INTRODUCTION
Pompe disease (PD; OMIM 232300) is a severe metabolic myopathy caused by the deficiency of acid alpha‐glucosidase (GAA; acid maltase, E.C.3.2.1.20), an enzyme responsible for breaking down glycogen to glucose within the acidic environment of lysosomes. The functional deficiency or complete absence of the enzyme results in accumulation of glycogen within this cellular compartment (Kroos et al,
2012; Van der Ploeg & Reuser,
2008). PD pathology is also characterized by secondary accumulation of autophagic debris (autophagic build‐up), typically found in skeletal muscle fibres (Raben et al,
2007a,
b,
2012). Although PD is a rare disorder (Martiniuk et al,
1998), it shares common pathological features with many other lysosomal storage disorders (LSDs), which encompass more than 60 nosological entities (Cox & Cachon‐Gonzalez,
2012).
GAA deficiency is a systemic disorder – distended glycogen‐filled lysosomes can be found in multiple tissues – but skeletal and cardiac muscles are particularly vulnerable to the accumulation of storage material. In the most serious infantile form, the disease manifests as profound weakness, hypertrophic cardiomyopathy, heart failure, feeding difficulties, respiratory infections and, if left untreated, causes death within the first year of life. In the attenuated phenotypes, characterized by later (childhood, juvenile or adult) onset, cardiac muscle is usually spared, but the illness remains a serious condition with progressive motor impairment, respiratory failure and premature death (Van der Ploeg & Reuser,
2008).
Enzyme replacement therapy (ERT) is now available for several LSDs including PD. Unfortunately, in many of these diseases the target tissues are not easily accessible to the replacement enzymes (Lachmann,
2011; Urbanelli et al,
2011). In PD, skeletal muscle – the major target for ERT – is particularly challenging to treat. Even with extremely high dosages of the drug, recombinant human GAA (rhGAA; alglucosidase alpha; Myozyme® and Lumizyme®, Genzyme Corporation, Framingham, MA), many patients experience limited clinical benefit or show signs of disease progression (Angelini & Semplicini,
2012; Kishnani et al,
2007; Schoser et al,
2008; Strothotte et al,
2010; Van den Hout et al,
2004; Van der Ploeg et al,
2010). Furthermore, the greatest success of ERT in PD – significantly improved survival of infants due to the restoration of cardiac function – comes at a price: many long‐term survivors suffer from debilitating myopathy, often more severe than in late‐onset cases.
Several factors contribute to the resistance of skeletal muscle to ERT. These include the sheer mass of muscle tissue, a relatively low density of the mannose‐6‐phosphate receptor that is responsible for the uptake of the enzyme, inefficient trafficking of the internalized enzyme to lysosomes, immune response to the recombinant enzyme in cross‐reactive immunologic material (CRIM)‐negative patients and the presence of large areas of autophagic build‐up alongside expanded lysosomes (Banugaria et al,
2011; Funk et al,
1992; Kishnani et al,
2010; Koeberl et al,
2012; Raben et al,
2002,
2010,
2012).
Significant efforts are aimed at improving the current therapy, such as the development of “second generation” recombinant enzymes with enhanced targeting properties (Maga et al,
2012; Tiels et al,
2012; Zhu et al,
2009), pharmacological chaperones that assist the folding of the mutant enzyme (Okumiya et al,
2007; Parenti et al,
2007) or increase the stability of the recombinant enzyme (Porto et al,
2009,
2012), and gene therapy (Byrne et al,
2011).
A recently proposed novel approach to therapy for LSDs – modulation of transcription factor EB (TFEB) – circumvents the problem of inefficient enzyme delivery by exploiting the ability of lysosomes to expel their content into the extracellular space, thus providing clearance of the stored material (Medina et al,
2011). It has been shown that overexpression of TFEB, a master regulator of lysosomal biogenesis and autophagy, leads to the generation of new lysosomes and increased numbers of autophagosomes in a variety of cell types (Sardiello et al,
2009; Settembre & Ballabio,
2011; Settembre et al,
2011). Furthermore, since TFEB has been shown to promote lysosomal–autophagosomal fusion (Settembre & Ballabio,
2011; Settembre et al,
2011), this approach, unlike most current efforts, has the potential to prevent or resolve autophagic build‐up.
