Stefano Schiaffino




Group Members

Lab Manager

Irene Moretti

Postdoctoral Fellow

Kenneth Dyar

Gene regulation in skeletal muscle


Field of Interest

Figure 1. The suprachiasmatic nucleus (SCN) of the hypothalamus controls the circadian rhythm of motor activity and feeding, both of which may affect the circadian rhythm of the muscle clock together with other SCN-controlled factors, such as hormones (dashed arrow). Circadian genes in skeletal muscle may be controlled by the intrinsic local clock or directly by various extrinsic factors, including activity and feeding.
Figure 1.
[click image to enlarge]

Our studies aim to identify the signaling pathways which control muscle gene regulation and affect muscle growth, fiber type specification and muscle metabolism, with particular reference to the effect of nerve activity in adult skeletal muscle. We use mostly genetic approaches, either transgenic models or in vivo transfection with constitutively active or dominant negative mutants of signal transducers. The dissection of the genetic mechanisms controlling the muscle phenotype may provide a better understanding of clinically relevant issues, including the beneficial effect of physical exercise, the role of skeletal muscle in metabolic diseases and the cause of muscle atrophy during aging. Our most recent work is focused on muscle gene regulation by circadian rhythms.

Summary of research activity

Muscle insulin resistance and metabolic dysregulation caused by muscle-specific knockout of the core clock gene Bmal1

Figure 2. Muscle-specific Bmal1 knockout. The left panel illustrates the decrease in insulin-stimulated 2-deoxyglucose uptake by isolated soleus muscles from muscle-specific Bmal1 knockout (KO) mice compared to controls (Ctrl). The right panel shows that the level of glucose oxidation in muscles from KO fed mice is significantly lower than that seen in control fed mice (C) and similar to that detected in muscles from control fasted mice (F).
Figure 2. Muscle-specific Bmal1 knockout.
[click image to enlarge]

Gene expression in skeletal muscle fibers, like in all cells in the body, is regulated by local intrinsic clocks and by extrinsic circadian factors, such as feeding and hormones, under the control of a central pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus (Figure 1). Circadian rhythms control metabolism and energy homeostasis, and disruption of either the central circadian pacemaker or peripheral clocks in liver, pancreas or adipose tissue results in metabolic dysregulation. However, the role of the skeletal muscle clock has never been explored. We have generated two mouse lines, including an inducible one, with muscle-specific ablation of the core clock gene Bmal1. Skeletal muscles from these mice showed impaired insulin-stimulated glucose uptake and reduced glucose oxidation (Figure 2). Pyruvate dehydrogenase (PDH) activity was reduced (Figure 3), due to altered expression of circadian genes Pdk4 and Pdp1, coding for the PDH kinase and phosphatase controlling PDH activity in skeletal muscle (Figure 4). The altered response to insulin in glucose uptake is apparently due to reduced GLUT4 protein levels and downregulation of the circadian gene Tbc1d1, coding for a Rab-GTPase involved in GLUT4 translocation (Figure 4). PDH inhibition leads to diversion of glycolytic intermediates to alternative metabolic pathways, as revealed by metabolome analysis. The impaired glucose metabolism induced by muscle-specific Bmal1 knockout suggest that a major physiological role of the muscle clock is to promote the transition from the fasting to the feeding phase, when glucose becomes the predominant fuel for skeletal muscle.

Figure 3. Pyruvate dehydrogenase (PDH) activity in gastrocnemius muscles from control and muscle-specific Bmal1 knockout mice across the day-night cycle (ZT0 = lights on, ZT12 = lights off; dark phase shown by shaded area). Right panel shows mean 24-hr PDH activity in muscles from Ctrl and KO mice (mean percent relative to control).
Figure 3.
[click image to enlarge]

Role of the myogenic regulatory factor Mrf4 in adult skeletal muscle

Mrf4 is a member of the bHLH family of myogenic regulatory factors, which also includes MyoD, Myf5 and myogenin. Mrf4 shows a biphasic pattern of expression, with transient expression in somites and subsequent reappearance in late fetal stages with progressive accumulation in the postnatal period. To address the role of Mrf4 in adult skeletal muscle, we examined the effect of knockdown in vivo. Muscle fibers transfected with plasmids coding for Mrf4 shRNAs showed increased muscle fiber size, suggesting that Mrf4 acts as a negative regulator of muscle growth (Fig. 5). Muscle atrophy induced by denervation was prevented by Mrf4 knockdown. The specificity of this effect was confirmed by rescue experiments with RNAi resistant Mrf4s (Fig. 6). Muscle hypertrophy induced by Mrf4 gene silencing is accompanied by upregulation of a large number of muscle-specific genes, including components of sarcomeric and membrane cytoskeleton, excitation-contraction coupling and energy metabolism. This response is associated with stimulation of MEF2 transcriptional activity and upregulation of MEF2 target genes, an effect specific for Mrf4, because myogenin knockdown in adult muscle leads to reduced MEF2 transcriptional activity.

