Mitochondrial function during muscle fiber type transition by a miR‐499/Fnip1/AMPK circuit

Contractile fiber type and mitochondrial function are two key factors of skeletal muscle function. However, the exact mechanism for coupling the two remains unknown. The genes encoding type I myosins Myh7/Myh7b regulate muscle fiber type switching by encoding their intronic miRNAs, miR-208b and miR-499. In a new study, Liu et al., 2016 use transgenic mice to show that miR-499 directly targets the gene encoding folliculin‐interacting protein‐1 (Fnip1), which  negatively regulates AMPK.  AMPK is a known activator of Ppargc1a (PGC-1a), which is a transcriptional co‐regulator of mitochondrial function. Targeting of Fnip1 by miR-499 drives a PGC-1a-dependent mitochondrial function, suggesting a mechanism to couple muscle fiber type and mitochondrial function.

The authors examined the expression of miR‐499 and miR‐208b during muscle fiber type transition by RT-qPCR and verified that the induced expression of Myh7/Myh7b genes matched the higher expression of miR‐208b and miR‐499. This correlated with a marked increase in mitochondrial respiration.

Using transgenic mice overexpressing miR‐499 in muscle (MCK‐miR‐499) the authors investigated the potential role of miR‐499 and miR‐208b, that function redundantly in vivo (van Rooij et al, 2009; Bell et al, 2010), in regulating mitochondrial function. The real‐time respiratory exchange ratio (RER), which indicates a tendency to preferentially oxidize fat over carbohydrate, was measured during an exercise protocol. MCK‐miR‐499 mice had a lower RER than controls indicating an enhanced exercise performance. In addition, levels of blood lactate after exercise were lower in transgenic mice. In line with the increased aerobic metabolism, mitochondrial respiration rates were significantly higher in MCK‐miR‐499 muscle compared to controls.

A broad array of mitochondrial-encoded genes, oxidative biomarkers and biomarkers associated with fatty acid uptake were all induced in MCK‐miR‐499 muscles compared to controls. Consistent with this, there was a marked increase in type I muscle fibers in the transgenic mouse muscle. Succinate dehydrogenase (SDH) activity, a marker of oxidative metabolism, was higher in the MCK‐miR‐499 muscle, while α‐glycerophosphate dehydrogenase (α‐GPDH) activity, a marker of glycolytic metabolism, was lower. This showed that miR‐499 reprograms muscle for increased mitochondrial oxidative capacity, as well as fiber type. Interestingly, Birt-Hogg-Dubé (BHD) syndrome renal tumours are also associated with up-regulation of mitochondrial-encoded genes (Klomp et al., 2010).

Using Ingenuity Pathways Analysis, the authors found that genes upregulated in MCK‐miR‐499 muscle were targets of PGC‐1α. RT-PCR confirmed that PGC‐1α mRNA levels were higher in MCK‐miR‐499 muscle along with PGC‐1α protein levels. To determine if the oxidative changes induced by miR‐499 were due to the levels of PGC‐1α the authors generated mutant mice where the PGC‐1α gene is disrupted in a miR‐499 transgenic background (499Tg/PGC‐1α mKO). Lack of PGC‐1α reduced the type I myofibers of MCK‐miR‐499 muscle, the induction of oxidative biomarker genes and the oxidative mitochondrial enzyme SDH activity. The miR‐499‐mediated enhancement of exercise capacity, the RER‐lowering effect during exercise and the reduction of lactate levels were also abolished in the absence of PGC‐1α showing that PGC‐1α is required for miR‐499‐mediated increase in mitochondrial oxidative metabolism.

Mice lacking Fnip1 have recently been shown to increase PGC‐1α signalling and oxidative metabolism (Hasumi et al, 2015, Reyes et al., 2015), the same was previously observed for FLCN deficiency (Hasumi et al., 2012), recapitulating the MCK‐miR‐499 mice phenotype. Fnip1 null mice recapitulated MCK‐miR‐499 mice metabolic phenotypes, including enhanced type I myofibers, capacity for SDH activity and mitochondrial respiration. Key regulators of aerobic metabolism such as AMPK/PGC‐1α levels and oxidative biomarkers induced in MCK‐miR‐499 muscle were similarly activated in Fnip1 null mice. AMPK inhibition abolished the Fnip1 induction of Ppargc1a mRNA showing the importance of AMPK in this mechanism. Both Fnip1 and FLCN have previously been shown to be vital for metabolic AMPK-dependent metabolic regulation (Park et al., 2012; Yan et al., 2014).

In summary, the authors unravel a novel mechanism for muscle fiber type coupled to its metabolic capacity. The myosin Myh7b gene encodes miR‐499, which directly inhibits Fnip1, leading to activation of AMPK‐PGC‐1α signalling and that triggers a muscle mitochondrial oxidative metabolism program. These findings show great therapeutic potential for a variety of metabolic diseases and muscular dystrophy.

Obtained from Liu et al., 2016
Obtained from Liu et al., (2016)
  • Liu J, Liang X, Zhou D, Lai L, Xiao L, Liu L, Fu T, Kong Y, Zhou Q, Vega RB, Zhu MS, Kelly DP, Gao X, & Gan Z (2016). Coupling of mitochondrial function and skeletal muscle fiber type by a miR-499/Fnip1/AMPK circuit. EMBO molecular medicine PMID: 27506764

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