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The enzymatic activity of sialidase Neu2 is inversely regulated during in vitro myoblast hypertrophy and atrophy
Biochemical and Biophysical Research Communications 370 (2008) 376–381
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The enzymatic activity of sialidase Neu2 is inversely regulated during in vitro myoblast hypertrophy and atrophy Alessandro Fanzani a,*, Roberta Giuliani a, Francesca Colombo a, Stefania Rossi a, Elena Stoppani a, Wim Martinet b, Augusto Preti a, Sergio Marchesini a a b
Department of Biomedical Sciences and Biotechnology, Unit of Biochemistry, University of Brescia, Viale Europa 11, 25123 Brescia, Italy Division of Pharmacology, University of Antwerp, Wilrijk, Belgium
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Article history: Received 10 March 2008 Available online 31 March 2008 Keywords: Neu2 Sialidases Hypertrophy Atrophy
a b s t r a c t Sialidase Neu2 is an exoglycosidase that removes terminal sialic acids from glycolipids and glycoproteins. In this study, we investigated Neu2 expression during muscle hypertrophy and atrophy. Neu2 mRNA and enzymatic activity were significantly increased in hypertrophic myofibers. A rise in Neu2 activity was observed after constitutive activation of AKT or Igf-1 treatment as well as in myoblasts treated with vasopressin or trichostatin, an inhibitor of histone deacetylases. In contrast, myofiber atrophy obtained by dexamethasone treatment or starvation triggered a significant loss of Neu2 activity and was paralleled by downregulation of Neu2 transcript levels. Overall, we may conclude that Neu2 enzymatic activity is causally linked to proper muscle differentiation and growth. Ó 2008 Elsevier Inc. All rights reserved.
The enzymatic activity of mammalian cytosolic sialidase Neu2 usually increases during formation and growth of myotubes [1– 3], suggesting that the modulation of sialylated glycoconjugates in the cytosol of myofibers contributes to proper differentiation. In line with this theory, Neu2 expression is stimulated predominantly via the PI3K/AKT/mTOR pathway [4], a key regulator of the myogenic program [5]. Insulin-like growth factor-1 (Igf-1) plays a predominant role in muscle growth [5,6], as its muscle-specific overexpression produces a remarkable muscle hypertrophy in transgenic mice [7]. Accordingly, conditional activation of AKT in muscle produces rapid hypertrophy [8]. Over the last few years, additional factors have been suggested to stimulate myofiber differentiation and hypertrophy through additional pathways. Among these, Arg8-vasopressin has been shown to stimulate myogenic differentiation by activation of both calcineurin and Ca2+/calmodulin-dependent kinase [9,10], whereas follistatin, a member of the TGF-b family, increases muscle cell size by increased recruitment of satellite cells into pre-existing myofibers [11]. In contrast, maintenance of muscle mass is often compromised under different physiological and pathological conditions that trigger sarcomere remodelling, leading in turn to myofiber atrophy. In this study, we employed different in vitro myoblast cell models to investigate Abbreviations: FBS, fetal bovine serum; HS, horse serum; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate buffered solution; PI3K, phosphoinositide-3 kinase; MyHC, myosin heavy chain. * Corresponding author. Fax: +39 030 3701157. E-mail address: [email protected] (A. Fanzani). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.03.111
the expression of sialidase Neu2 in relation to myofiber hypertrophy and atrophy. Materials and methods Materials. All reagents were from Sigma–Aldrich, if not indicated otherwise. Cell culture and pharmacological treatments. Myoblasts were maintained at 37 °C and 5% CO2 in DMEM supplemented with 20% FBS and 100 lg/mL penicillin–streptomycin. To induce differentiation, preconfluent C2C12 cells were cultured in DMEM supplemented with 2% HS. This medium was changed daily. To induce atrophy, C2C12 myotubes were starved in glucose- and amino acid-deprived medium (10 mM Hank’s/Hepes buffer (pH 7.4)) or, alternatively, treated with 100 lM dexamethasone. Differentiation of rat L6E9 myoblasts was induced when preconfluent cells were shifted to DMEM supplemented with 1% FBS, whereas hypertrophy was obtained after administration of 10 ng/mL Igf-1 after one day of differentiation. Preconfluent rat L6C5 myoblasts were differentiated in DMEM supplemented with 1% fatty acid-free BSA, whereas hypertrophy was obtained after treatment with 0.1 lM Arg8-vasopressin (AVP). Alternatively, L6C5 myoblasts were treated with 50 nM trichostatin (TSA) for 16–18 h in high-serum medium, and then allowed to undergo hypertrophy in DMEM supplemented with 1% FBS. The myoblasts were also treated with the following pharmacological agents: PI3K inhibitor LY294002 (10 lM), mTOR inhibitor rapamycin (5 ng/ml), calcineurin inhibitor cyclosporin A (2.5 lM) and the Ca2+/calmodulin-dependent kinase inhibitor KN562 (8 lM). To quantify the myofiber size, 10 fields were randomly chosen and 10 myotubes were measured per field. The average diameter per myotube was the mean of 10 measurements taken along the length of the myotube. The fusion index was determined by counting the number of nuclei in a single myofiber. Ten myotubes in ten different microscopic fields were chosen to calculate the average number of nuclei. Sialidase assay. The enzymatic activity of cytosolic sialidase was assayed as previously described [3,4]. Briefly, ultracentrifuged cytosolic fractions of myotubes were assayed using a mixture containing 60 nmol of the synthetic substrate 4-