Here, we have characterized the effect of TFEB overexpression on lysosomal and autophagic accumulation in previously described and newly developed PD models. Expression of TFEB in myotubes and muscle fibres resulted in lysosomal docking/fusion with the plasma membrane, lysosomal exocytosis and a dramatic reduction of intra‐lysosomal glycogen accumulation. In addition, TFEB overexpression in PD muscle alleviated autophagic pathology by promoting the formation and removal of autophagolysosomes. Thus, modulation of TFEB activity holds promise for the development of a better therapy for this devastating disorder.
DISCUSSION
We have shown that TFEB efficiently triggers lysosomal exocytosis and promotes cellular clearance in muscle cells, isolated live muscle fibres, and the whole muscle of PD mice.
The process of regulated lysosomal exocytosis – which underlies TFEB‐mediated cellular clearance – was initially ascribed to a subset of secretory lysosomes (or lysosome‐related organelles) that reside in particular cell types, such as haematopoietic cells or melanocytes (Blott & Griffiths,
2002). However, it has been shown that conventional lysosomes can also undergo the same process of docking to the plasma membrane, followed by fusion with the membrane and a discharge of lysosomal content (Andrews,
2000). This synaptotagmin‐regulated Ca
2+‐dependent process is considered to be critical for the repair of plasma membrane lesions (Rao et al,
2004).
Interestingly, lysosomal exocytosis has been implicated as a mechanism of cellular clearance in cell models of several LSDs, such as metachromatic leukodystrophy and mucolipidosis type I (Klein et al,
2005; Yogalingam et al,
2008). Furthermore, it has been shown that lysosomal exocytosis is the mechanism by which 2‐hydroxypropyl‐β‐cyclodextrin – an approved drug for yet another LSD, Niemann Pick Type C (NPC) disease – alleviates cholesterol storage in NPC cells (Chen et al,
2010). Similarly, δ‐tocopherol was shown to improve the phenotypes in several LSD cell models by stimulating Ca
2+‐mediated lysosomal exocytosis (Xu et al,
2012).
However, it was not until the discovery of TFEB that the stimulation of lysosomal exocytosis and biogenesis became a therapeutic option for LSDs and other disorders with accumulation of abnormal proteins (Medina et al,
2011; Sardiello et al,
2009; Settembre & Ballabio,
2011; Settembre et al,
2011). Overexpression of TFEB induced lysosomal exocytosis in a mouse model of multiple sulfatase deficiency (MSD) and in cells derived from the murine models of mucopolysaccharidosis type IIIA (MPS‐IIIA), neuronal ceroid lipofuscinoses, and MSD (Medina et al,
2011). The effect of TFEB was also demonstrated in fibroblasts from a PD patient (Medina et al,
2011), but it remained unclear whether TFEB would have similar effects in terminally differentiated multinucleated muscle cells and PD muscle, the tissue that presents the greatest challenge for ERT.
In this study, we have demonstrated that the number of large glycogen‐filled lysosomes and the amount of accumulated glycogen – the hallmarks of PD – were significantly reduced in both cultured myotubes and muscle fibres, thus providing strong evidence of TFEB potential as a therapeutic target in PD.
The simplicity of detecting hugely expanded lysosomes and glycogen in PD myotubes made this cell model particularly attractive: the effects of TFEB were easily monitored and evident within days. In TFEB‐treated myotubes, the lysosomal marker LAMP1 – a protein typically present inside the cells – was seen on the outer plasma membrane. Furthermore, the release of lysosomal acid phosphatase from TFEB‐treated cells provided additional evidence that lysosomal clearance proceeds through exocytosis in muscle cells, consistent with findings in other cell types (Medina et al,
2011). The cell system also allowed us to expose a potential problem with high TFEB activity; a constitutively active form of TFEB, in which S211 is mutated to alanine (Martina et al,
2012), invariably resulted in morphological changes of myotubes, perhaps because of massive expulsion of the lysosomes.
Live fibre experiments employing time‐lapse confocal microscopy provided a unique opportunity to track lysosomal movements and fusion, to estimate the motility of lysosomes, and to examine the interactions between lysosomes and autophagosomes following TFEB activation and nuclear translocation. Similar to what was observed in the cell system, in vivo TFEB delivery efficiently triggered lysosomal exocytosis and promoted cellular clearance, as evidenced by a significant reduction in glycogen levels and the number of large lysosomes in PD muscle. Thus, lysosomal pathology in PD muscle can be corrected by TFEB overexpression.
