Autophagy Is Required to Maintain Muscle Mass
Eva Masiero,1,2,4Lisa Agatea,2Cristina Mammucari,4Bert Blaauw,2,3Emanuele Loro,2Masaaki Komatsu,5
Daniel Metzger,6Carlo Reggiani,3Stefano Schiaffino,2,4and Marco Sandri1,2,4,*
1Dulbecco Telethon Institute, via Orus 2, 35129 Padova, Italy
2Venetian Institute of Molecular Medicine, via Orus 2, 35129 Padova, Italy
3Department of Human Anatomy and Physiology
4Department of Biomedical Science
University of Padova, viale Colombo 3, Padova, Italy
5Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
6Centre National de la Recherche Scientifique, INSERM, Illkirch-Cedex, France
The ubiquitin-proteasome and autophagy-lysosome
pathways are the two major routes for protein and
organelle clearance. In skeletal muscle, both systems
are under FoxO regulation and their excessive activa-
tion induces severe muscle loss. Although altered au-
tophagy has been observed in various myopathies,
the specific role of autophagy in skeletal muscle has
not been determinedbyloss-of-function approaches.
Here, we report that muscle-specific deletion of a
crucial autophagy gene, Atg7, resulted in profound
Atg7 null muscles showed accumulation of abnormal
mitochondria, sarcoplasmic reticulum distension,
disorganization of sarcomere, and formation of aber-
rant concentric membranous structures. Autophagy
and fasting. Thus, autophagy flux is important to
tophagy can contribute to myofiber degeneration and
mulation of abnormal mitochondria and inclusions.
Macroautophagy, hereafter referred to as autophagy, is a highly
conserved homeostatic process carrying out degradation of
cytoplasmic components including damaged organelles, toxic
protein aggregates and intracellular pathogens (Mizushima
et al., 2008). Autophagy takes place at basal levels in all eukary-
otic cells, turning over long-lived macromolecules and large
supramolecular structures including whole organelles to rejuve-
nate their function. In addition, autophagy can be upregulated
during metabolic, genotoxic, or hypoxic stress conditions and
acts as an adaptive mechanism essential for cell survival. Skel-
etal muscle is a major site of metabolic activity—and the most
abundant tissue in the human body, accounting for about 40%
of the total body mass. Being the largest protein reservoir,
muscle serves as a source of amino acids to be utilized for
energy production by various organs during catabolic periods
(Lecker et al., 2006). For instance, amino acids generated from
muscle protein breakdown are utilized by the liver to produce
glucose and to support acute phase protein synthesis (Lecker
et al., 2006). Protein degradation in skeletal muscle, like in all
the mammalian cells, is controlled by the two major proteolytic
Both degradation pathways are activated in a number of cata-
bolic disease states, including cancer, AIDS, diabetes, and heart
and renal failure and contribute to muscle loss and weakness.
The two systems are controlled by a transcriptional program
that upregulates few critical and rate-limiting enzymes (Sandri,
2008). We have recently identified FoxO transcription factors
as the main coordinators of the two proteolytic pathways by
inducing several autophagy-related genes as well as the two
muscle-specific ubiquitin ligases atrogin-1 and MuRF1 (Mam-
mucari et al., 2007; Sandri et al., 2004). While ubiquitin-preotea-
some dependent degradation has been deeply investigated and
its contribution to muscle loss has been already well docu-
mented, the role of autophagy in regulating muscle mass has
just started to be studied. Excessive activation of autophagy
aggravates muscle wasting (Dobrowolny et al., 2008; Mammu-
portion of cytoplasm, proteins, and organelles. Conversely, inhi-
bition of lysosome-dependent degradation causes myopathies
like Pompe and Danon diseases, and autophagy inhibition is
thought to play a role in many myopathies with inclusions or
with abnormal mitochondria (Levine and Kroemer, 2008; Temiz
et al., 2009). However, the exact role of autophagy in physiology
of skeletal muscle has never been addressed. Thus, defining the
role of autophagy in skeletal muscle homeostasis is critical for
understanding the pathogenesis of different diseases and for
developing new therapies against muscle loss. To clarify this
issue we have generated conditional knockout for Atg7 gene
to block autophagy specifically in skeletal muscle.
