Gene regulation in skeletal and cardiac muscle

Field of Interest

Our studies aim to identify the signaling pathways which mediate the effect of nerve activity in adult skeletal muscle, and specifically the pathways which control muscle growth and fiber type specification. We use mostly genetic approaches, either transgenic models or in vivo transfection with constitutively active or dominant negative mutants of signal transducers. We predict that the dissection of the mechanisms controlling the muscle phenotype should provide a better understanding of clinically relevant issues, including the beneficial effect of physical exercise and the cause of muscle atrophy during aging.

Summary of research activity

Calcineurin-NFAT signalling in skeletal muscle

Figure 1. Nucleocytoplasmic shuttling of NFATc1-GFP visualized in muscles of living mice. Tibialis anterior muscle transiently expressing NFATc1-GFP was observed in situ by using two-photon microscopy, either during application or after suspension of low-frequency stimulation. (A,B) Micrographs depicting maximum intensity projections of either 15 confocal sections of individual fibers taken at the indicated time points after start (A) or suspension of low frequency stimulation (B); bar, 25 m. (C) Graphs show the mean fluorescence intensity of 30 (import, n=3 fibers) or 49 nuclei (export, n=3 fibers). Data (mean ± s.e.m.) were obtained from background-corrected sum plots and were either normalized to the 60-minute values (import, left panel) or 0-minute values (export, right panel). Bar, 5 micron. For further details see Tothova et al, J Cell Sci 2006.

Figure 1. Nucleocytoplasmic shuttling of NFATc1-GFP visualized in muscles of living mice. Tibialis anterior muscle transiently expressing NFATc1-GFP was observed in situ by using two-photon microscopy, either during application or after suspension of low-frequency stimulation. (A,B) Micrographs depicting maximum intensity projections of either 15 confocal sections of individual fibers taken at the indicated time points after start (A) or suspension of low frequency stimulation (B); bar, 25 m. (C) Graphs show the mean fluorescence intensity of 30 (import, n=3 fibers) or 49 nuclei (export, n=3 fibers). Data (mean ± s.e.m.) were obtained from background-corrected sum plots and were either normalized to the 60-minute values (import, left panel) or 0-minute values (export, right panel). Bar, 5 micron. For further details see Tothova et al, J Cell Sci 2006.
[click image to enlarge]

Our lab has previously shown that the calcineurin-NFAT pathway acts as a nerve activity sensor and controls activity-dependent myosin switching and fiber type specification (Serrano et al, PNAS 2001; McCullagh et al, PNAS 2004). More recently, we focused on two aspects of NFAT signaling in skeletal muscle:
1. NFATc1 nuclear translocation in response to specific nerve activity patterns (Tothova et al, 2006). NFATc1 is localized predominantly in the nuclei of slow muscle fibers while it is mainly cytoplasmic in fast fibers. Continuous low frequency stimulation, tipical of slow motor neurons, induces a rapid NFATc1 nuclear import in fast fibers, as determined by direct in vivo analyses with two-photon microscopy (Figure 1).

2. The role of NFAT isoforms in activity-dependent muscle fiber type specification. The NFAT family consists of four transcription factors, NFATc1, c2, c3, and c4. To determine the role of each isoform, we have carried out an in vivo analysis of NFAT isoform-specific silencing by RNAi in adult muscles (Calabria et al, 2009).

We are also using isolated Flexor Digitorum Brevis (FDB) muscle fibers from adult mice to determine the relative role of calcium derived from RyR and IP3R stores on NFATc1 nuclear translocation. Our findings indicate that calcium derived from RyR channels but not from IP3R channels is involved in NFATc1 nuclear translocation in mature muscle fibers.

Akt/PKB activation prevents activity-dependent muscle damage in dystrophin-deficient mdx mice

Figure 2. (A) Schematic representation of the generation of a muscle-specific inducible Akt transgenic line and its crossing with mdx mice. These mice were obtained by crossing a transgenic line expressing the Cre recombinase under a skeletal muscle-specific promoter (mlc1f) with a line expressing activated Akt fused with a mutated estrogen receptor only after the deletion of an upstream DNA sequence by Cre (caAkt:ER-mCre mice). We crossed these mice with the dystrophin-deficient mdx mice and induced Akt activation in adult mice with tamoxifen. (B) In vivo recordings during eccentric contractions reveal a much greater force drop (measured as percent of initial force after 20 eccentric contractions in vivo) in mdx compared to wild-type muscles. (C) Eccentric contractions in oil- and tamoxifen-treated Akt-mdx muscles show that the force drop is reduced to levels of wild-type mice by Akt activation. From Blaauw et al, 2008.