A. Fanzani et al. / Biochemical and Biophysical Research Communications 370 (2008) 376–381 methylumbelliferyl N-acetylneuraminic acid and 100 lg of BSA in 0.2 mL of 50 mM sodium acetate buffer (pH 5.8). After 3 h of incubation at 37 °C, the reaction was terminated by addition of 0.8 mL of 0.25 M glycine buffer (pH 10.4), and the amount of 4-methylumbelliferone released was determined fluorometrically with an excitation wavelengh of 365 nm and an emission of 450 nm. A pH curve has been set up for each experiment to determine whether the enzymatic activity assayed in the cytosolic fraction had the optimum in the typical range of cytosolic sialidases, usually between 5.6 and 6. Plasmid construction and stable trasfections. The pBABE vectors harboring a myristoylated or a kinase-dead AKT (mutated at the ATP binding site K179M) [6] were used to transfect C2C12 myoblasts. Rat Neu2 cDNA was amplified using pCDNANeu2 plasmid as template [3] and primers 50 -CGGGATCCCGATGGAGACCTGCCCCG TCCTCCAGAAA-30 and 50 -CGGGATCCCGTCAAGCGTAGTCTGGGACGTCGTATGGGTAC CCCTGAGCACCATGTACTGTGGG-30 containing BamHI restriction sites and the hemagglutinin (HA) epitope in the reverse primer. The resultant PCR product was BamHI digested and cloned in the similarly opened pBabe vector, yielding plasmid pBabe/HA-Neu2 used to transfect C2C12 myoblasts. Stable transfectants were obtained after 10–15 days of selection in medium containing the antibiotic puromycin (2 lg/mL). RT-PCR analysis. Total RNA was obtained by Tri-reagent extraction, digested with 1 U of DNase (DNA-free, Ambion), and retrotranscribed (2 lg) with 400 U of MMLV-RT (Promega). The primers (250 nM) were as follows: mouse Neu2 sialidase forward primer (50 -CGAGCCAGCAAGACGGATGAG-30 ) and reverse (50 -GGCTCTACAAGCTTACTCAC TACCCGG-30 ) were used for 29 cycles of PCR; rat Neu2 sialidase forward primer (50 -CCGTCCAGGACCTCACAGAG-30 ) and reverse (50 -TCACTGAGCACCATGTACTG-30 ) were used for 30 cycles of PCR; rat follistatin forward primer (50 -CTCTTCAAGTGG ATGATTTTC-30 ) and reverse (50 -ACAGTAGGCATTATTGGTCTG-30 ) were used for 30 rounds of PCR; mouse Murf-1 forward primer (50 -GGTGCCTACTTGCTCCTTGT-30 ) and reverse (50 -CTGGTGGCTATTCTCCTTGG-30 ) were used for 27 rounds of PCR; mouse Atrogin forward primer (50 -CGACCTGCCTGTGTGCTTAC-30 ) and reverse (50 CTTGCGAATCTGCCTCTCTG-30 ) were used for 28 cycles of PCR. Gene expression levels were normalized to gapdh mRNA expression by 23 rounds of PCR. Western blot analysis. Samples were separated by SDS–PAGE under reducing conditions and transferred to nitrocellulose membranes according to standard procedures. The following primary antibodies were used: anti-myogenin (clone F5D) and anti-GATA-2 (clone CG2-96) from Santa Cruz Biotechnology, anti-MyHC from the Hybridoma Bank (University of Iowa), anti-phospho-AKT (Ser473) and antiphospho-p70S6K (Thr389) from Cell Signalling Technology, anti-HA, and anti-tubulin from Sigma–Aldrich. After incubation with horseradish peroxidase-conjugated secondary antibodies (Chemicon), immunocomplexes were visualized using enhanced chemiluminescence reagent (Chemicon). Degradation of long-lived proteins. Bulk degradation of long-lived proteins was determined according to a method previously reported [12]. Briefly, cells were plated into 35 mm dishes and cultured in cysteine/methionine free media containing 5 lCi L-[35S] cysteine/methionine (GE Healthcare) for 6 h at 37 °C. Unincorporated radioisotopes and degraded amino acids released from short-lived proteins were removed by rinsing three times with PBS. Cells were then chased with the culture medium containing 10% FBS and 2 mM cold cysteine/methionine. After 15 h incubation, at which time short-lived proteins were being degraded, the chase medium was replaced with serum-containing DMEM (control) or Hank’s/Hepes buffer. After incubation at 37 °C for 6 h, the medium was harvested and 100% trichloroacetic acid (TCA) was added to 10% final concentration. The samples were centrifuged at 12,000g for 10 min and the acid-soluble radioactivity was measured by liquid scintillation counting. Meanwhile, the cells were fixed by adding 1 ml of 10% TCA directly to the culture dishes, washed with 10% TCA and dissolved in 1 ml of 0.2 N NaOH. Radioactivity in the samples was measured similarly. The percentage protein degradation was calculated by dividing the amount of acid-soluble radioactivity in the culture medium by the sum of acid-soluble and acid-precipitable radioactivities. Electron microscopy. Samples were fixed in 0.1 M sodium cacodylate-buffered (pH 7.4) 2.5% glutaraldehyde solution for 2 h and postfixed in 0.1 M sodium cacodylate-buffered (pH 7.4) 1% OsO4 solution for 1 h. After dehydration in an ethanol gradient (70% ethanol [20 min], 96% ethanol [20 min], 100% ethanol [2 20 min]), samples were incubated with propylenoxid (2 10 min), impregnated with a mixture of propylenoid/LX-112 (Ladd Research Industries, 1:1) and embedded in LX112. Ultrathin sections were stained with uranyl acetate and lead citrate. Sections were examined in a Jeol-100 CX II TEM at 80 kV. Immunofluorescence microscopy. The myotubes grown on 12 mm glass coverslips were coated with 20 lg/mL laminin (Roche), fixed with ice cold methanol and then incubated with rat monoclonal anti-HA antibody (clone 3F10, Roche) followed by an anti-rat Alexa Fluor 594-conjugated secondary antibody (Molecular Probes). Alternatively, cells were incubated with anti-MyHC antibody (Hybridoma Bank, University of Iowa) followed by a biotinylated anti-mouse antibody and Alexa Fluor 488 streptavidin-conjugate (dilution 1:800). Fluorescent staining of myotubes was observed under an Axiovert S100 microscope (Zeiss). Statistics. All data are expressed as means ± SEM. Statistical significance was determined using t-Student analysis. A p value of <0.05 was considered significant.