Figure 4. Muscle-specific Bmal1 knockout causes the downregulation of two circadian genes, Tbc1d1, coding for a Rab-GTPase involved in insulin-dependent GLUT4 translocation to the plasma membrane, and Pdp1, coding for the PDH phosphatase that activates PDH activity. Transcript levels were determined by qPCR across the day-night cycle, protein levels were analyzed by Western blotting (WB) at ZT20. The resulting blocks in glucose metabolism are shown in the scheme on the right.
Figure 4.
[click image to enlarge]

Future research plans

Activity-dependent control of circadian rhythms in muscle gene expression

In skeletal muscle, gene expression is controlled by nerve activity through different signaling pathways that control both muscle fiber size and the fiber type profile (Schiaffino & Reggiani, Physiol Rev 2011; Schiaffino et al., FEBS J 2013). However, although “locomotor activity” has been traditionally a major readout of the circadian systems in both flies and mice, the relation of contractile activity and the muscle clock is not known.

Figure 5. Myofiber hypertrophy induced by Mrf4 knockdown in rat soleus muscle. Muscles were transfected by electroporation with plasmids coding for LacZ shRNAs as a control (left panel) or Mrf4 shRNAs (right panel) and co-transfected with GFP. Sections of muscles at 14 days after transfection were stained for dystrophin to visualize fiber profiles. Transfected fibers are identified by GFP fluorescence.
Figure 5.
[click image to enlarge]
Physical exercise is not an appropriate experimental system to investigate the specific role of activity in muscle gene regulation, because exercise produces a variety of systemic physiological responses, such as hormonal changes and increased body temperature, which are known to affect both peripheral clocks, including the muscle clock, and the central pacemaker. Therefore we have decided to use an alternative approach by investigating the effect of complete inactivity induced by denervation on circadian gene expression in fast and slow mouse muscles. For comparison, we have examined the effect of feeding using a restricted feeding protocol, with access to food restricted to the light phase, and the effect of the intrinsic muscle clock, using the muscle-specific Bmal1 knockout model. Our preliminary studies indicate the calcineurin-NFAT pathway, previously identified in our lab as a major activity sensor in muscle cells (Serrano et al, PNAS 2001; McCullagh et al., PNAS 2004; Tothova et al., J Cell Sci 2006), is also involved in mediating the activity-dependent regulation of a number of circadian muscle genes. On the other hand, we found that the oscillations of muscle clock genes are only slightly affected by denervation.

Role of the muscle clock in the transition from the resting to the activity phase of the circadian cycle

Figure 6. Left panel : HEK 293 cells were transfected with rat Mrf4 (rMrf4) and co-transfected with a control pSUPER vector that generates shRNAs against LacZ, or a vector that generate shRNAs against Mrf4. GFP was co-transfected to determine transfection efficiency. Note efficient knockdown of rMrf4 expression with the rMrf4-specific sequences, whereas human Mrf4 (hMrf4) is resistant to RNAi because of a single mismatch in the sequence recognized by shRNAs. Right panel: In adult transfected soleus muscles, the increase in fiber size induced by Mrf4 RNAi is prevented by co-transfection with RNAi-resistant hMrf4.
Figure 6.
[click image to enlarge]

The changes in glucose metabolism induced by Bmal1 muscle-specific knockout indicate that the muscle clock contributes to the metabolic adaptations that occur during the transition from the fasting to the feeding phase of the circadian cycle (see above). By comparing the gene expression profile of Bmal1 knockout and control muscles, we found that other adaptive changes in gene expression controlled by the muscle clock take place during the day/night cycle, which appear to reflect the transition from the resting to the activity phase. We are presently investigating the signaling pathways and transcription factors controlled by the muscle clock, including those dependent on adrenergic stimulation, which may be involved in the adaptation of skeletal muscle fibers to this specific transition phase.