Figure 2. (A) Schematic representation of the generation of a muscle-specific inducible Akt transgenic line and its crossing with mdx mice. These mice were obtained by crossing a transgenic line expressing the Cre recombinase under a skeletal muscle-specific promoter (mlc1f) with a line expressing activated Akt fused with a mutated estrogen receptor only after the deletion of an upstream DNA sequence by Cre (caAkt:ER-mCre mice). We crossed these mice with the dystrophin-deficient mdx mice and induced Akt activation in adult mice with tamoxifen. (B) In vivo recordings during eccentric contractions reveal a much greater force drop (measured as percent of initial force after 20 eccentric contractions in vivo) in mdx compared to wild-type muscles. (C) Eccentric contractions in oil- and tamoxifen-treated Akt-mdx muscles show that the force drop is reduced to levels of wild-type mice by Akt activation. From Blaauw et al, 2008.
[click image to enlarge]

We have previously shown that in vivo transfection with a constitutively active mutant of Akt/PKB leads to muscle hypertrophy (Pallafacchina et al, PNAS 2002). We have now used an inducible muscle-specific Akt1 transgenic model to determine whether Akt activation is able to prevent activity-dependent muscle damage in mdx skeletal muscle fibers (Blaauw et al, 2008). The increased force drop induced by lengthening contractions in mdx muscles was completely prevented by Akt activation. Microarray and PCR analyses indicate that Akt activation induces up-regulation of genes coding for proteins associated with Z-disks and costameres, and for proteins with anti-oxidant or chaperone function. The protein levels of utrophin and dysferlin are also increased by Akt activation.

Autophagy in skeletal muscle: control by FoxO3 and role in organelle turnover

(in collaboration with M. Sandri)

We have established that FoxO3 transcription factor is required for the induction of autophagy in skeletal muscle in vivo (Mammucari et al, 2007; Zhao et al, 2007). These findings, together with our previous studies (Sandri et al, Cell 2004), support the notion that FoxO3 controls the two major systems of protein breakdown in skeletal muscle, the ubiquitin-proteasome and autophagy-lysosome pathways. To determine the role of autophagy in skeletal muscle, we generated mice with targeted muscle-specific inactivation of the autophagy gene, Atg7 (Masiero et al, 2009). Atg7 null muscles were atrophic and showed accumulation of abnormal mitochondria and formation of aberrant concentric membranous structures. Thus, the continuous operation of the autophagy machinery is important to maintain myofiber integrity.

Novel/ancient myosins in mammalian skeletal muscle

We have identified two novel myosin heavy chain (MYH) selectively expressed in mammalian extraocular muscles and in the intrafusal fibers of muscle spindles . The genes coding for these MYHs are conserved in evolution. MYH14 (also called MYH7b) corresponds to the MYH coding for chicken slow myosin 2 and is expressed in the slow-tonic fibers of mammalian extraocular muscles. MYH15 corresponds to the MYH coding for ventricular myosin in frogs and chicken: this is a striking case of evolutionary tinkering, whereby a myosin gene used to control cardiac pumping function in amphibians is used to control eyeball movement in mammals. These findings complete the inventory of the MYHs present in mammalian striated muscles.

On-going activities and future research plans

Activity-dependent and -independent circadian rhythms in skeletal muscle

In mammals, the hypothalamic suprachiasmatic nucleus (SCN) acts as a master pacemaker that controls circadian rhythms of behavior and metabolism and synchronizes the circadian clocks in peripheral tissues. In skeletal muscle, the SCN has a double role, as it entrains both the muscle cell-autonomous circadian clock and the circadian rhythm of locomotor activity. However, it is not known whether motor activity and muscle clock gene rhythms are coupled together and controlled by the same entrainment signals. We are examining the role of neural and non neural signals in circadian activity and clock gene expression patterns in skeletal muscle, focusing on NFAT transcription factors, which are known to act as activity sensors, and on the core clock genes Bmal1, Per1 and Per2.

Role of the myogenic regulatory factor Mrf4 in adult skeletal muscle

Mrf4 is the fourth member of the MyoD family of myogenic regulatory factors (MRFs), which includes also Myf5 and myogenin. In contrast to the other MRFs, whose expression level is high in the embryo and declines to low levels in adult muscle, Mrf4 shows a biphasic pattern of expression, with a transient expression in somites and a subsequent reappearance in fetal muscle with progressive accumulation in postnatal stages. A major unresolved issue is the role of Mrf4 in adult skeletal muscle. To address this question, we are performing RNAi experiments in vivo to determine the effect of Mrf4 transcript depletion on the muscle phenotype, with particular reference to muscle growth and fiber type specification. Appropriate rescue experiments are being performed to validate the results of RNAi tests.

Synoptic CV

Honours

Selected VIMM Publications

• 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.
• 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.
• 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.
• 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.

VIMM Publications

• 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.
• 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.
• 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.
• 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.
• 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

Contact

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