The development of a new PD mouse model, the GFP‐LC3:GAA−/− strain, allowed us to address the effect of TFEB on autophagic accumulation – the major secondary abnormality in PD skeletal muscle (Raben et al,
2007a,
b,
2012). Defects in autophagy, a major lysosome‐dependent degradative system (Yang & Klionsky,
2010)], significantly contribute to the pathophysiology of several lysosomal storage diseases (Cao et al,
2006; Cox & Cachon‐Gonzalez,
2012; Fukuda et al,
2006a,
b; Liao et al,
2007; Lieberman et al,
2012; Settembre et al,
2008). In PD skeletal muscle, the autophagic defect is particularly striking, manifesting as a massive build‐up which poses an additional problem for ERT.
Remarkably, none of the TFEB‐transfected fibres had large areas of autophagic build‐up, which are so prominent in most non‐transfected PD fibres. The lack of typical build‐up suggests that TFEB may have rescued autophagic pathology in PD muscle. The mechanism of this rescue is not clear, but the unexpected surge in autophagy – a dramatic increase in the number of LC3‐positive autophagosomes during hours of time‐lapse microscopy – provided a clue. TFEB‐treated fibres exhibited a significant increase in fusion between LC3‐ and LAMP1‐positive vesicles, an appearance of multiple LC3/LAMP1‐positive autophagolysosomes, and docking of these double‐positive structures to the plasma membrane. These data suggest that TFEB may relieve autophagic build‐up by stimulating the formation and secretion of autophagolysosomes – a product of lysosomal‐autophagosomal fusion.
We hypothesize that lysosomal exocytosis may in fact not be a purely lysosomal event, but rather a process involving autophagy and secretion of autophagolysosomes. Our data on the much attenuated effects of TFEB on lysosomal velocity and clearance in autophagy‐deficient PD mice support this idea. Lysosomal function depends on the ability of lysosomal membranes to fuse with other vesicles' membranes and with the plasma membrane (Luzio et al,
2007). Abnormal accumulation of cholesterol in the endolysosomal membranes in cell models of two neurodegenerative LSDs (MSD and MPS‐IIIA) was shown to sequester SNARE receptors – essential components of the membrane fusion machinery – in cholesterol‐enriched membrane regions, resulting in a significantly reduced ability of lysosomes to fuse with vesicles of endocytic and autophagic pathways (Fraldi et al,
2010). Perhaps the merging of lysosomal and autophagosomal membranes is required for successful fusion with the plasma membrane and for fully efficient TFEB‐mediated cellular clearance.
Two kinases, mTORC1 and ERK, have been implicated in TFEB regulation in different cells (Martina et al,
2012; Roczniak‐Ferguson et al,
2012; Settembre et al,
2011,
2012). The regulation of TFEB in skeletal muscle remains an open question, which is beyond the scope of this paper. An unexpected activation of ERK in TFEB‐overexpressing cells could be an attempt to down‐regulate TFEB and restore homeostasis; it is also possible that activation of ERK could be a downstream event. Whatever the case, the search for compounds which can activate TFEB in muscle would benefit the development of a TFEB‐mediated therapeutic approach. In thinking about TFEB as a target for potential therapeutic intervention, one can speculate that a single event of TFEB‐mediated clearance of the stored substrate and autophagic material may provide prolonged relief since the rate of muscle glycogen accumulation in late‐onset PD is relatively slow. Furthermore, ERT may function more efficiently in TFEB‐treated muscle, particularly because of the reduction in autophagic build‐up. A combination of TFEB‐targeted therapy with ERT, perhaps at much lower dosages than those currently employed (and therefore at reduced cost), could then maintain muscle tissue in normal or nearly normal condition. In the meantime, pre‐clinical studies are needed to fully evaluate the consequences of TFEB overexpression on muscle function and to exclude long‐term toxicity.
Finally, the potential applications of our newly developed models, PD myotubes and GFP‐LC3:GAA−/− mice, extend beyond testing the effects of TFEB. For example, an
in vitro PD model is much needed for large‐scale drug testing. Recently, several attempts have been made to develop such a model: these include the generation of cardiomyocyte‐like cells from human induced pluripotent stem (iPS) cells (Huang et al,
2011), muscle cells from reprogrammed murine iPS cells (Kawagoe et al,
2011), and murine muscle cells transduced by viral constructs containing immortalizing genes (Douillard‐Guilloux et al,
2009; Takikita et al,
2009). Our new PD muscle cell lines have several advantages: they are highly myogenic, derived from individual clones and, most importantly, they maintain the capacity to differentiate when the immortalizing gene, shown to perturb myogenic program (Douillard‐Guilloux et al,
2009; Mouly et al,
1996), is inactivated. These cell lines replicate lysosomal pathology but fail to recapitulate autophagic build‐up. Possible explanations for this failure – common to all
in vitro PD models – are the relatively short survival time of myotubes in culture and the need for nutrient‐rich conditions (Fig S3C). Additional studies are needed to find a way to “trick” these cells into displaying the autophagic accumulation that is so striking in Pompe muscle fibres.