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Results and discussion In vitro myoblast differentiation and hypertrophy enhance Neu2 enzymatic activity In this study, we analyzed the Neu2 enzymatic activity in myoblasts undergoing differentiation and hypertrophy upon different stimuli. Different pathways have been characterized to promote myoblast fusion and growth; in particular, the PI3K/AKT/mTOR pathway plays a pivotal role during myofiber growth [5,6], as its conditional activation in skeletal muscle induces rapid hypertrophy in transgenic mice [8]. Accordingly, the constitutive activation of AKT (caAKT) by expression of a myristoylated AKT form [6] led to sustained hypertrophy of C2C12 myoblasts, as revealed by morphological analysis (Fig. 1A) and myofiber size measurements (Fig. 1B, top); in caAKT myofibers, an increased phosphorylation of AKT and its direct downstream target p70S6K were detected via Western blotting as compared to control (Fig. 1B, bottom), whereas Neu2 transcript levels were upregulated (Fig. 1B, bottom). Under these conditions, a strong increase of sialidase enzymatic activity was detectable in the cytosol of hypertrophic myotubes already two days after differentiation (Fig. 1C). Inhibition of mTOR activity in caAKT myoblasts after treatment with rapamycin abolished the myofiber hypertrophy (not shown) and caused a significant loss of Neu2 activity (Fig. 1C). Also expression of a kinase inactive form of AKT (kiAKT) in C2C12 cells reduced myofiber size (not shown) [6] and downregulated Neu2 activity (Fig. 1C), indicating that the activation of the AKT/mTOR pathway stimulates sustained Neu2 enzymatic activity that may accompany muscle hypertrophy. We further analyzed Neu2 activity in L6E9 myoblasts, a cell model which has been well characterized to develop hypertrophy upon Igf-1 treatment [13]. Igf-1 induced already at day 3 a pronounced myofiber hypertrophy as compared to untreated L6E9 myoblasts (Fig. 1D). This effect was accompanied by elevated expression of myogenin as detected via Western blotting and by increased transcription of Neu2 mRNA (Fig. 1E), followed by increased Neu2 enzymatic activity (Fig. 1F). Because PI3K and calcineurin activity are both required to switch myoblasts from a proliferative to a myogenic condition [14,11], co-administration of Igf-1 with the PI3K inhibitor LY294002 (LY) or the calcineurin inhibitor cyclosporin A (CsA) reverted the effects induced by Igf1 and resulted in loss of Neu2 activity (Fig. 1F). To obtain additional evidence that increased Neu2 expression may depend on calcineurin activity in myotubes, we stimulated L6C5 myoblasts with Arg8vasopressin (AVP), a neurohypophyseal peptide that by interacting with V1 type receptors triggers hypertrophy mainly through the activation of calcineurin and Ca2+/calmodulin-dependent kinase [9,10]. Myoblasts AVP-treated for 6 days formed larger myotubes as compared to control cells and showed two typical hallmarks of muscle hypertrophy [13], the reorganization of nuclei into nuclear rings (Fig. 1G) and increased expression of the transcription factor GATA-2 (Fig. 1H). Under these conditions, AVP treatment upregulated Neu2 transcription significantly (Fig. 1H) and triggered a progressive, long-lasting (6 days) increase in Neu2 activity (Fig. 1I) that was suppressed after single or simultaneous administration of two specific inhibitors of calcineurin and Ca2+/calmodulin-dependent kinase from day 3 to 6 (d3–d6) (Fig. 1J), a time point during which the myogenic program was already started. Finally, we tested whether Neu2 sialidase activity was modulated during myofiber hypertrophy caused by an augmented recruitment of myoblasts into pre-existing myofibers. In particular, follistatin is considered to play a pivotal role in this process through a mechanism that involves the inhibition of classes I–II histone deacetylases (HDAC) [11,15,16]. After administration of trichostatin
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Fig. 1. In vitro myoblast hypertrophy enhanced Neu2 enzymatic activity. (A) Parental and stably transfected caAKT C2C12 myoblasts were differentiated for 2 days to visualize the myofiber size. (B) Quantification of myofiber diameter (top) and Western blot analysis of AKT and P70S6K phosphorylation followed by Neu2 transcript RT-PCR analysis (bottom) in parental and caAKT myoblasts after 2 days of differentiation. (C) Neu2 enzymatic activity was assayed in parental and caAKT myotubes, in the absence or presence of rapamycin (5 ng/mL). In addition, Neu2 activity was determined in C2C12 stably transfected with a kinase inactive form of AKT (kiAKT). (D) Morphology of rat L6E9 myoblasts subjected to Igf-1 (10 ng/ml). (E) Western blot analysis of myogenin expression and RT-PCR analysis of Neu2 transcript in Igf-1 treated and untreated myoblasts. (F) Neu2 enzymatic activity assayed in untreated or Igf-1 treated myoblasts at day three, in the absence or presence of the PI3K inhibitor LY (10 lM) or the calcineurin inhibitor CsA (2.5 lM). (G) Treatment of L6C5 myoblasts with 0.1 lM Arg8-vasopressin (AVP) triggered hypertrophy. (H) Expression of GATA-2 by Western blot and Neu2 by RT-PCR analysis in AVP-treated and untreated myotubes. (I) Neu2 enzymatic activity in AVP-treated and control myoblasts. (J) Neu2 activity in myofibers treated with AVP (6 days) in the presence or absence of CsA (2.5 lM) and/or KN562 (8 lM) administered from day 3 to 6 (d3–d6). (K) Treatment of L6C5 myoblasts with 50 nM TSA triggered hypertrophy (day 3). (L) Fusion index of control and TSA-treated L6C5 myotubes. (M) RT-PCR analysis of follistatin and Neu2 in untreated and TSA-treated L6C5 myoblasts. (N) Neu2 activity in control untreated and TSA-treated L6C5 myoblasts. (*P < 0.05 vs control, **P < 0.05 vs *).
A. Fanzani et al. / Biochemical and Biophysical Research Communications 370 (2008) 376–381
(TSA), an inhibitor of HDAC, the size of L6C5 myofibers was considerably augmented (Fig. 1K) after 3 days as compared to normally differentiated cells, and this effect was mediated by increased cell recruitment, as shown by the fusion index (Fig. 1L). Accordingly,
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TSA treatment triggered a remarkable increase of follistatin expression in L6C5 myoblasts as compared to untreated cells, followed by a rise in Neu2 transcript levels (Fig. 1M). Neu2 activity was significantly increased 5 days after TSA treatment as compared
Fig. 2. In vitro myoblast atrophy decreased Neu2 transcript and enzymatic activity. C2C12 myoblasts were differentiated for 5 days and then treated with dexamethasone (dex) for 48 h or incubated in glucose- and amino acid-free medium (starv) for 15 h to induce muscle atrophy. (A) Mean myotube size shown is relative to control, dexamethasone (dex) and starved (starv) myotubes. (B) Transmission electron microscopy of C2C12 myotubes (day 5) starved in 10 mM Hank’s/Hepes buffer (pH 7.4) for 12 h. Control myotubes displayed a normal cell morphology, whereas starvation triggered the formation of autophagic vesicles (boxed area, shown at higher magnification in the right panel). (C) Degradation of long-lived proteins in control and starved myotubes. (D) Semiquantitative RT-PCR analysis for detection of Atrogin, Murf-1, and Neu2 transcripts. The mRNA expression profiles were normalized by gapdh expression. (E) Neu2 enzymatic activity was assayed in atrophic myofibers (dex and starv) as compared to control. (F) C2C12 myotubes stably transfected with HA-Neu2 protein were subjected to either dexamethasone or starvation treatment. HA-Neu2 expression was analyzed via Western blotting together with MyHC protein expression. (G) HA-Neu2 and MyHC expression were analyzed by fluorescence microscopy in control and atrophic myofibers (dex and starv). Scale bar = 100 lm (*P < 0.05 vs control).