Synoptic CV

2011–presentProfessor Emeritus, University of Padua
2000–presentVIMM Principal Investigator
1988–2002Director, Consiglio Nazionale delle Ricerche (CNR) Center of Muscle Biology
1986–1987Visiting Scientist, INSERM U 127, Hôpital Lariboisière, Paris, France
1981–2010Professor of General Pathology, School of Medicine, Univ. of Padua
1971–1981Associate Professor of General Pathology, School of Medicine, Univ. Padua
1965–1971Assistant Professor of General Pathology, School of Medicine, Univ. Padua
1963MD, University of Modena, Italy


Honours

2012Member of the Selection Committee of the Pathophysiology Section, French National Research Agency (ANR)
2011Review panel on "Exercise and metabolism", Danish Council for Independent Research/Medical Sciences
2009Member of the Scientific Council, Association Française contre les Myopathies (AFM)
2005National member of the Accademia dei Lincei, Rome
2005Member of the Scientific Committee, Enciclopedia Medica Italiana

Selected VIMM Publications

  • Ciciliot S, Rossi AC, Dyar KA, Blaauw B, Schiaffino S (2013) Muscle type and fiber type specificity in muscle wasting. Int. J. Biochem. Cell Biol. 45:2191-9.
  • Blaauw B, Del Piccolo P, Rodriguez L, Hernandez Gonzalez VH, Agatea L, Solagna F, Mammano F, Pozzan T, Schiaffino S (2012) No evidence for inositol 1,4,5-trisphosphate-dependent Ca2+ release in isolated fibers of adult mouse skeletal muscle. J. Gen. Physiol. 140:235-41.
  • Schiaffino S (2012) Tubular aggregates in skeletal muscle: just a special type of protein aggregates? Neuromuscul. Disord. 22:199-207.
  • Schiaffino S, Reggiani C (2011) Fiber types in mammalian skeletal muscles. Physiol. Rev. 91:1447-531.