In the interim, the newly generated GFP‐LC3:GAA−/− mouse model provides an excellent tool to monitor autophagic accumulation in live fibres, in muscle bundles, and even in muscle of a living mouse [
i.e. by intravital imaging (our unpublished data)]. In addition to using these mice to address TFEB's effects, we have utilized the model to examine intracellular trafficking of rhGAA and to explore possible mechanisms of autophagic build‐up. The near absence of lysosomal–autophagosomal fusion and strikingly low expression of LAMP1 in the build‐up area suggest inefficient local lysosomal biogenesis. LAMPs are considered to be of key importance for lysosomal movement, maturation of autophagosomes and lysosomal exocytosis (Saftig & Klumperman,
2009). Therefore, these low local LAMP1 concentrations in the build‐up area may have far reaching consequences. The build‐up process appears to begin with the failure of a subset of lysosomes to fuse with autophagosomes, perhaps because of lysosomal rupture during muscle contractions. It is possible that this fusion defect and the paucity of the newly formed lysosomes create conditions that favour expansion of the autophagic build‐up. With the capacity to resolve both of these deficits by promoting lysosomal–autophagosomal fusion and biogenesis, TFEB is uniquely suited to remedy autophagic and lysosomal pathologies in PD.
MATERIALS AND METHODS
Antibodies: rat anti‐LAMP1 (1DB4), mouse anti‐cytochrome c, and mouse anti‐MyoD (MoAb 5.8A) (BD Pharmingen, San Diego, CA); rabbit anti‐LC3B and mouse anti‐Flag (M2) (Sigma–Aldrich, St. Louis, MO); mouse anti‐myogenin (F5D) (Dako, Glostrup, Denmark); mouse anti‐myosin heavy chain (Clontech, Mountain View, CA); rabbit anti‐caspase‐3, rabbit anti‐p44/42 MAPK (Erk1/2), rabbit anti‐phospho‐p44/42 MAPK (Erk1/2; Thr202/Thr204), rabbit anti‐phospho‐4EBP1 (Thr37/46), and rabbit anti‐Histone H3 (D1H2) (Cell Signaling, Beverly, MA). Rabbit anti‐alpha tubulin (Abcam, Cambridge, MA) or mouse anti‐vinculin antibody (Sigma–Aldrich) was used as a loading control for western blot. Alexa Fluor‐conjugated secondary antibodies (Life Technologies, Grand Island, NY) were used for immunostaining and western blot analysis. Alexa Fluor 546 labelled recombinant human GAA (6.2 mg/ml) was provided by Genzyme Corp. (Framingham, MA).
Plasmids: plasmid containing the full‐length 3xFlag‐tagged human TFEB (referred to as Flag‐TFEB) was described previously (Sardiello et al,
2009); plasmid containing the full‐length rat LAMP1 (mCherry‐LAMP1) was obtained from Dr. Kristien Zaal (Light Imaging Section, Office of Science & Technology, NIAMS, NIH, Bethesda, MD, USA); plasmid‐containing GFP‐TFEB was described previously (Martina et al,
2012).
Generation of mice and isolation of single live myofibres
The generation of GFP‐LC3:GAA−/−, immorto:GAA−/−, and immorto:GFP‐LC3:GAA−/− mice is described in the Supporting Information. Single live myofibres from gastrocnemius and extensor digitorum longus (EDL) muscles from 3 to 4 month‐old mice were isolated as described (Rosenblatt et al,
1995). Isolation of single live fibres from FDB muscle was done with some modifications of the previously published protocol (Raben et al,
2010) (Supporting Information). Live fibres were either monitored by time‐lapse microscopy or used for labelling acidic organelles [LysoTracker® Red DND‐99 (Life Technologies), 100 nM] or mitochondria [MitoTracker® Red CMXRos (Life Technologies), 100 nM]. Alternatively, fibres were fixed in 2% paraformaldehyde for 30 min and immunostained with anti‐cytochrome c and anti‐LAMP1 antibodies. Both live and fixed fibres were analysed on a LSM 510 confocal microscope (Carl Zeiss, Göttingen, Germany; see Supporting Information) equipped with Plan‐Neofluar 40X oil immersion objective.