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to untreated cells (Fig. 1N), thus after the increase in myofiber size mediated by increased satellite recruitment. This finding suggests that Neu2 is involved in growth and maintaining of myofiber size after muscle fusion. Overall we may conclude that Neu2 enzymatic activity usually reaches maximal activity when muscle growth occurs. Two different stimuli trigger myofiber growth: (i) increased protein synthesis due to sustained AKT and calcineurin activation and (ii) increased myoblast recruitment, a mechanism which is important during the repair of muscle myofibers after muscle injury. Myotube atrophy caused by dexamethasone or serum starvation treatment impairs Neu2 activity Muscle atrophy usually occurs under physiological and pathological conditions such as ageing, muscle unloading, cancer, AIDS, denervation and dystrophies. Under these conditions, increased protein breakdown and diminished protein synthesis in turn lead to unbalanced steady-state levels of proteins triggering a reduction in myofiber size [17]. To investigate whether Neu2 expression was modulated in atrophic myofibers, terminally differentiated C2C12 myotubes were treated with 100 lM dexamethasone (dex) for 48 h or starved in glucose- and amino acid-deprived medium for 15 h (starv). As shown in Fig. 2A, the diameter of the myofibers was significantly reduced in both conditions suggesting that an atrophic program occurs in myoblasts. Usually the bulk of intracellular proteins during muscle atrophy is degraded by two main proteolytic systems: the endosome–lysosome system which degrades cytoplasmic components engulfed by autophagic vacuoles through the activity of acidic cathepsins [18], and the proteasome 26S complex which triggers degradation of ubiquitinated proteins via an ATP-dependent system [19–21]. We first detected two hallmarks of autophagy in starved atrophic myofibers, namely the formation of autophagic vacuoles visualized by transmission electron microscopy (Fig. 2B), and a significant rise in the degradation of long-lived proteins (Fig. 2C). In addition, the ATP-proteasome-dependent pathway was activated both in starved and dex-treated myofibers, as shown by increased expression of two predictive atrophy-related E3 ubiquitin ligases, Atrogin and Murf-1 (Fig. 2D) [20,21]. Under both conditions, Neu2 mRNA levels were downregulated (Fig. 2D). Also Neu2 enzymatic activity was significantly decreased in C2C12 atrophic myotubes (Fig. 2E), with the most pronounced effect after starvation. To test whether downregulation of Neu2 activity was dependent on protein loss, C2C12 myotubes stably transfected with HA-Neu2 were subjected to either dexamethasone treatment or starvation. Western blots showed that HA-Neu2 protein was downregulated as compared to control myotubes at day 5 (Fig. 2F), and this effect was well paralleled by downregulation of MyHC (Fig. 2F), a marker of terminal muscle differentiation. Moreover, when we analyzed the expression of both endogenous MyHC and transfected HA-Neu2 in myotubes by immunofluorescence, we found a diffuse staining pattern of both these proteins along the cytosol of myofibers (Fig. 2G). Indeed, the fluorescence intensity was much lower in atrophic myofibers as compared to control (Fig. 2G), and the reduction in fluorescence was more evident in the case of starvation. These data suggest that a specific impairment of Neu2 sialidase expression occurs in atrophic myotubes under starvation and dexamethasone treatments. Of note, overexpression of Neu2 was not able to rescue the morphology of atrophic myofibers because protein loss under these conditions is broad and not Neu2-specific. Overall, further studies are needed to evaluate how Neu2 loss is accomplished during myofiber atrophy, either via increased protein breakdown or via reduced protein synthesis, two main processes that usually occur during
the sarcomere remodelling to regulate the protein half-life. Although the decrease of PI3 K/AKT/mTOR pathway is a well-documented effect during myofiber atrophy [22] that might play a role in diminishing Neu2 protein synthesis, the specific mechanism by which Neu2 breakdown potentially occurs is actually under investigation. Acknowledgments We thank Antonio Musarò and Sergio Adamo (University of Rome) for providing L6E9 and L6C5 myoblasts. This work was supported by grants from 60% MIUR to A.F. and to S.M. and by grants from ‘‘Amici per il cuore” to S.M. W. Martinet is a postdoctoral fellow of the Fund for Scientific Research (FWO)-Flanders (Belgium). References [1] T. Miyagi, K. Konno, Y. Emori, H. Kawasaki, K. Suzuki, A. Yasui, S. Tsuik, Molecular cloning and expression of cDNA encoding rat skeletal muscle cytosolic sialidase, J. Biol. Chem. 268 (1993) 26435–26440. [2] K. Sato, T. Miyagi, Involvement of an endogenous sialidase in skeletal muscle cell differentiation, Biochem. Biophys. Res. Commun. 221 (1996) 826–830. [3] A. Fanzani, R. Giuliani, F. Colombo, D. Zizioli, M. Presta, A. Preti, S. Marchesini, Overexpression of cytosolic sialidase Neu2 induces myoblast differentiation in C2C12 cells, FEBS Lett. 547 (2003) 183–188. [4] A. Fanzani, F. Colombo, R. Giuliani, A. Preti, S. Marchesini, Insulin-like growth factor 1 signaling regulates cytosolic sialidase Neu2 expression during myoblast differentiation and hypertrophy, FEBS J. 273 (2006) 3709– 3721. [5] S.C. Bodine, T.N. Stitt, M. Gonzalez, W.O. Kline, G.L. Stover, R. Bauerlein, E. Zlotchenko, A. Scrimgeour, J.C. Lawrence, D.J. Glass, G.D. Yancopoulos, Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo, Nat. Cell Biol. 3 (2001) 1014– 1019. [6] C. Rommel, S.C. Bodine, B.A. Clarke, R. Rossman, L. Nunez, T.N. Stitt, G.D. Yancopoulos, D.J. Glass, Mediation of IGF-1-induced skeletal myotube hypertrophy by PI(3)K/Akt/mTOR and PI(3)K/Akt/GSK3 pathways, Nat. Cell Biol. 3 (2001) 1009–1013. [7] A. Musarò, K. McCullagh, A. Paul, L. Houghton, G. Dobrowolny, M. Molinaro, E.R. Barton, H.L. Sweeney, N. Rosenthal, Localized Igf-1 transgene expression sustains hypertrophy and regeneration in senescent skeletal muscle, Nat. Genet. 27 (2001) 195–200. [8] K.M. Lai, M. Gonzalez, W.T. Poueymirou, W.O. Kline, E. Na, E. Zlotchenko, T.N. Stitt, A.N. Economides, G.D. Yancopoulos, D.J. Glass, Conditional activation of akt in adult skeletal muscle induces rapid hypertrophy, Mol. Cell Biol. 24 (2004) 9295–9304. [9] B.M. Scicchitano, L. Spath, A. Musaro, M. Molinaro, S. Adamo, C. Nervi, AVP induces myogenesis through the transcriptional activation of the myocyte enhancer factor 2, Mol. Endocrinol. 