Generation of Muscle-Specific Atg7 Knockout Mice
We crossed Atg7-floxed mice (Atg7f/f) with a transgenic line ex-
pressing Cre recombinase under the control of a myosin light
mice, which are hereafter referred to as Atg7?/?. PCR analysis
Cell Metabolism 10, 507–515, December 2, 2009 ª2009 Elsevier Inc. 507
confirmed deletion of floxed sequence in genomic DNA from
undetectable in muscles of homozygous mice and considerably
Atg7 protein are due to endothelial cells, fibroblasts, macro-
phages, and blood cells. Efficient inhibition of autophagy in skel-
slow muscles (Figures 1B and S1A). LC3 exists in two forms: the
free mature form (LC3I) and the faster lipidated LC3 (LC3II). The
tion to phospholipids was completely blocked. LC3 and p62
proteins are known to be sequestered into the autophagosomes
and lostwhenautophagosomesfusewith lysosomes.Thus, their
increase indicates an efficient inhibition of autophagy. Con-
versely, Atg7 protein was detected and LC3I to LC3II conversion
over, immunohistochemical analyses showed the presence of
p62 aggregates in myofibers of Atg 7?/?mice (Figure 1D). To
further confirm that autophagic vesicles formation was blocked,
we transfected adult skeletal muscle with YFP-LC3, and 1 week
later we starved the mice (Mammucari et al., 2007). Ablation of
Atg7 in fasted muscle completely abolished the formation of
YFP-LC3 positive autophagosomes in myofibers (Figure 1E).
Altogether, these findings validate our genetic mouse model of
muscle-specific inhibition of the autophagy system.
Autophagy Inhibition Induces Muscle Atrophy,
Loss-of-Force Production, and Morphological
Features of Myopathy
The resulting Atg7?/?mice were indistinguishable in appearance
from age-matched control Atg7+/+mice. However, the growth
curve showed a slight reduction of body growth, which started
to differ from control after about 40 days from birth (Figure S2).
Morphological analysis of adult muscles revealed degenerative
changes, including vacuolated and centrally nucleated myofiber,
and a general decrease in myofiber size at 2 months of age
Figure 1. Generation of Muscle-Specific Atg7-Knockout (Atg7?/?) Mice
(A) Upper panel, genotyping of the Atg7f/fmice. Lower panel, PCR analysis with genomic DNA from gastrocnemius muscle. One of the two PCR primers is inside
the floxed region. Absence of a PCR product revealed an efficient Cre-mediated recombination of lox-P sites.
(B) Impaired LC3 lipidation and accumulation of p62 protein in Atg7?/?muscles. Muscle homogenates were immunoblotted with antibodies against Atg7, LC3,
(C) Immunoblot analysis of Atg7 and LC3 in homogenates from different tissues.
(D) Immunohistochemistry for p62 showed aggregates in Atg7?/?muscles but not in Atg7f/fmice.
(E)Autophagosome formationinducedby fasting issuppressedinAtg7?/?mice.MusclesofAtg7f/fand Atg7?/?weretransfected by electroporationwithplasmid
coding for YFP-LC3. Eight days later, mice were fasted for 24 hr before sacrifice. Myofibers expressing YFP-LC3 were analyzed by fluorescent microscopy.
Autophagy in Muscle
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Atg7 -/-Atg7 f/f
cross sectional area (%)
Figure 2. Morphological and Functional Changes in Muscles of Atg7?/?Mice Reveal Muscle Dysfunction and Features of Myopathy
(A) H&E staining showing a general decrease in myofibers size and different features of muscle degeneration (white arrows), including central nuclei and vacu-
(B) Quantification of cross-sectional area (CSA) of myofibers. Values are mean ± SEM of data from five mice in each group.
(C) Upregulation of the critical atrophy-related and muscle specific genes in adult skeletal muscle of Atg7?/?. RNA was extracted from TA muscles, and quan-
titative PCR analysis was performed in triplicates using specific oligonucleotides (see Table S1). Data were normalized to the b-actin content and expressed as
fold increase over levels of Atg7f/fmuscles, data are mean ± SEM. (*p < 0.001).
(D) Force measurements performed in vivo showed that Atg7?/?led to a profound decrease in force generation especially of maximal force generated during
tetanic contraction. The force is still significantly reduced even when the absolute tetanic force is normalized for the muscle weight (n = 5); data are mean ± SEM.
(E) Age aggravated the impairment of force production.
(F) Electron micrographs of Atg7?/?EDL muscles.