In vivo electroporation of skeletal muscle and enzyme replacement therapy
Electric pulse‐mediated gene transfer into FDB muscle was performed as described (DiFranco et al,
2009). Three to four month‐old GAA−/−, GFP‐LC3:GAA−/−, or muscle‐specific autophagy‐deficient GAA−/− (MLCcre:Atg7F/F:GAA−/−; referred to as Atg7:GAA DKO) mice were used for the experiments (Table
1). The animals were sacrificed 4–6 days after the procedure. Live FDB fibres were isolated and analysed by time‐lapse confocal microscopy. Alexa‐Fluor 546 labelled recombinant human GAA (rhGAA) was administered into 3–4 month‐old GFP‐LC3:GAA−/− mice at a dosage of 100 mg/kg twice with a 24 h interval. Mice were sacrificed 24 h after the last injections. Single fibres, isolated from EDL and gastrocnemius muscle, were analysed by confocal microscopy.
Fibroblast and myoblast culture systems
Primary skin fibroblasts, derived from two unrelated patients with severe infantile PD, were obtained from Coriell Cell Repositories (Camden, New Jersey, USA). Clinical diagnosis was confirmed by genetic analysis. Fibroblasts were maintained in DMEM (high glucose) supplemented with 10% (v/v) FBS, 1× P/S/l‐Glutamine. Adult mouse fibroblasts were isolated from the tail tip according to a standard procedure.
Immortalized PD cell lines were derived from immortoGAA−/− and immortoGFP‐LC3:GAA−/− mice. Single fibres from EDL muscle of 3–4 month‐old mice were plated in each well (∼10 fibres per well) of a Matrigel‐coated six‐well plate in plating medium (10% horse serum in DMEM, 0.5% chick embryo extract, and 1× P/S/
l‐Glutamine) at 37°C, 5% CO
2. After 3 days, when the satellite cells began to migrate away from fibres, most of the medium (3/4) was replaced with proliferation medium (20% foetal bovine serum, 10% horse serum, 1% chick embryo extract, 1× P/S/
l‐Glutamine in high glucose DMEM). Myogenic colonies were isolated with cloning cylinders (Corning, Corning, NY) on day 5 or 6. Several individual myogenic clones were isolated and analysed for their proliferation capacity and the ability to differentiate into myotubes. To induce the expression of H‐2K
b‐tsA58 SV40 large T‐antigen for immortalization of the satellite cells and myoblasts, recombinant IFN‐γ (Life Technologies) was added to the proliferation medium (100 units/ml). The cells were then incubated at 33°C in an atmosphere of 5% CO
2 (Jat et al,
1991). When myoblasts became nearly confluent, the medium was changed to differentiation medium (DMEM containing 2% horse serum, 0.5% chick embryo extract, 1× P/S/
l‐Glutamine) and the cells were moved to 37°C in an atmosphere of 5% CO
2. Myotubes began to form within 2 days. Glucose concentration was either 1 g/L (low) or 4.5 g/L (high). Myotubes survived in culture for 2–3 weeks. A wild type immortalized muscle cell line served as the control.
Infection of myotubes with adenovirus expressing TFEB and immunofluorescence microscopy
Adenoviruses expressing either WT or mutant (S211A) TFEB were described previously (Martina et al,
2012). Myotubes were infected with either adenovirus (Ad‐null) or adenovirus expressing WT TFEB (Ad‐TFEB) or S211A TFEB (Ad‐TFEBmt) for 24, 48 or 72 h. Myotubes were fixed in 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) for 15 min at room temperature, washed twice in PBS, and permeabilized in 0.2% Triton X‐100 (Sigma–Aldrich, St. Louis, MO). Immunostaining with LAMP1, LC3 or Flag antibodies was done using M.O.M. kit (Vector Laboratories, Burlingame, CA) as previously described (Raben et al,
2009). The cell nuclei were stained with 2 µg/ml Hoechst 33342 (Life Technologies) in PBS for 10 min. After staining, the cells were imaged on a Carl Zeiss LSM 510 confocal microscope with a 40× or 63× oil immersion objective.