16 (2002) 1407–1416. [10] B.M. Scicchitano, L. Spath, A. Musaro, M. Molinaro, N. Rosenthal, C. Nervi, S. Adamo, Vasopressin-dependent myogenic cell differentiation is mediated by both Ca2+/calmodulin-dependent kinase and calcineurin pathways, Mol. Biol. Cell. 16 (2005) 3632–3641. [11] S. Iezzi, M. Di Padova, C. Serra, G. Caretti, C. Simone, E. Maklan, G. Minetti, P. Zhao, E.P. Hoffman, P.L. Puri, V. Sartorelli, Deacetylase inhibitors increase muscle cell size by promoting myoblast recruitment and fusion through induction of follistatin, Dev. Cell 6 (2004) 673–684. [12] S. Furuta, E. Hidaka, A. Ogata, S. Yokota, T. Kamata, Ras is involved in the negative control of autophagy through the class I PI3-kinase, Oncogene 23 (2004) 3898–3904. [13] A. Musarò, N. Rosenthal, Maturation of the myogenic program is induced by postmitotic expression of insulin-like growth factor I, Mol. Cell. Biol. 19 (1999) 3115–3124. [14] P. Kaliman, F. Viñals, X. Testar, M. Palacín, A. Zorzano, Phosphatidylinositol 3kinase inhibitors block differentiation of skeletal muscle cells, J. Biol. Chem. 271 (1996) 19146–19151. [15] A. Pisconti, S. Brunelli, M. Di Padova, C. De Palma, D. Deponti, S. Baesso, V. Sartorelli, G. Cossu, E. Clementi, Follistatin induction by nitric oxide through cyclic GMP: a tightly regulated signaling pathway that controls myoblast fusion, J. Cell Biol. 172 (2006) 233–244. [16] S. Iezzi, G. Cossu, C. Nervi, V. Sartorelli, P.L. Puri, Stage-specific modulation of skeletal myogenesis by inhibitors of nuclear deacetylases, Proc. Natl. Acad. Sci. USA 99 (2002) 7757–7762. [17] D.J. Glass, Signalling pathways that mediate skeletal muscle hypertrophy and atrophy, Nat. Cell Biol. 5 (2003) 87–90. [18] D. Bechet, A. Tassa, D. Taillandier, L. Combaret, D. Attaix, Lysosomal proteolysis in skeletal muscle, Int. J. Biochem. Cell Biol. 37 (2005) 2098– 2114. Review.
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The enzymatic activity of sialidase Neu2 is inversely regulated during in vitro myoblast hypertrophy and atrophy Alessandro Fanzani a,*, Roberta Giuliani a, Francesca Colombo a, Stefania Rossi a, Elena Stoppani a, Wim Martinet b, Augusto Preti a, Sergio Marchesini a a b
Department of Biomedical Sciences and Biotechnology, Unit of Biochemistry, University of Brescia, Viale Europa 11, 25123 Brescia, Italy Division of Pharmacology, University of Antwerp, Wilrijk, Belgium
a r t i c l e
i n f o
Article history: Received 10 March 2008 Available online 31 March 2008 Keywords: Neu2 Sialidases Hypertrophy Atrophy
a b s t r a c t Sialidase Neu2 is an exoglycosidase that removes terminal sialic acids from glycolipids and glycoproteins. In this study, we investigated Neu2 expression during muscle hypertrophy and atrophy. Neu2 mRNA and enzymatic activity were significantly increased in hypertrophic myofibers. A rise in Neu2 activity was observed after constitutive activation of AKT or Igf-1 treatment as well as in myoblasts treated with vasopressin or trichostatin, an inhibitor of histone deacetylases. In contrast, myofiber atrophy obtained by dexamethasone treatment or starvation triggered a significant loss of Neu2 activity and was paralleled by downregulation of Neu2 transcript levels. Overall, we may conclude that Neu2 enzymatic activity is causally linked to proper muscle differentiation and growth. Ó 2008 Elsevier Inc. All rights reserved.
The enzymatic activity of mammalian cytosolic sialidase Neu2 usually increases during formation and growth of myotubes [1– 3], suggesting that the modulation of sialylated glycoconjugates in the cytosol of myofibers contributes to proper differentiation. In line with this theory, Neu2 expression is stimulated predominantly via the PI3K/AKT/mTOR pathway [4], a key regulator of the myogenic program [5]. Insulin-like growth factor-1 (Igf-1) plays a predominant role in muscle growth [5,6], as its muscle-specific overexpression produces a remarkable muscle hypertrophy in transgenic mice [7]. Accordingly, conditional activation of AKT in muscle produces rapid hypertrophy [8]. Over the last few years, additional factors have been suggested to stimulate myofiber differentiation and hypertrophy through additional pathways. Among these, Arg8-vasopressin has been shown to stimulate myogenic differentiation by activation of both calcineurin and Ca2+/calmodulin-dependent kinase [9,10], whereas follistatin, a member of the TGF-b family, increases muscle cell size by increased recruitment of satellite cells into pre-existing myofibers [11]. In contrast, maintenance of muscle mass is often compromised under different physiological and pathological conditions that trigger sarcomere remodelling, leading in turn to myofiber atrophy. In this study, we employed different in vitro myoblast cell models to investigate Abbreviations: FBS, fetal bovine serum; HS, horse serum; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate buffered solution; PI3K, phosphoinositide-3 kinase; MyHC, myosin heavy chain. * Corresponding author. Fax: +39 030 3701157. E-mail address: [email protected] (A. Fanzani). 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.03.111
the expression of sialidase Neu2 in relation to myofiber hypertrophy and atrophy. Materials and methods Materials. All reagents were from Sigma–Aldrich, if not indicated otherwise. Cell culture and pharmacological treatments. Myoblasts were maintained at 37 °C and 5% CO2 in DMEM supplemented with 20% FBS and 100 lg/mL penicillin–streptomycin. To induce differentiation, preconfluent C2C12 cells were cultured in DMEM supplemented with 2% HS. This medium was changed daily. To induce atrophy, C2C12 myotubes were starved in glucose- and amino acid-deprived medium (10 mM Hank’s/Hepes buffer (pH 7.4)) or, alternatively, treated with 100 lM dexamethasone. Differentiation of rat L6E9 myoblasts was induced when preconfluent cells were shifted to DMEM supplemented with 1% FBS, whereas hypertrophy was obtained after administration of 10 ng/mL Igf-1 after one day of differentiation. Preconfluent rat L6C5 myoblasts were differentiated in DMEM supplemented with 1% fatty acid-free BSA, whereas hypertrophy was obtained after treatment with 0.1 lM Arg8-vasopressin (AVP). Alternatively, L6C5 myoblasts were treated with 50 nM trichostatin (TSA) for 16–18 h in high-serum medium, and then allowed to undergo hypertrophy in DMEM supplemented with 1% FBS. The myoblasts were also treated with the following pharmacological agents: PI3K inhibitor LY294002 (10 lM), mTOR inhibitor rapamycin (5 ng/ml), calcineurin inhibitor cyclosporin A (2.5 lM) and the Ca2+/calmodulin-dependent kinase inhibitor KN562 (8 lM). To quantify the myofiber size, 10 fields were randomly chosen and 10 myotubes were measured per field. The average diameter per myotube was the mean of 10 measurements taken along the length of the myotube. The fusion index was determined by counting the number of nuclei in a single myofiber. Ten myotubes in ten different microscopic fields were chosen to calculate the average number of nuclei. Sialidase assay. The enzymatic activity of cytosolic sialidase was assayed as previously described [3,4]. Briefly, ultracentrifuged cytosolic fractions of myotubes were assayed using a mixture containing 60 nmol of the synthetic substrate 4-