Autophagy in Muscle
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staining as a consequence of membrane permeabilization, sug-
gesting the presence of rare necrotic events, which, however do
not modify creatine kinase blood levels. Myosin composition
was not affected in Atg7?/?muscles (Figures S3 and S4). The
frequency of centrally nucleated fibers slowly increased with
age (Figure S5A). Importantly myofiber degeneration is not
caused by alteration in dystrophin expression and localization
(Figure S6). Quantification of cross-sectional area showed a
2B and S7). Further characterization displayed no difference
between fiber types; both glycolitic and oxidative fibers undergo
muscle atrophy (Figure S8). The muscle-to-body weight ratio
was also decreased, suggesting an important waste of muscle
tissue (Figure S9). Loss of muscle mass is controlled by a tran-
scriptional program that requires activation of a subset of genes
named atrophy-related genes or atrogenes (Lecker et al., 2004).
Thus, we monitored the level of expression of atrogenes involved
uitin ligases atrogin-1 and MuRF1, as well as genes involved in
different catabolic pathways, were upregulated in Atg7?/?
muscles at basal state (Figures 2C and S10). The upregulation
of the ubiquitin ligases is associated with FoxO1 dephosphoryla-
tion and activation (Figure S11). Interestingly, proteasomal func-
tion is not impaired in Atg7 null muscles but instead is increased
(Figure S12). Inhibition of autophagy led also to induction of
apoptosis (Figure S12). Altogether, these results suggest that
deletion of Atg7 triggers compensatory upregulation of ubiquitin
proteasome system and activation of apoptosis, which con-
tribute, at least partially, to muscle loss.
Next, we asked whether muscle atrophy is accompanied by
showed a marked reduction in absolute force independently of
gender (Figure 2D). Importantly, when the absolute force was
normalized for the muscle mass, the resulting specific force
was still significantly decreased. Thus, not only do the muscles
become smaller but there is a general impairment in force trans-
mission that leads to profound weakness. Importantly, force
drop was age dependent since 5-month-old males showed
a more important decrease in specific force, when compared
to age-matched control littermates, than 2-month-old mice
(Figure 2E). To understand the important impairment in force
generation, we performed electron-microscopy studies. Several
changes were detected in Atg7?/?muscles including misalign-
ment of Z-line, accumulation of big abnormal mitochondria
which in some cases span from one to the next Z line, presence
of swollen mitochondria, sarcoplasmic reticulum distension, and
formation of aberrant concentric membranous structure (Figures
Atg5-deficient hearts (Komatsu et al., 2005; Nakai et al., 2007).
The alteration of mitochondrial morphology is associated with
oxidative stress, as revealed by increased protein carbonylation
and expression of antioxidant genes, but apparently not to
energy unbalance, since AMPK was not activated (Figures
S14A–S14C). In addition, the changes of sarcoplasmic reticulum
are related with a markedly increased phosphorylation, and
therefore inhibition, of the translation initiation factor eIF2a,
which is known to lead to suppression of ribosome assembly
and protein synthesis. Altogether, the phosphorylation of eIF2a
and the increase of the endoplasmic reticulum (ER) chaperone,
BiP/GRP78, are consistent with an unfolding protein response
Inhibition of Autophagy Exacerbates Muscle Loss
and Degeneration in Catabolic Conditions
Next, we wanted to clarify the contribution and the role of
autophagy under conditions of muscle wasting. We used two
models of muscle atrophy, fasting and denervation, and we
compared Atg7 null muscles with controls. Inhibition of autoph-
agy did not prevent muscle loss and activation of the atrophy-
related program in denervated muscles. On the contrary, au-
tophagy-deficient animals lost significantly more muscle mass
than control ones (Figure 3A). Expression of several atrophy-
related genes, including MuRF1, cathepsin L, and Bnip3l, were
more upregulated in atrophying muscles of Atg7?/?muscles,
which suggest a more important activation of the atrophy
program (Figures 3B and S16). Morphological observations
showed different features of myopathy in denervated Atg7?/?
