CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Activity-dependent control of fiber type profile in skeletal muscle

Calcineurin-NFAT signalling in skeletal muscle

 

Previous studies in our lab had shown that calcineurin signaling has an important role in mediating the effect of nerve activity in modulating the fiber type-specific gene programs in regenerating skeletal muscle without affecting muscle fiber size (Serrano et al, PNAS 2001). We subsequently focused on the transcription factor NFAT, a major calcineurin dowstream effector. Using both gain-of-function and loss-of-function approaches in vivo, we found that NFAT is involved in nerve activity-dependent remodeling of the muscle phenotype, specifically in the activation of the slow gene program (McCullagh et al., PNAS 2004). NFAT transcriptional activity, monitored with NFAT-dependent reporters, is higher in slow than fast muscles, is decreased by denervation in slow muscles and is increased by electrical stimulation of denervated muscles with low frequency impulse patterns, mimicking the firing pattern of slow motor neurons. In addition, transfection with VIVIT, a specific inhibitor of calcineuring-mediated NFAT activation, blocks the activation of the slow gene program in regenerating slow muscles and the maintenance of the slow program in adult slow muscle. Finally, a constitutively active NFATc1 mutant stimulates the myosin heavy chain (MyHC) slow and inhibits the MyHC-fast gene promoter in adult muscle (Fig. 1) and is able to induce MyHC-slow expression in regenerating muscle. These results support the notion that calcineurin-NFAT signalling acts as nerve activity sensor in skeletal muscle and controls nerve activity-dependent fast/slow fiber type switching.

 

More recently, we addressed the issue of the relative role of different NFAT isoforms (NFATc1, c2, c3 and c4). We observed that nerve activity controls the subcellular localization of NFATc1 in vivo. NFATc1 is localized predominantly in the nuclei of slow muscle fibers but is mainly cytoplasmic in fast fibers. Low frequency stimulation, tipical of slow motor neurons, induces a rapid NFATc1 nuclear import in fast fibers (Fig. 2), whereas denervation induces nuclear export in slow fibers. NFATc1 nuclear import/export is a rapid event, as determined by direct in vivo analyses in living mice with two-photon microscopy (Tothova et al, J Cell Sci 2006). In contrast, another NFAT isoform, NFATc4, appears to be constitutively nuclear in both innervated and denervated fast and slow muscles. The role of the various NFATs was analyzed using specific siRNAs in co-transfection experiments with MyHC promoter-reporter constructs (Murgia et al, PNAS 2009). Interestingly, NFATc4 silencing was found to inhibit selectively the fast MyHC-2B promoter, that was unaffected by silencing other NFATs, whilst NFATc2 and c3 silencing inhibits the fast MyHC-2A and MyHC-2X promoters, that are unaffected by NFATc1 silencing. These results suggest that NFATs may also control the phenotype of fast muscle fibers and that fiber type specificity may be maintained by sequentially recruiting NFAT isoforms to the nucleus in predictable combinations, possibly in response to graded calcium changes induced by specific nerve activity patterns.

CaMKII, NFAT and AMPK signaling in the control of GLUT4 gene transcription

In addition to the calcineurin-NFAT pathway, two other signaling pathways have been implicated in the activity-dependent regulation of muscle genes. We focused on the regulation of the GLUT4 gene, which is known to be an important target of both insulin and muscle contraction. Indeed, exercise increases muscle GLUT4 gene transcription, and overexpression of GLUT4 in skeletal muscle has been shown to promote glucose uptake and counteract insulin resistance in diabetic mice. To determine the relative role of three signaling pathways, CaMKII, calcineurin and AMPK on GLUT4 gene regulation, we have used an in vivo transfection approach. Plasmids coding for protein inhibitors of CaMKII or calcineurin were co-transfected in slow and fast muscles with a GLUT4 enhancer-reporter construct, either in normal mice or in mice expressing a dominant negative AMPK mutant. We found that the three pathways redundantly control the GLUT4 enhancer, presumably affecting MEF2 transcriptional activity, a point of convergence of different signaling pathways on muscle gene regulation (Murgia et al, J Physiol 2009).

Novel/ancient myosins in mammalian skeletal muscles

The mammalian genome contains three ancient sarcomeric myosin heavy chain (MYH) genes, MYH14/7b, MYH15 and MYH16, in addition to the two well characterized clusters of skeletal and cardiac MYHs. MYH16 is expressed in jaw muscles of carnivores, however the expression pattern of MYH14 and MYH15 is not known. We have found that in rat and mouse, MYH14 and miR-499 transcripts are detected in heart, slow muscles and extraocular (EO) muscles, whereas MYH15 transcripts are detected exclusively in EO muscles. However, MYH14 protein is detected only in a minor fibre population in EO muscles, corresponding to slow-tonic fibres, and in bag fibres of muscle spindles. MYH15 protein is present in most fibres of the orbital layer of EO muscles and in the extracapsular region of bag fibres. The identification of the expression pattern of MYH14 and MYH15 brings to completion the inventory of the MYH isoforms involved in sarcomeric architecture of skeletal muscles and provides an unambiguous molecular basis to study the contractile properties of slow tonic fibres in mammals (Rossi et al, submitted).

Publications

  • Calabria E, Ciciliot S, Moretti I, Garcia M, Picard A, Dyar KA, Pallafacchina G, Tothova J, Schiaffino S, Murgia M (2009) NFAT isoforms control activity-dependent muscle fiber type specification. Proc. Natl. Acad. Sci. U.S.A. 106:13335-40.
  • Murgia M, Jensen TE, Cusinato M, Garcia M, Richter EA, Schiaffino S (2009) Multiple signalling pathways redundantly control glucose transporter GLUT4 gene transcription in skeletal muscle. J. Physiol. (Lond.) 587:4319-27.
  • Schiaffino S, Sandri M, Murgia M (2007) Activity-dependent signaling pathways controlling muscle diversity and plasticity. 22:269-78.
  • Tothova J, Blaauw B, Pallafacchina G, Rudolf R, Argentini C, Reggiani C, Schiaffino S (2006) NFATc1 nucleocytoplasmic shuttling is controlled by nerve activity in skeletal muscle. J. Cell. Sci. 119:1604-11.
  • McCullagh KJ, Calabria E, Pallafacchina G, Ciciliot S, Serrano AL, Argentini C, Kalhovde JM, Lømo T, Schiaffino S (2004) NFAT is a nerve activity sensor in skeletal muscle and controls activity-dependent myosin switching. Proc. Natl. Acad. Sci. U.S.A. 101:10590-5.

Grants

European Commission, VI Programme: MYORES (Network of Excellence) "Multi-organismic Approach to Study Normal and Aberrant Muscle Development, Function and Repair". (5 yrs: 2005-2009). € 510.000,00

Collaborations

  • Terje Lomo, Department of Physiology, University of Oslo, Norway.
  • Erik Richter, Copenhagen Muscle Research Centre, Department of Exercise and Sport Sciences, University of Copenhagen, Denmark.

 

PI photo

Stefano Schiaffino

Contact information

email  E-mail

email  049 7923232

Participating staff

Marta Murgia
Assistant Professor

Stefano Ciciliot
Postdoc

Irene Moretti
Postdoc

Kenneth Dyar
Postdoc

Alberto C. Rossi
PhD student