1. Circadian rhythms in skeletal muscle (Dyar et al, Mol Metab 2014; Dyar et al, Mol Metab 2015)
Circadian rhythms control metabolism and energy homeostasis, but the role of the intrinsic muscle clock has not been explored. We generated conditional and inducible mouse lines with muscle-specific ablation of the core clock gene Bmal1. Skeletal muscles from these mice showed impaired insulin-stimulated glucose uptake with reduced protein levels of GLUT4, the insulin-dependent glucose transporter, and TBC1D1, a Rab-GTPase involved in GLUT4 translocation. Pyruvate dehydrogenase (PDH) activity was also reduced due to altered expression of the circadian genes Pdk4 and Pdp1, coding for PDH kinase and phosphatase, respectively. PDH inhibition leads to reduced glucose oxidation and diversion of glycolytic intermediates to alternative metabolic pathways, as revealed by metabolome analysis (in collaboration with Paolo Sassone Corsi). The impaired glucose metabolism induced by muscle-specific Bmal1 knockout suggests that a major physiological role of the muscle clock is to prepare for the transition from the rest/fasting phase to the active/feeding phase, when glucose becomes the predominant fuel for skeletal muscle.
Changes in glucose metabolism induced by muscle-specific knockout of the core clock gene Bmal1. In control mice glucose uptake in skeletal muscle is enhanced by insulin at the transition from the rest/fasting to the active/feeding phase, and PDH activity is increased by upregulation of PDP1 and downregulation of PDK4. In Bmal1 mKO mice, insulin-dependent glucose uptake is impaired due to decreased GLUT4 and TBC1D1 protein levels, and PDH activity is reduced due to downregulation of PDP1 and upregulation of PDK4. As a result of reduced HK2 and PDH activity induced by loss of Bmal1, glucose metabolism is channeled to alternative pathways, including the polyol, pentose phosphate and glucuronic acid pathways, as shown by metabolome analysis.
2. A novel pathway to boost muscle growth and prevent muscle wasting: the MRF4-MEF2 axis (Moretti et al, Nat Commun 2016)
The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. We have found that knockdown of MRF4 in adult muscle causes hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after MRF4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by MRF4 RNAi. The MRF4 RNAi-dependent increase in fiber size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofiber hypertrophy. The nuclear localization of the MEF2 co-repressor HDAC4 is impaired by MRF4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia.
MRF4 knockdown in adult skeletal muscle causes muscle hypertrophy, which is inhibited by dominant negative MEF2 and is accompanied by upregulation of MEF2-dependent muscle genes and activation of MEF2 reporters. MRF4 is exclusively expressed in skeletal muscle, not in heart or any other tissue, thus providing a unique tissue-specific target for the identification of treatments aimed at preventing muscle wasting during aging and cachexia.
3. Mitochondrial specialization revealed by single muscle fiber proteomics (Murgia et al, EMBO Rep 2015; Murgia et al, Cell Rep 2017)
In collaboration with Marta Murgia and Matthias Mann we have analyzed the proteome of single skeletal muscle fibers using a highly sensitive mass-spectrometry-based proteomic workflow developed in Mann’s laboratory. This study revealed significant differences in the mitochondrial proteome of the four major fiber types present in mouse skeletal muscle. A major result has been the demonstration of the differential distribution of the two mitochondrial isocitrate dehydrogenases, IDH2 and IDH3. Type 1/slow fibers contain high levels of IDH2 and relatively low levels of IDH3, whereas fast 2X and 2B fibers show an opposite expression pattern. The findings suggest that in skeletal muscle IDH2 functions in the forward direction of the Krebs cycle and that substrate flux along the cycle occurs predominantly via IDH2 in type 1 fibers and via IDH3 in 2X and 2B fibers. IDH2-mediated conversion of isocitrate to α-ketoglutarate leads to the generation of NADPH, which is critical to buffering the H2O2 produced by the respiratory chain. Nicotinamide nucleotide transhydrogenase (NNT), the other major mitochondrial enzyme involved in NADPH generation, is also more abundant in type 1 fibers. We suggest that the continuously active type 1 fibers are endowed with a more efficient H2O2 scavenging capacity to cope with the higher levels of reactive oxygen species production.
More recently, we have developed a highly sensitive single muscle fiber proteomics workflow to study human aging and found that the senescence of slow and fast muscle fibers is characterized by diverging metabolic and protein quality control adaptations (Murgia et al, 2017). Whereas mitochondrial content declines with aging in both fiber types, glycolysis and glycogen metabolism are upregulated in slow but downregulated in fast muscle fibers. Slow fibers upregulate a subset of actin and myosin chaperones whereas an opposite change happens in fast fibers. These changes in metabolism and sarcomere quality control may be related to the ability of slow, but not fast, muscle fibers to maintain their mass during aging.
Left panel: scheme of the Krebs cycle with highlighted IDH2 and IDH3 pathways involved in the isocitrate to α-ketoglutarate conversion. Bottom: relative abundance of IDH2 and IDH3 subunits in the different fiber types, as derived from single muscle fiber proteomics (relative values normalized to OXPHOS proteins). Note that IDH3 subunits, like most other Krebs cycle enzymes, show highest values in type 2X fibers, whereas IDH2 is especially abundant in type 1 fibers.
Right panel: scheme showing some of the mitochondrial systems involved in reactive oxygen species (ROS) scavenging. Superoxide anions O2-•, which are formed during aerobic respiration, are converted to H2O2 by mitochondrial superoxide dismutase (SOD2) and then to water by peroxidases (Prx), that use reduced glutathione (GSH). GSH levels are maintained by glutathione reductase (GR), with NADPH levels being continuously replenished by the activity of IDH2 and nicotinamide nucleotide transhydrogenase (NNT). Bottom: single muscle fiber proteomics shows that both IDH2 and NNT are especially abundant in type 1/slow fibers and expressed at much lower levels in fast type 2X/2B fibers (relative values normalized to OXPHOS proteins). (Modified from Schiaffino et al, 2015).