muscles, including presence of abnormal myonuclei, accumula-
tion of hematoxylin-positive inclusions, and vacuolated area,
which were present only in autophagy-deficient muscles (Fig-
ures 3C and S17). Electron microscopy revealed the presence
of concentric membranous structures embedded into an elec-
tron-opaque amorphous material (Figure 3D). Interestingly, p62
aggregates were increased in size and number in denervated
myofibers of Atg7?/?mice compared to innervated muscles
(Figure 3E). The p62-positive aggregates were also positive for
ubiquitin (Figures 3F and S18). Accordingly, p62 and ubiquiti-
nated proteins greatly accumulate in detergent soluble and
insoluble fractions of autophagy-deficient denervated muscles
(Figures 3G and S19). Thus, autophagy inhibition does not
preserve muscle mass during catabolic conditions and, surpris-
ingly, exacerbates muscle loss during denervation.
and Atg7+/+muscles showed similar upregulation of atrophy-
related genes, which reflects no major difference in the changes
of Akt phosphorylation and downstream targets (Figures 4A and
4B). However, the morphological features of muscle degenera-
tion were more evident. Many small flattened or irregularly
shaped fibers containing hematoxylin-positive inclusions as
well as fibers with fragmented and vacuolated cytosol appeared
in Atg7?/?muscles during fasting (Figure 4C). Electron micros-
copy revealed an increase of the concentric membranous struc-
tures (Figure 4D). However, fasted muscles never showed the
amorphous material detected in denervated muscles. Interest-
in fed Atg7?/?muscles and certainly never reached the size of
those observed in denervated muscles (Figure 4E). Indeed,
p62 did not accumulate in detergent-soluble and -insoluble frac-
tions of starved muscles (Figure S20). Thus, autophagy plays
different roles and importance in different conditions of muscle
loss but seems to be always crucial for maintaining normal
homeostasis of muscle mass in physiological and pathological
Deletion of Atg7 Gene in Adulthood Triggers
Muscle Loss and Weakness
To further check the role of autophagy in adulthood, we gener-
ated tamoxifen-inducible muscle-specific Atg7 knockout mice.
Autophagy in Muscle
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Muscle loss (% of control)
Atg7-/-f / f 7g tA f / f 7g tA
Muscle loss (% of control)
Figure 3. Denervation Aggravates Morphological Abnormalities and Muscle Loss in Atg7?/?Muscles
(A) Quantification of muscle loss after 2 weeks from denervation. CSA of innervated and denervated fibers was measured. Muscle loss is expressed as
percentage of decrease of cross-sectional areas of denervated fibers versus innervated ones. More than 1000 fibers per each muscle were counted (n = 4);
data are mean ± SEM (*p < 0.001).
(B) Enhanced upregulation of the atrophy-related genes in denervated skeletal muscles of Atg7?/?mice. Data are mean ± SEM (*p < 0.01).
(C) H&E staining showing accumulation of hematoxylin-positive structures, vacuolated areas, and abnormal nuclei (white arrows) in denervated Atg7?/?.
(D) Electron micrographs of denervated Atg7?/?showing aberrant concentric membranous structures dispersed between amorphous electron opaque material.
(E) Immunostaining for anti-p62 showed that p62 aggregates increased in size and number in denervated muscle of Atg7?/?.
(F) Double imunofluorescence staining reveals the colocalization of p62 and ubiquitin.
(G) Increase of ubiquitinated proteins and of p62 in Atg7?/?muscles during denervation. Detergent-soluble (Sup) and -insoluble (Pellet) fractions of control and
denervated muscles were immunoblotted against ubiquitin and p62. Data are representative of three different experiments.
Autophagy in Muscle
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concon fasted fasted
40 120 160
cross sectional area (%)
40µ µm40µ µm
Figure 4. Atg7 Deficiency in Fasting and in a Tamoxifen-Inducible Muscle-Specific Atg7-Knockout Mice
(A) Upregulation of the atrophy-related genes in fasted skeletal muscles of Atg7?/?mice.
(B) Immunoblotting for insulin-dependent pathway in fed (F) and starved (S) muscles of Atg7f/fand Atg7?/?mice.
nuclei, vacuoles, and loss of plasma membrane integrity.
Autophagy in Muscle
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Immunoblotting analyses confirmed the Atg7 deletion and the
concomitant block of autophagy revealed by p62 accumulation
and by inhibition of LC3 lipidation in glycolytic and oxidative
muscles (Figures 4F and S1B). Morphological analyses showed
the presence of structural alterations that are identical to those
observed in non-inducible Atg7?/?muscle. Succinate dehydro-
genase staining revealed accumulation of abnormal mitochon-
dria in small atrophic fibers (Figure 4G). However, centrally
nucleated fibers were more abundant after acute Atg7 deletion
than in non-inducible autophagy-deficient muscles (Figure S5B).