A. Fanzani et al. / Biochemical and Biophysical Research Communications 370 (2008) 376–381 methylumbelliferyl N-acetylneuraminic acid and 100 lg of BSA in 0.2 mL of 50 mM sodium acetate buffer (pH 5.8). After 3 h of incubation at 37 °C, the reaction was terminated by addition of 0.8 mL of 0.25 M glycine buffer (pH 10.4), and the amount of 4-methylumbelliferone released was determined fluorometrically with an excitation wavelengh of 365 nm and an emission of 450 nm. A pH curve has been set up for each experiment to determine whether the enzymatic activity assayed in the cytosolic fraction had the optimum in the typical range of cytosolic sialidases, usually between 5.6 and 6. Plasmid construction and stable trasfections. The pBABE vectors harboring a myristoylated or a kinase-dead AKT (mutated at the ATP binding site K179M) [6] were used to transfect C2C12 myoblasts. Rat Neu2 cDNA was amplified using pCDNANeu2 plasmid as template [3] and primers 50 -CGGGATCCCGATGGAGACCTGCCCCG TCCTCCAGAAA-30 and 50 -CGGGATCCCGTCAAGCGTAGTCTGGGACGTCGTATGGGTAC CCCTGAGCACCATGTACTGTGGG-30 containing BamHI restriction sites and the hemagglutinin (HA) epitope in the reverse primer. The resultant PCR product was BamHI digested and cloned in the similarly opened pBabe vector, yielding plasmid pBabe/HA-Neu2 used to transfect C2C12 myoblasts. Stable transfectants were obtained after 10–15 days of selection in medium containing the antibiotic puromycin (2 lg/mL). RT-PCR analysis. Total RNA was obtained by Tri-reagent extraction, digested with 1 U of DNase (DNA-free, Ambion), and retrotranscribed (2 lg) with 400 U of MMLV-RT (Promega). The primers (250 nM) were as follows: mouse Neu2 sialidase forward primer (50 -CGAGCCAGCAAGACGGATGAG-30 ) and reverse (50 -GGCTCTACAAGCTTACTCAC TACCCGG-30 ) were used for 29 cycles of PCR; rat Neu2 sialidase forward primer (50 -CCGTCCAGGACCTCACAGAG-30 ) and reverse (50 -TCACTGAGCACCATGTACTG-30 ) were used for 30 cycles of PCR; rat follistatin forward primer (50 -CTCTTCAAGTGG ATGATTTTC-30 ) and reverse (50 -ACAGTAGGCATTATTGGTCTG-30 ) were used for 30 rounds of PCR; mouse Murf-1 forward primer (50 -GGTGCCTACTTGCTCCTTGT-30 ) and reverse (50 -CTGGTGGCTATTCTCCTTGG-30 ) were used for 27 rounds of PCR; mouse Atrogin forward primer (50 -CGACCTGCCTGTGTGCTTAC-30 ) and reverse (50 CTTGCGAATCTGCCTCTCTG-30 ) were used for 28 cycles of PCR. Gene expression levels were normalized to gapdh mRNA expression by 23 rounds of PCR. Western blot analysis. Samples were separated by SDS–PAGE under reducing conditions and transferred to nitrocellulose membranes according to standard procedures. The following primary antibodies were used: anti-myogenin (clone F5D) and anti-GATA-2 (clone CG2-96) from Santa Cruz Biotechnology, anti-MyHC from the Hybridoma Bank (University of Iowa), anti-phospho-AKT (Ser473) and antiphospho-p70S6K (Thr389) from Cell Signalling Technology, anti-HA, and anti-tubulin from Sigma–Aldrich. After incubation with horseradish peroxidase-conjugated secondary antibodies (Chemicon), immunocomplexes were visualized using enhanced chemiluminescence reagent (Chemicon). Degradation of long-lived proteins. Bulk degradation of long-lived proteins was determined according to a method previously reported [12]. Briefly, cells were plated into 35 mm dishes and cultured in cysteine/methionine free media containing 5 lCi L-[35S] cysteine/methionine (GE Healthcare) for 6 h at 37 °C. Unincorporated radioisotopes and degraded amino acids released from short-lived proteins were removed by rinsing three times with PBS. Cells were then chased with the culture medium containing 10% FBS and 2 mM cold cysteine/methionine. After 15 h incubation, at which time short-lived proteins were being degraded, the chase medium was replaced with serum-containing DMEM (control) or Hank’s/Hepes buffer. After incubation at 37 °C for 6 h, the medium was harvested and 100% trichloroacetic acid (TCA) was added to 10% final concentration. The samples were centrifuged at 12,000g for 10 min and the acid-soluble radioactivity was measured by liquid scintillation counting. Meanwhile, the cells were fixed by adding 1 ml of 10% TCA directly to the culture dishes, washed with 10% TCA and dissolved in 1 ml of 0.2 N NaOH. Radioactivity in the samples was measured similarly. The percentage protein degradation was calculated by dividing the amount of acid-soluble radioactivity in the culture medium by the sum of acid-soluble and acid-precipitable radioactivities. Electron microscopy. Samples were fixed in 0.1 M sodium cacodylate-buffered (pH 7.4) 2.5% glutaraldehyde solution for 2 h and postfixed in 0.1 M sodium cacodylate-buffered (pH 7.4) 1% OsO4 solution for 1 h. After dehydration in an ethanol gradient (70% ethanol [20 min], 96% ethanol [20 min], 100% ethanol [2 20 min]), samples were incubated with propylenoxid (2 10 min), impregnated with a mixture of propylenoid/LX-112 (Ladd Research Industries, 1:1) and embedded in LX112. Ultrathin sections were stained with uranyl acetate and lead citrate. Sections were examined in a Jeol-100 CX II TEM at 80 kV. Immunofluorescence microscopy. The myotubes grown on 12 mm glass coverslips were coated with 20 lg/mL laminin (Roche), fixed with ice cold methanol and then incubated with rat monoclonal anti-HA antibody (clone 3F10, Roche) followed by an anti-rat Alexa Fluor 594-conjugated secondary antibody (Molecular Probes). Alternatively, cells were incubated with anti-MyHC antibody (Hybridoma Bank, University of Iowa) followed by a biotinylated anti-mouse antibody and Alexa Fluor 488 streptavidin-conjugate (dilution 1:800). Fluorescent staining of myotubes was observed under an Axiovert S100 microscope (Zeiss). Statistics. All data are expressed as means ± SEM. Statistical significance was determined using t-Student analysis. A p value of <0.05 was considered significant.