Fluorescent glycogen detection, LDH measurements, in situ detection of apoptotic cells, surface LAMP1 analysis, and acid phosphatase assay
Lysosomal glycogen in live cells was detected by the incorporation of 2‐NBDG, a
d‐glucose fluorescent derivative (2‐deoxyglucose), into glycogen as described (Louzao et al,
2008). The amount of LDH released into the medium was assayed using the LDH‐Cytotoxicity Assay Kit II (Abcam) according to the manufacturer's instructions. Apoptotic cells were detected with TUNEL using the ApopTag® Fluorescein Direct
In Situ Apoptosis Detection Kit (Millipore, Billerica, MA). Lysosomal exocytosis in TFEB‐treated cell cultures was evaluated by the surface LAMP1 assay as previously described (Medina et al,
2011) and by the measurement of acid phosphatase released into the medium. The acid phosphatase assay was performed in cultures treated with Ad‐TFEB for 2 days according to the standard procedure (Supporting Information, Table S1).
Western blot
Cells were homogenized in RIPA buffer [PBS containing 1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS and Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology, Danvers, MA)] and centrifuged for 30 min at 13,000 rpm at 4°C. Alternatively, for nuclear/cytosolic fractionation, cells were lysed in 0.5% Triton X‐100 buffer [50 mM Tris–HCl, 0.5% Triton X‐100, 137.5 mM NaCl, 10% glycerol, 5 mM EDTA and protease and phosphatase inhibitors] and centrifuged at 3000 rpm at 4°C for 5 min. The supernatant constituted the cytosolic fraction. The pellet was then washed with 0.5% Triton X‐100 buffer, re‐suspended in Triton X‐100 buffer with 0.5% SDS, sonicated, and centrifuged at 13,000 rpm for 15 min at 4°C. The supernatant constituted the nuclear fraction. Protein concentrations were measured using the Bio‐Rad Protein Assay (Bio‐Rad Laboratories, Inc., Hercules, CA). Western blots were performed according to standard procedures. Blots were scanned on an Odyssey Infrared Imager (LI‐COR Biosciences, Lincoln, NE).
Intramuscular injection of AAV‐TFEB, muscle staining and glycogen assay, and electron microscopy (EM)
Six 1‐month‐old GAA−/− mice were injected with a total dose of 1011 GC of AAV2/1 vector preparation into three sites of the right gastrocnemius (three injections of 30 µl each) using a Hamilton syringe. Equivalent doses of AAV2/1CMV‐EGFP or equal volumes of PBS were injected into the contralateral muscle. Animals were sacrificed 45 days after injection and perfused with PBS.
Gastrocnemii, free from neighbouring muscles and connective tissue, were isolated and analysed. Part of the sample was used to test the levels of TFEB expression by RT‐PCR. For morphologic analysis, muscles were frozen in liquid nitrogen‐cooled isopentane. Immunostaining of 10 µm sections was performed using anti‐LAMP1 (1:500; Abcam, Cambridge, MA, USA) and anti‐Caspase‐3 Active (1:500; R&D Systems, Minneapolis, MN, USA) antibodies. Apoptotic cells were detected with TUNEL using the Fluorescein In Situ Cell Death Detection Kit (Roche, Basel, Switzerland) according to the supplier's protocol. Muscle sections were mounted with Vectashield, and photographs were taken using a fluorescence microscope Zeiss (Thornwood, NY) Axioplan 2 integrated with the AxioCam MR camera. Haematoxylin–eosin and PAS staining were performed according to standard procedures.
Glycogen concentration was assayed in muscle lysates by measuring the amount of glucose released after digestion with amyloglucosidase (Aspergillus niger) using a commercial kit (BioVision, Milpitas, CA, USA). Data were expressed as µg of glycogen/mg of protein. For EM, muscle tissue was fixed in 1% glutaraldehyde in 0.2 M HEPES buffer and post‐fixed in uranyl acetate and OsO4. After dehydration through a graded series of ethanol and propilenoxide, the tissue was embedded in the Epoxy resin (Epon 812, Sigma–Aldrich) and polymerized at 60°C for 72 h. EM images of thin sections were acquired using a FEI Tecnai‐12 electron microscope (FEI, Eindhoven, Netherlands) equipped with a VELETTA CCD digital camera (Soft Imaging Systems GmbH, Munster, Germany). Quantification of the number of lysosome‐like organelles and their dimensions as well as the number of autophagosomes was performed in 50 fields (of 5 µm2 dimensions) distributed randomly through the thin sections containing different fibres.
Quantitative analyses are presented in the Supporting Information. Data in text are given as mean ± SE. Kolmogorov–Smirnov test was used for comparison of cumulative distributions of lysosomal size in myotubes and muscle fibres; Wilcoxon rank sum test was used for comparison of median values. Student's t‐test was used for all other comparisons. Differences were considered significant at p < 0.05.
Animal care and experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the European Union Directive 86/609 regarding the protection of animals used for experimental purposes.