Indeed, when we measured muscle mass we found that autoph-
agy inhibition triggered muscle wasting. Quantification of cross-
sectional area showed a 20% decrease in myofiber size (Fig-
ure 4H), which was accompanied by a decrease in absolute
and specific force (Figure 4I).
Our results indicate that basal autophagy plays a beneficial role
incontrolling muscle mass.Lack ofautophagyaffects theorgan-
elle shaping machinery and leads to accumulation of atypical
giant mitochondria and dilated sarcoplasmic reticulum. How-
ever, accumulation of abnormal organelles is not always harmful
for cellular function. In fact, ablation of p62 in liver-specific au-
tophagy-deficient mice suppresses pathological phenotypes
including severe hepatomegaly, inflammation, and leakage of
hepatic enzymes despite accumulation of abnormal organelles
in the mice (Komatsu et al., 2007). Therefore, at least in autoph-
agy-deficient liver, the presence of degenerated mitochondria
might be hardly attributed to the phenotypes. Conversely, loss
of p62 in neural-specific Atg7?/?mice does not suppress the
pathology. Neurons from both single Atg7?/?and Atg7/p62
double knockout accumulate large number of abnormal organ-
elles in the axon terminals, suggesting that an appropriate turn-
over of organelles in axon terminal is essential for neuronal
homeostasis (Komatsu et al., 2007). Therefore, we can conclude
that pathogenesis of cellular dysfunction and degeneration
during autophagy inhibition differs among tissues and cell types.
In muscle, the persistence of dysfunctional organelle seems to
be important for the activation of catabolic pathways, which
results in muscle atrophy and weakness. In our model, alteration
in sarcoplamic reticulum reflects an unfolding protein response
that suppresses protein synthesis while mitochondrial damage
generates oxidative stress and apoptosis. Also, the induction
of Bnip3, which promotes mitochondrial fragmentation and mi-
tophagy, in Atg7?/?muscles might contribute to caspase activa-
tion and apoptosis by affecting permeability transition pore
opening. The control of mitochondrial function seems to be
crucial for preventing a cascade of signals that lead to muscle
atrophy (Sandri et al., 2006). The accumulation of aged and dys-
functional mitochondria and their potential negative role for cell
survival has been recently underlined by different genetic
evidences. For instance, it has been shown that dysfunctional
lem dystrophies (Angelin et al., 2007; Irwin et al., 2003; Merlini
et al., 2008). Similarly, erythroid cells lacking Bnip3l show persis-
tence of mitochondria, due to a block of autophagy, which
causes premature cell death and anemia (Sandoval et al.,
2008). It is unclear whether our data of oxidative stress in
Atg7?/?muscle is mainly caused by accumulation of dysfunc-
tional mitochondria due to a defect in mitophagy, as recently
described in autophagy-deficient cells (Tal et al., 2009), or
whether it is secondary to p62 aggregates (Mathew et al.,
2009). Moreover Atg7?/?muscles showed activation of ER
chaperones, such as BiP, as well as the phosphorylation of
eIF2a, suggesting an ongoing unfolded protein response. The
failure of protein-folding quality control in Atg7?/?mice induces
endoplasmic reticulum stress, which can generate ROS, and
suppression of protein synthesis, which can contribute to
Recently muscle-specific Atg5?/?mice have been generated
(Raben et al., 2008), and their phenotype is similar though not
identical to that of Atg7?/?mice. Both knockouts show muscle
loss, protein aggregates, and accumulation of abnormal
membranous structures. The main difference between the two
studies is related to the lack-of-force impairment reported in
Atg5?/?animals. However, muscle force in Atg5?/?mice was
evaluated by an indirect test, the wire-hang test, which can be
affected by many variables including fatigue, whereas we per-
formed a direct physiological analysis of force measurement
on gastrocnemius muscles. In conclusion, our results suggest
that in skeletal muscle defects in organelle removal generate
a signaling cascade, which induces profound muscle loss and
weakness. It has been shown that the efficiency of autophagic
degradation declines during aging, leading to accumulation of
intracellular waste products (Salminen and Kaarniranta, 2009).
Our results suggest that impaired autophagy may contribute to
aging sarcopenia. Thus, to combat sarcopenia, it is important
to maintain autophagy flux to rejuvenate organelles and to
prevent accumulation of dysfunctional mitochondria and ER
membranes, as well as to block excessive protein breakdown.