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Results and discussion In vitro myoblast differentiation and hypertrophy enhance Neu2 enzymatic activity In this study, we analyzed the Neu2 enzymatic activity in myoblasts undergoing differentiation and hypertrophy upon different stimuli. Different pathways have been characterized to promote myoblast fusion and growth; in particular, the PI3K/AKT/mTOR pathway plays a pivotal role during myofiber growth [5,6], as its conditional activation in skeletal muscle induces rapid hypertrophy in transgenic mice [8]. Accordingly, the constitutive activation of AKT (caAKT) by expression of a myristoylated AKT form [6] led to sustained hypertrophy of C2C12 myoblasts, as revealed by morphological analysis (Fig. 1A) and myofiber size measurements (Fig. 1B, top); in caAKT myofibers, an increased phosphorylation of AKT and its direct downstream target p70S6K were detected via Western blotting as compared to control (Fig. 1B, bottom), whereas Neu2 transcript levels were upregulated (Fig. 1B, bottom). Under these conditions, a strong increase of sialidase enzymatic activity was detectable in the cytosol of hypertrophic myotubes already two days after differentiation (Fig. 1C). Inhibition of mTOR activity in caAKT myoblasts after treatment with rapamycin abolished the myofiber hypertrophy (not shown) and caused a significant loss of Neu2 activity (Fig. 1C). Also expression of a kinase inactive form of AKT (kiAKT) in C2C12 cells reduced myofiber size (not shown) [6] and downregulated Neu2 activity (Fig. 1C), indicating that the activation of the AKT/mTOR pathway stimulates sustained Neu2 enzymatic activity that may accompany muscle hypertrophy. We further analyzed Neu2 activity in L6E9 myoblasts, a cell model which has been well characterized to develop hypertrophy upon Igf-1 treatment [13]. Igf-1 induced already at day 3 a pronounced myofiber hypertrophy as compared to untreated L6E9 myoblasts (Fig. 1D). This effect was accompanied by elevated expression of myogenin as detected via Western blotting and by increased transcription of Neu2 mRNA (Fig. 1E), followed by increased Neu2 enzymatic activity (Fig. 1F). Because PI3K and calcineurin activity are both required to switch myoblasts from a proliferative to a myogenic condition [14,11], co-administration of Igf-1 with the PI3K inhibitor LY294002 (LY) or the calcineurin inhibitor cyclosporin A (CsA) reverted the effects induced by Igf1 and resulted in loss of Neu2 activity (Fig. 1F). To obtain additional evidence that increased Neu2 expression may depend on calcineurin activity in myotubes, we stimulated L6C5 myoblasts with Arg8vasopressin (AVP), a neurohypophyseal peptide that by interacting with V1 type receptors triggers hypertrophy mainly through the activation of calcineurin and Ca2+/calmodulin-dependent kinase [9,10]. Myoblasts AVP-treated for 6 days formed larger myotubes as compared to control cells and showed two typical hallmarks of muscle hypertrophy [13], the reorganization of nuclei into nuclear rings (Fig. 1G) and increased expression of the transcription factor GATA-2 (Fig. 1H). Under these conditions, AVP treatment upregulated Neu2 transcription significantly (Fig. 1H) and triggered a progressive, long-lasting (6 days) increase in Neu2 activity (Fig. 1I) that was suppressed after single or simultaneous administration of two specific inhibitors of calcineurin and Ca2+/calmodulin-dependent kinase from day 3 to 6 (d3–d6) (Fig. 1J), a time point during which the myogenic program was already started. Finally, we tested whether Neu2 sialidase activity was modulated during myofiber hypertrophy caused by an augmented recruitment of myoblasts into pre-existing myofibers. In particular, follistatin is considered to play a pivotal role in this process through a mechanism that involves the inhibition of classes I–II histone deacetylases (HDAC) [11,15,16]. After administration of trichostatin
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Fig. 1. In vitro myoblast hypertrophy enhanced Neu2 enzymatic activity. (A) Parental and stably transfected caAKT C2C12 myoblasts were differentiated for 2 days to visualize the myofiber size. (B) Quantification of myofiber diameter (top) and Western blot analysis of AKT and P70S6K phosphorylation followed by Neu2 transcript RT-PCR analysis (bottom) in parental and caAKT myoblasts after 2 days of differentiation. (C) Neu2 enzymatic activity was assayed in parental and caAKT myotubes, in the absence or presence of rapamycin (5 ng/mL). In addition, Neu2 activity was determined in C2C12 stably transfected with a kinase inactive form of AKT (kiAKT). (D) Morphology of rat L6E9 myoblasts subjected to Igf-1 (10 ng/ml). (E) Western blot analysis of myogenin expression and RT-PCR analysis of Neu2 transcript in Igf-1 treated and untreated myoblasts. (F) Neu2 enzymatic activity assayed in untreated or Igf-1 treated myoblasts at day three, in the absence or presence of the PI3K inhibitor LY (10 lM) or the calcineurin inhibitor CsA (2.5 lM). (G) Treatment of L6C5 myoblasts with 0.1 lM Arg8-vasopressin (AVP) triggered hypertrophy. (H) Expression of GATA-2 by Western blot and Neu2 by RT-PCR analysis in AVP-treated and untreated myotubes. (I) Neu2 enzymatic activity in AVP-treated and control myoblasts. (J) Neu2 activity in myofibers treated with AVP (6 days) in the presence or absence of CsA (2.5 lM) and/or KN562 (8 lM) administered from day 3 to 6 (d3–d6). (K) Treatment of L6C5 myoblasts with 50 nM TSA triggered hypertrophy (day 3). (L) Fusion index of control and TSA-treated L6C5 myotubes. (M) RT-PCR analysis of follistatin and Neu2 in untreated and TSA-treated L6C5 myoblasts. (N) Neu2 activity in control untreated and TSA-treated L6C5 myoblasts. (*P < 0.05 vs control, **P < 0.05 vs *).