VIMM Publications

  • Schiaffino S, Mammucari C (2011) Regulation of skeletal muscle growth by the IGF1-Akt/PKB pathway: insights from genetic models. Skelet Muscle 1:4.
  • Schiaffino S (2010) Fibre types in skeletal muscle: a personal account. Acta Physiol (Oxf) 199:451-63.
  • Rossi CA, Pozzobon M, Ditadi A, Archacka K, Gastaldello A, Sanna M, Franzin C, Malerba A, Milan G, Cananzi M, Schiaffino S, Campanella M, Vettor R, De Coppi P (2010) Clonal characterization of rat muscle satellite cells: proliferation, metabolism and differentiation define an intrinsic heterogeneity PLoS One 5:.
  • Ciciliot S, Schiaffino S (2010) Regeneration of mammalian skeletal muscle. Basic mechanisms and clinical implications. Curr. Pharm. Des. 16:906-14.
  • Rossi AC, Mammucari C, Argentini C, Reggiani C, Schiaffino S (2010) Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles J Physiol 588:353-364.
  • Masiero E, Agatea L, Mammucari C, Blaauw B, Loro E, Komatsu M, Metzger D, Reggiani C, Schiaffino S, Sandri M (2009) Autophagy is required to maintain muscle mass Cell Metab 10:507-515.
  • Masiero E, Agatea L, Mammucari C, Blaauw B, Loro E, Komatsu M, Metzger D, Reggiani C, Schiaffino S, Sandri M (2009) Autophagy is required to maintain muscle mass Cell Metab 10:507-515.
  • Blaauw B, Agatea L, Toniolo L, Canato M, Quarta M, Dyar KA, Danieli-Betto D, Betto R, Schiaffino S, Reggiani C (2010) Eccentric contractions lead to myofibrillar dysfunction in muscular dystrophy J Appl Physiol 108:105-111.
  • Blaauw B, Canato M, Agatea L, Toniolo L, Mammucari C, Masiero E, Abraham R, Sandri M, Schiaffino S, Reggiani C (2010) Inducible activation of Akt increases skeletal muscle mass and force without satellite cell activation FASEB J 23:3896-3905.
  • 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 106:13335-13340.
  • 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 587:4319-4327.
  • Zaglia T, Dedja A, Candiotto C, Cozzi E, Schiaffino S, Ausoni S (2009) Cardiac interstitial cells express GATA4 and control dedifferentiation and cell cycle re-entry of adult cardiomyocytes. J. Mol. Cell. Cardiol. 46:653-62.
  • Blaauw B, Mammucari C, Toniolo L, Agatea L, Abraham R, Sandri M, Reggiani C, Schiaffino S (2008) Akt activation prevents the force drop induced by eccentric contractions in dystrophin-deficient skeletal muscle. Hum. Mol. Genet. 17:3686-96.
  • Schiaffino S, Mammucari C, Sandri M (2008) The role of autophagy in neonatal tissues: just a response to amino acid starvation? Autophagy 4:727-30.
  • Mammucari C, Schiaffino S, Sandri M (2008) Downstream of Akt: FoxO3 and mTOR in the regulation of autophagy in skeletal muscle. Autophagy 4:524-6.
  • Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, Lecker SH, Goldberg AL (2007) FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab. 6:472-83.
  • Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del Piccolo P, Burden SJ, Di Lisi R, Sandri C, Zhao J, Goldberg AL, Schiaffino S, Sandri M (2007) FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab. 6:458-71.
  • Di Lisi R, Picard A, Ausoni S, Schiaffino S (2007) GATA elements control repression of cardiac troponin I promoter activity in skeletal muscle cells. BMC Mol. Biol. 8:78.
  • Schiaffino S, Sandri M, Murgia M (2007) Activity-dependent signaling pathways controlling muscle diversity and plasticity. 22:269-78.
  • Dedja A, Zaglia T, Dall'Olmo L, Chioato T, Thiene G, Fabris L, Ancona E, Schiaffino S, Ausoni S, Cozzi E (2006) Hybrid cardiomyocytes derived by cell fusion in heterotopic cardiac xenografts. FASEB J. 20:2534-6.
  • 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.
  • Bortoluzzi S, Scannapieco P, Cestaro A, Danieli GA, Schiaffino S (2006) Computational reconstruction of the human skeletal muscle secretome. Proteins 62:776-92.
  • Ausoni S, Zaglia T, Dedja A, Di Lisi R, Seveso M, Ancona E, Thiene G, Cozzi E, Schiaffino S (2005) Host-derived circulating cells do not significantly contribute to cardiac regeneration in heterotopic rat heart transplants. Cardiovasc. Res. 68:394-404.
  • Kalhovde JM, Jerkovic R, Sefland I, Cordonnier C, Calabria E, Schiaffino S, Lømo T (2005) "Fast" and "slow" muscle fibres in hindlimb muscles of adult rats regenerate from intrinsically different satellite cells. J. Physiol. (Lond.) 562:847-57.
  • 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.
  • Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K, Schiaffino S, Lecker SH, Goldberg AL (2004) Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell 117:399-412.
  • Sandri C, Di Lisi R, Picard A, Argentini C, Calabria E, Myklak K, Scartezzini P, Schiaffino S (2004) Heart morphogenesis is not affected by overexpression of the Sh3bgr gene mapping to the Down syndrome heart critical region. Hum. Genet. 114:517-9.
  • Moreno H, Serrano AL, Santalucía T, Gumá A, Cantó C, Brand NJ, Palacin M, Schiaffino S, Zorzano A (2003) Differential regulation of the muscle-specific GLUT4 enhancer in regenerating and adult skeletal muscle. J. Biol. Chem. 278:40557-64.
  • Salamon M, Millino C, Raffaello A, Mongillo M, Sandri C, Bean C, Negrisolo E, Pallavicini A, Valle G, Zaccolo M, Schiaffino S, Lanfranchi G (2003) Human MYO18B, a novel unconventional myosin heavy chain expressed in striated muscles moves into the myonuclei upon differentiation. J. Mol. Biol. 326:137-49.
  • Schiaffino S, Serrano A (2002) Calcineurin signaling and neural control of skeletal muscle fiber type and size. Trends Pharmacol. Sci. 23:569-75.
  • Pallafacchina G, Calabria E, Serrano AL, Kalhovde JM, Schiaffino S (2002) A protein kinase B-dependent and rapamycin-sensitive pathway controls skeletal muscle growth but not fiber type specification. Proc. Natl. Acad. Sci. U.S.A. 99:9213-8.
  • Serrano AL, Murgia M, Pallafacchina G, Calabria E, Coniglio P, Lømo T, Schiaffino S (2001) Calcineurin controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth. Proc. Natl. Acad. Sci. U.S.A. 98:13108-13.