Generation of Muscle-Specific Atg7?/?Mice
and In Vivo Transfection Experiments
Generation of muscle-specific Atg7?/?mice is described in Supplemental
Data. In vivo transfection experiments were performed by intramuscular injec-
tion of plasmid DNA in tibialis anterior (TA) muscle followed by electroporation
(D) Electron micrograph of fasted Atg7?/?muscles.
(E) Immunostaining for p62 showed positive aggregates in Atg7?/?muscles.
were collected and analyzed.
(G) H&E staining showing a general decrease in myofiber size and accumulation of hematoxylin positive inclusions. SDH staining on serial sections showed an
accumulation of abnormal mitochondria.
(H) Quantification of CSA of myofibers. Values are mean ± SEM of data from five mice in each group, at least 1000 fibers for each muscles were measured
(*p < 0.001).
(I) Force measurements performed in vivo, data are mean ± SEM (n = 5).
Autophagy in Muscle
Cell Metabolism 10, 507–515, December 2, 2009 ª2009 Elsevier Inc. 513
as described (Mammucari et al., 2007). Muscles were removed at 8 days after
transfectionandfrozeninliquidnitrogen forsubsequentanalyses. Denervation
as control. Muscles were collected 3 days after denervation for gene-expres-
sion studies and 14 days after denervation for morphological analyses.
Total RNA was prepared from TA muscles using Promega SV Total RNA Isola-
tion Kit. Complementary DNA generated with Invitrogen SuperScript III
Reverse Transcriptase was analyzed by quantitative real-time RT-PCR using
QIAGEN QuantiTect SYBR Green PCR Kit. All data were normalized to b-actin.
The oligonucleotide primers used are shown in Table S1.
Frozen gastrocnemius muscles were powdered by pestle and mortar and
lysed in a buffer containing 50 mM Tris pH 7.5, 150 mM NaCl, 10 mM
MgCl2, 0.5 mM DTT, 1 mM EDTA, 10% glycerol, 2% SDS, 1% Triton X-100,
RocheCompleteProtease InhibitorCocktail, 1mMPMSF,1mMNaVO3,5mM
NaF and 3 mM b-glycerophosphate. The samples were immunoblotted as
previously described (Sandri et al., 2004) and visualized with SuperSignal
West Pico Chemiluminescent substrate (Pierce). Blots were stripped using
Restore Western Blotting Stripping Buffer (Pierce) according to the manufac-
turer’s instructions and reprobed if necessary. Detergent-soluble and -insol-
uble fractions were obtained according to (Hara et al., 2006). A list of anti-
bodies is shown in Supplemental Data.
Histology, Fluorescence Microscopy, and Electron Microscopy
rescence microscope as described (Mizushima et al., 2004). Cryosections of
TA were stained for H&E, for SDH, PAS, anti-ubiquitin, and anti-p62. CSA
was performed on TA as described (Blaauw et al., 2008; Mammucari et al.,
2007). For electron microscopy, we used conventional fixation-embedding
procedures based on glutaraldehyde-osmium fixation and Epon embedding.
Measurements of Muscle Force In Vivo
Muscle force was measured in a living animal as previously described (Blaauw
et al., 2008). Briefly gastrocnemius muscle contractile performance was
measured in vivo in anaesthetized mice using a 305B muscle lever system
(Aurora Scientific, Inc.). Contraction was elicited by electrical stimulation of
the sciatic nerve. Force developed by plantar flexor muscles was calculated
by dividing torque by the lever arm length (taken as 2.1 mm).
The Supplemental Data include 20 figures, Supplemental Experimental Proce-
This work was supported by grants from Agenzia Spaziale Italiana (OSMA
project) to M.S. and S.S., from Telethon (S04009), AFM (14135), the Italian
Ministry of Education, University and Research (PRIN 2007) and Compagnia
San Paolo to M.S., from the European Union (MYOAGE, contract: 223576 of
FP7 to M.S. and S.S.), from the Japan Science and Technology Agency to
M.K. Atg7 antibody was a generous gift of Dr. T. Ueno. We gratefully acknowl-
edged S. Burden for the gift of MLC1f-Cre mice and the FP6 EXGENESIS Inte-
grated Project to S.S.
Received: March 29, 2009
Revised: August 9, 2009
Accepted: October 6, 2009
Published: December 1, 2009
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