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(TSA), an inhibitor of HDAC, the size of L6C5 myofibers was considerably augmented (Fig. 1K) after 3 days as compared to normally differentiated cells, and this effect was mediated by increased cell recruitment, as shown by the fusion index (Fig. 1L). Accordingly,
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TSA treatment triggered a remarkable increase of follistatin expression in L6C5 myoblasts as compared to untreated cells, followed by a rise in Neu2 transcript levels (Fig. 1M). Neu2 activity was significantly increased 5 days after TSA treatment as compared
Fig. 2. In vitro myoblast atrophy decreased Neu2 transcript and enzymatic activity. C2C12 myoblasts were differentiated for 5 days and then treated with dexamethasone (dex) for 48 h or incubated in glucose- and amino acid-free medium (starv) for 15 h to induce muscle atrophy. (A) Mean myotube size shown is relative to control, dexamethasone (dex) and starved (starv) myotubes. (B) Transmission electron microscopy of C2C12 myotubes (day 5) starved in 10 mM Hank’s/Hepes buffer (pH 7.4) for 12 h. Control myotubes displayed a normal cell morphology, whereas starvation triggered the formation of autophagic vesicles (boxed area, shown at higher magnification in the right panel). (C) Degradation of long-lived proteins in control and starved myotubes. (D) Semiquantitative RT-PCR analysis for detection of Atrogin, Murf-1, and Neu2 transcripts. The mRNA expression profiles were normalized by gapdh expression. (E) Neu2 enzymatic activity was assayed in atrophic myofibers (dex and starv) as compared to control. (F) C2C12 myotubes stably transfected with HA-Neu2 protein were subjected to either dexamethasone or starvation treatment. HA-Neu2 expression was analyzed via Western blotting together with MyHC protein expression. (G) HA-Neu2 and MyHC expression were analyzed by fluorescence microscopy in control and atrophic myofibers (dex and starv). Scale bar = 100 lm (*P < 0.05 vs control).
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to untreated cells (Fig. 1N), thus after the increase in myofiber size mediated by increased satellite recruitment. This finding suggests that Neu2 is involved in growth and maintaining of myofiber size after muscle fusion. Overall we may conclude that Neu2 enzymatic activity usually reaches maximal activity when muscle growth occurs. Two different stimuli trigger myofiber growth: (i) increased protein synthesis due to sustained AKT and calcineurin activation and (ii) increased myoblast recruitment, a mechanism which is important during the repair of muscle myofibers after muscle injury. Myotube atrophy caused by dexamethasone or serum starvation treatment impairs Neu2 activity Muscle atrophy usually occurs under physiological and pathological conditions such as ageing, muscle unloading, cancer, AIDS, denervation and dystrophies. Under these conditions, increased protein breakdown and diminished protein synthesis in turn lead to unbalanced steady-state levels of proteins triggering a reduction in myofiber size [17]. To investigate whether Neu2 expression was modulated in atrophic myofibers, terminally differentiated C2C12 myotubes were treated with 100 lM dexamethasone (dex) for 48 h or starved in glucose- and amino acid-deprived medium for 15 h (starv). As shown in Fig. 2A, the diameter of the myofibers was significantly reduced in both conditions suggesting that an atrophic program occurs in myoblasts. Usually the bulk of intracellular proteins during muscle atrophy is degraded by two main proteolytic systems: the endosome–lysosome system which degrades cytoplasmic components engulfed by autophagic vacuoles through the activity of acidic cathepsins [18], and the proteasome 26S complex which triggers degradation of ubiquitinated proteins via an ATP-dependent system [19–21]. We first detected two hallmarks of autophagy in starved atrophic myofibers, namely the formation of autophagic vacuoles visualized by transmission electron microscopy (Fig. 2B), and a significant rise in the degradation of long-lived proteins (Fig. 2C). In addition, the ATP-proteasome-dependent pathway was activated both in starved and dex-treated myofibers, as shown by increased expression of two predictive atrophy-related E3 ubiquitin ligases, Atrogin and Murf-1 (Fig. 2D) [20,21]. Under both conditions, Neu2 mRNA levels were downregulated (Fig. 2D). Also Neu2 enzymatic activity was significantly decreased in C2C12 atrophic myotubes (Fig. 2E), with the most pronounced effect after starvation. To test whether downregulation of Neu2 activity was dependent on protein loss, C2C12 myotubes stably transfected with HA-Neu2 were subjected to either dexamethasone treatment or starvation. Western blots showed that HA-Neu2 protein was downregulated as compared to control myotubes at day 5 (Fig. 2F), and this effect was well paralleled by downregulation of MyHC (Fig. 2F), a marker of terminal muscle differentiation. Moreover, when we analyzed the expression of both endogenous MyHC and transfected HA-Neu2 in myotubes by immunofluorescence, we found a diffuse staining pattern of both these proteins along the cytosol of myofibers (Fig. 2G). Indeed, the fluorescence intensity was much lower in atrophic myofibers as compared to control (Fig. 2G), and the reduction in fluorescence was more evident in the case of starvation. These data suggest that a specific impairment of Neu2 sialidase expression occurs in atrophic myotubes under starvation and dexamethasone treatments. Of note, overexpression of Neu2 was not able to rescue the morphology of atrophic myofibers because protein loss under these conditions is broad and not Neu2-specific. Overall, further studies are needed to evaluate how Neu2 loss is accomplished during myofiber atrophy, either via increased protein breakdown or via reduced protein synthesis, two main processes that usually occur during
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