Additional Publications

  • Di Lisi R, Millino C, Calabria E, Altruda F, Schiaffino S, Ausoni S (1998) Combinatorial cis-acting elements control tissue-specific activation of the cardiac troponin I gene in vitro and in vivo. J. Biol. Chem. 273:25371-80.
  • Gautel M, Fürst DO, Cocco A, Schiaffino S (1998) Isoform transitions of the myosin binding protein C family in developing human and mouse muscles: lack of isoform transcomplementation in cardiac muscle. Circ. Res. 82:124-9.
  • Schiaffino S, Reggiani C (1996) Molecular diversity of myofibrillar proteins: gene regulation and functional significance. Physiol. Rev. 76:371-423.
  • Schiaffino S, Gorza L, Sartore S, Saggin L, Ausoni S, Vianello M, Gundersen K, Lømo T (1989) Three myosin heavy chain isoforms in type 2 skeletal muscle fibres. J. Muscle Res. Cell. Motil. 10:197-205.
  • Schiaffino S, Samuel JL, Sassoon D, Lompré AM, Garner I, Marotte F, Buckingham M, Rappaport L, Schwartz K (1989) Nonsynchronous accumulation of alpha-skeletal actin and beta-myosin heavy chain mRNAs during early stages of pressure-overload--induced cardiac hypertrophy demonstrated by in situ hybridization. Circ. Res. 64:937-48.
  • Bottinelli R, Schiaffino S, Reggiani C (1991) Force-velocity relations and myosin heavy chain isoform compositions of skinned fibres from rat skeletal muscle. J. Physiol. (Lond.) 437:655-72.

Selected Seminars

2013SummerSchool on "Basic Muscle Sciences", Max-Delbruck-Centre for Molecular Medicine, Berlin, June
August Krogh Symposium "Metabolism in the extreme and extreme metabolism", Copenhagen, November
Symposium on "Muscle and matrix", Borupgaard, Snekkersten, Denmark, December
2012Conference on "The Biomedical Basis of Elite Performance", London, March
Symposium on "The aging human muscle", Copenhagen, August
Keynote lecture, European Muscle Conference, Rhodes (Greece), September
Conferences in Cell Biology, Universitè Pierre et Marie Curie, Paris, September
2011Symposium "Exercise physiology: cellular and molecular aspects", Annual Meeting of the German Physiological Society, Regensburg, March
8th International Conference on "Amino Acid/Protein Metabolism in Health and Disease", Santa Margherita Ligure (Genova), April
2nd EMBO Congress on "The molecular and cellular mechanisms regulating skeletal muscle development and regeneration", Wiesbaden, May
Conference on "Molecular mechanisms of skeletal muscle maintenance, wasting and repair", Ascona, September
2009IUPS 2009, Symposium "Energy sensing and metabolic signaling in skeletal muscle", Kyoto (Japan), 27 July - 1 August 2009
2008Seminar, Institut Pasteur, Paris (France), 1 December 2008
Meeting "From muscle remodeling to biotherapies" (Homage to Ketty Schwartz), Paris (France), 29 November 2008
EMBO Conference on Myogenesis, Girona (Spain), 24-29 September 2008
Meeting "Molecular Mechanisms Modulating Skeletal Muscle Mass & Function", Cold Spring Harbor, New York (USA), 6-9 April 2008
2007European Muscle Conference, Session "Muscle plasticity" (Chairman & speaker), Stockholm (Sweden), 9-12 September 2007
Symposium "Muscle Plasticity", 86th Congress of the German Physiological Society, Hannover (Germany), 26 March 2007
2006Seminar, Cajal Institute, Madrid (Spain), 15 December 2006
Experimental Biology 2006, American Physiological Society Symposium "Cellular and Molecular Signals Regulating Plasticity of Skeletal Muscle", San Francisco (USA), 1-6 April 2006
International Meeting "Frontiers in Myogenesis", Atlanta (USA)
2005EMBO/FEBS Workshop "Molecular and Cellular Mechanisms underlying Skeletal Muscle Formation and Repair", Fontevraud (France)
XXXV International Congress of Physiological Sciences, San Diego (USA)
2004Gordon Research Conference on "Myogenesis", Il Ciocco, Lucca (Italy) 16-21 May 2004
Killam Seminar, Montreal Neurological Institute, Mc Gill University, Montreal (Canada)

Contact

email Stefano Schiaffino
Venetian Institute of Molecular Medicine
Via Orus 2
35129 Padua — Italy