuel oxidation and ATP synthesis, fiber-type mix, and vascularity (1
). The inducible transcriptional coactivator PGC-1α was shown previously to coordinately trigger these programs in muscle-specific transgenic mice (13
). Thereafter, muscle transgenes for PPARβ/δ and ERRγ, known transcription factor targets of PGC-1, were shown to exhibit a trained muscle phenotype, including increased expression of well-established PPAR/ERR target genes involved in mitochondrial energy transduction and oxidative phosphorylation, together with an increased proportion of type I fibers (14
). We sought to delineate the regulatory circuitry linking the activation of energy metabolic genes with the fiber-type program by taking advantage of the markedly different muscle phenotypes of the MCK-PPARα and MCK-PPARβ/δ lines. Using a combination of gene expression and miRNA profiling, we found that ERRγ/ERRβ and miR-208b/miR-499 levels were increased in parallel with expression of type I fiber genes in the muscle of MCK-PPARβ/δ mice. In striking contrast, miR-208b/miR-499 levels were suppressed in MCK-PPARα muscle, together with a lack of induction of ERRγ and ERRβ and suppression of the type I program. These results were intriguing given the recent discovery that miR-208b and miR-499 are embedded in slow-twitch MHC genes and serve to activate slow-twitch fiber program by downregulating the expression of transcriptional repressors, including Sox6
, which, in turn, suppress slow-twitch genes (19
We found that ERRγ serves a critical role in this regulatory network by directly activating transcription of the Myh7
) and Myh7b
) genes via highly conserved ERR-responsive elements. The expression of ERRγ and the related ERRβ (but not ERRα) is elevated in MCK-PPARβ/δ muscle through mechanisms that remain unknown. Interestingly, the results of our studies using myotubes in culture suggest that activation of the type I fiber program by PPARβ/δ occurs via a ligand-independent mechanism (data not shown). The mouse ERRγ has at least two different promoters. We have found that the activity of both promoters is increased in MCK-PPARβ/δ muscle, with the transcript driven by the downstream promoter exhibiting the greatest increase in MCK-PPARβ/δ muscle (data not shown). Using informatics search approaches, we have not been able to identify conserved PPAR binding motifs (37
) in potential regulatory regions in the DNA sequence spanning from approximately –100 kb upstream to approximately 50 kb downstream of the ERRγ downstream promoter transcription initiation site. Thus, we conclude that activation of ERRγ by PPARβ/δ likely occurs via an indirect mechanism. In addition, it is possible that PPARβ/δ and ERRγ interact at other levels, including, but not limited to, cooperating (directly or indirectly) to activate transcription of type I fiber genes. Our results also suggest that the ERRγ/β-driven muscle fiber-type program does not require the transcriptional coactivator PGC-1α, which has been shown to regulate fiber type, likely in response to endurance training (13
). However, it would seem likely that with certain physiological conditions, such as exercise, induction of PGC-1 could further enhance the mechanism described herein by coactivating PPARβ/δ. Taken together, our results suggest the existence of a regulatory network, as shown in Figure .
Model of nuclear receptor/miRNA circuit in the coordinate control of energy metabolism and muscle fiber type.
Muscle fiber-type proportion is determined by both fundamental developmental mechanisms and postnatal physiological stimuli, such as exercise training (1
). The tissue-selective promoters used in the generation of gain-of-function (PPAR transgene) and loss-of-function (Esrrg/Esrrb
gene knockout) mouse lines for this study are both active during prenatal development, suggesting that the mechanisms described may be capable of driving fundamental fiber-type determination. However, the results of our CLFS studies in cell culture indicate that this nuclear receptor/miRNA regulatory circuit is inducible. Interestingly, we found that ERRγ is required for the CLFS-triggered induction of this program. In addition, our survey of the human muscle samples also demonstrated that the ERRγ/miR-499 circuit was activated in the “active” group. Specifically, ESRRG
and miR-499 levels were higher in muscle samples obtained from trained “active” individuals compared with those of sedentary individuals. We also found that the expression of ESRRG
and miR-499 was strongly correlated with fiber-type proportion, expression of MYH7
genes, and measures of enhanced exercise performance (including VO2max
). However, we did not see similar correlations with PPARD
expression in humans. This could relate to species differences. Alternatively, the programs directed by PPARA
in humans may not be manifest in the “active”-versus-sedentary group comparison, because this pathway is primarily involved in other genetic programs (e.g., fundamental developmental programs). Given that we did not conduct a longitudinal training study in humans or mice, our results do not allow us to determine whether this regulatory pathway is involved in the response to endurance training or determination of fundamental fiber type during development. Obviously, these are not mutually exclusive roles, and future studies will be necessary to further delineate the function of this pathway in response to physiological inputs.
The upstream regulatory mechanisms that link inputs, such as developmental cues or exercise, to the network defined here are unknown. Evidence has emerged that exercise induces a switch in the relative abundance of transcriptional corepressor complexes, such as the nuclear receptor corepressor 1/histone deacetylase (NCoR1/HDAC) (44
) complex, and coactivator complexes containing inducible factors, such as PGC-1α (13
), at the regulatory regions of key genes that control muscle mitochondrial function and fiber type. Interestingly, NCoR1/HDAC– and PGC-1α–containing cofactor complexes are known to corepress or coactivate ERRs (44
) and PPARs (44
), respectively, thereby suggesting a mechanism whereby the pathways described here could be dynamically modulated. In addition, the transcriptional coregulator MED13 was recently shown to control metabolism downstream of miR-208a in heart, suggesting additional potential control mechanisms (49
Surprisingly, in contrast to miR-499, miR-208b levels were not higher in the “active” muscle compared with those in control. The reason for this difference compared with the results in the mouse lines is not clear. This could reflect a species-specific posttranscriptional regulatory effect. Indeed, evidence is emerging that RNA processing, including but not limited to alternative splicing and RNA degradation, are highly active and regulated in muscle. It is tempting to speculate that such mechanisms are active in human muscle such that the miR-208b steady-state levels are not increased in humans.
In summary, we have identified a gene regulatory pathway, involving nuclear receptor and miRNA signaling, which is involved in the coordinate control of muscle energy metabolism and fiber type. Increased proportion of type I fibers and enhanced mitochondrial function are linked to improved glucose tolerance and insulin sensitivity (6
). Conversely, obesity and chronic disease states, such as heart failure, result in detrained muscle, with reduced numbers of oxidative muscle fibers and diminished fuel-burning capacity. Therefore, the regulatory network described here shows promise as a candidate target for new therapeutic approaches aimed at coordinately increasing muscle type I fibers and energy metabolic capacity.