Constitutive Activation of MAPK Cascade in

Acute Quadriplegic Myopathy

Simone Di Giovanni, MD,1 Annamaria Molon, PhD,1 Aldobrando Broccolini, MD,2 Gisela Melcon, MD,1
Massimiliano Mirabella, MD,2 Eric P. Hoffman, PhD,1 and Serenella Servidei, MD2

Acute quadriplegic myopathy (AQM; also called “critical illness myopathy”) shows acute muscle wasting and weakness
and is experienced by some patients with severe systemic illness, often associated with administration of corticosteroids
and/or neuroblocking agents. Key aspects of AQM include muscle atrophy and myofilament loss. Although these features
are shared with neurogenic atrophy, myogenic atrophy in AQM appears mechanistically distinct from neurogenic atro-
phy. Using muscle biopsies from AQM, neurogenic atrophy, and normal controls, we show that both myogenic and
neurogenic atrophy share induction of myofiber-specific ubiquitin/proteosome pathways (eg, atrogin-1). However, AQM
patient muscle showed a specific strong induction of transforming growth factor (TGF)–␤/MAPK pathways. Atrophic
AQM myofibers showed coexpression of TGF-␤ receptors, p38 MAPK, c-jun, and c-myc, including phosphorylated
active forms, and these same fibers showed apoptotic features. Our data suggest a model of AQM pathogenesis in which
stress stimuli (sepsis, corticosteroids, pH imbalance, osmotic imbalance) converge on the TGF-␤ pathway in myofibers.
The acute stimulation of the TGF-␤/MAPK pathway, coupled with the inactivity-induced atrogin-1/proteosome pathway,
leads to the acute muscle loss seen in AQM patients.
Ann Neurol 2004;55:195–206

Acute quadriplegic myopathy (AQM) or critical illness
myopathy (CIM) is a subacute muscle disorder charac-
terized by generalized progressive muscle weakness and
atrophy that occurs in critically ill patients. Its patho-
genesis is multifactorial and is associated with a patient
history of sepsis, severe pulmonary disorders, major
surgery, multiorgan failure, intensive care, and expo-
sure to corticosteroids and neuroblocking agents.1–3
Electromyography has excluded a primary neurogenic
process and instead points to a primary myogenic dis-
ease.4 Diagnosis is possible with muscle biopsy, in
which histopathology shows a marked myogenic atro-
phy5 with or without selective loss of myosin filaments.
Classic necrosis is a rare event and limited to a minor-
ity of fibers. Thus, the presence of severe subacute at-
rophy in the absence of evidence of denervation or
nerve disease is strongly suggestive of a diagnosis of
AQM.
The molecular chain of events leading to myofiber
atrophy has been poorly understood. Ubiquitin-
mediated proteolysis and both cytoplasmic (calpains)
and lysosomal-dependent (cathepsins) proteolytic path-
ways have been associated with muscle wasting in
AQM patients and with cell atrophy in both in vivo
and in vitro models.6 –12 Lack of muscle excitability
due to sodium channel inactivation also has been de-
scribed in a subset of patients and in an animal model
of AQM.13,14 Gene expression changes in animal mod-
els of AQM and CIM also have been reported and
showed repression of specific Na channels15 and induc-
tion of proteolytic machinery at the RNA transcription
level.16,17
We have described previously the occurrence of ap-
optotic features in atrophic fibers in human muscle bi-
opsies from AQM patients, as shown by DNA frag-
mentation in situ by terminal deoxynucleotidyltrans-
ferase–mediated dUTP nick end labeling (TUNEL),
and nuclear margination and condensation by electron
microscopy.18 Many of the fibers were found to show
strong expression of calpain, cathepsin B, and caspase
3.18 We also have shown that these coordinated
changes occurred specifically in AQM muscles as op-
posed to biopsies from neurogenic myofiber atrophy.
These data suggested that stimulation of proapoptotic
pathways may be part of the underlying pathogenesis
of AQM.

From the 1Center for Genetic Medicine, Children’s National Med-
ical Center and Genetics Program, George Washington University,
Washington, DC; and 2Institute of Neurology, Catholic University,
Rome, Italy.
Received Aug 5, 2003, and in revised form Sep 15. Accepted for
publication Sep 15, 2003.
Address correspondence to Dr Hoffman, Center for Genetic Medi-
cine, Children’s National Medical Center, Washington, DC 20010.
E-mail: ehoffman@cnmcresearch.org
This article is a US Government work and, as such, is in the public do-
main in the United States of America.

Published 2003 by Wiley-Liss, Inc., through Wiley Subscription Services 195

 To provide a more complete picture of the biochem-
ical pathways activated in AQM, we defined a molec-
ular fingerprint unique to myogenic atrophy (AQM) as
opposed to neurogenic atrophy by genomewide mi-
croarray analysis of patient muscle biopsies. We show
that unsupervised data analysis is easily able to differ-
entiate myogenic atrophy from neurogenic atrophy via
expression profiles, and that constitutive stimulation of
the proapoptotic transforming growth factor (TGF)–␤/
MAPK pathway underlies myogenic atrophy. We also
show that the muscle-specific S␬p1-Cullin-F-box pro-
tein (SCF) ubiquitin ligase component, atrogin-1
(FBx32), is strongly induced in AQM, consistent with
the significant loss of myofilaments in this disorder.
We propose a model for AQM in which a combination
of cellular stress, inactivity of muscle, and drug stimu-
lation (corticosteroids) all converge on the TGF-␤/
MAPK and atrogin-1 pathways, leading to acute stim-
ulation of apoptosis and myofilament loss.
Patients and Methods
Patients and Biochemical and Morphological Analysis
of Muscle Biopsies
Diagnostic muscle biopsies were obtained, with informed
consent, from five patients, affected by acute quadriplegic
myopathy (Table 1). Muscle biopsies from all patients were
studied by standard histochemistry for morphological assess-
ment, and for enzyme biochemistry for acid and neutral
maltase. Disease control muscle biopsies were five patients
with neurogenic disorders, including motor neuron disease,
lower motor neuron disease, spinal muscle atrophy type III,
and chronic axonal neuropathy. Seven normal control mus-
cles were from patients with asymptomatic elevations of cre-
atine kinase (hyperCKemia), with no evidence of histological
changes on muscle biopsy.
TUNEL, electron microscopy, and immunocytochemistry
for apoptosis-related proteins caspase 1, caspase 3, caplain,
and cathepsin B were done as previously described18 (see Ta-
ble 1).
Expression Profiling
Expression profiling was done as we have described previ-
ously.19 In brief, RNA was extracted from each frozen mus-
Table 1. Summary of Clinical, Electrophysiological, and Muscle Pathological Features of AQM patients
Pt. Age Surgery/
No. (yr) ICU Systemic Illness Anesthesia Corticosteroids Weakness
1 66 No COPD-diabetes No
2 66 Yes Sepsis-MD Yes
3 77 No Lymphoma-hyper No
cortisol.
4 64 Yes Sepsis-ARDS-MODS No
5 76 Yes Sepsis-MODS Yes
ICU ⫽ intensive care unit; EMG ⫽ electromyogram; CK ⫽ creatine kinase; COPD ⫽ chronic obstructive pulmonary disease; n.v. ⫽ normal
values; MD ⫽ myelodisplasia. ARDS ⫽ acute respiratory distress syndrome; MODS ⫽ multiorgan distress syndrome; AQM ⫽ acute quan-
titative myopathy.
196 Annals of Neurology Vol 55 No 2 February 2004
cle biopsy using TRIzol reagent (GIBCO BRL, Gaithers-
burg, MD). Seven milligrams of total RNA from each tissue
sample (17 samples total) was processed accordingly to pro-
tocol (Affymetrix) to obtain fragmented cRNA that was hy-
bridized on the chip.
Microarray (genechip) Quality Control, Data
Scrubbing, and Statistical Analysis
We used stringent quality control methods as previously
published19 and detailed on our Web site: (http://
microarray.cnmcresearch.org/pgaoutline-qcofsamples.asp).
Three of the neurogenic atrophy subject biopsies did not
meet our usual threshold for “present” calls. We attributed
this to the severe atrophy and fibrosis shown by muscle of
these patients. The scaling factors determinations were done
using default Affymetrix algorithms (MAS 5) with a target
intensity of chip sector fluorescence to 800. Both preampli-
fication (s1) and postamplification with streptavidin/phyco-
erythrin (s2) scans were done, and the scans were compared
by scatterplots and correlation coefficients.19 Saturated probe
sets showing evidence of saturation of the PMT in s2 were
eliminated with our custom Array Data Manipulation soft-
ware. We have shown recently that use of Affymetrix MAS
5.0 signal intensity values, together with a “present call”
noise filter achieves an excellent signal to noise balance for
human muscle relative to other probe set analysis methods
(RMA).20 Data analyses were limited to probe sets that
showed one or more “present” (P “calls”) in the 17 genechip
profiles in our complete data set. Experiment normalization
was performed by normalizing gene chips as described.19
Normalized data then were compared for differential gene
expression analysis between AQM patients and the two con-
trol groups. Genes that showed a Welch analysis of variance
(ANOVA) t test with p value less than 0.05 between groups
were retained for further analysis. Initial data analysis also
included a fold change filter of greater than 1.5 (50% dif-
ference) increase or decrease relative to control groups (Af-
fymetrix MAS 5.0). Although a p value of less than 0.05
alone would give many false-positives, the combination of
present call filters, fold change thresholds, and p value
thresholds, eliminates most false-positives that are obtained
with only p values less than 0.05 (all confirmed by other
methods). We also confirmed nearly all expression changes at
the mRNA and/or protein level using independent tech-
niques.
Apoptotic
Atrophy/ Myofiber Signs
EMG CK Atrophy (% fibers)
Yes ⫹⫹⫹ Myopathy 1/10 n.v. ⫹⫹⫹ 70
Yes ⫹⫹⫹ Myopathy 1/2 n.v. ⫹⫹⫹ 80
No ⫹⫹⫹ Myopathy 1/5 n.v. ⫹⫹ 50
Yes ⫹⫹⫹ Myopathy n.v. ⫹⫹ 50
Yes ⫹⫹ Myopathy 1/20 n.v. ⫹⫹ 40
Real-time Reverse Transcription Polymerase
Chain Reaction
We studied gene expression in muscle from AQM patients
and normal controls, by real-time polymerase chain reaction
(PCR) for both validation purposes (gadd45 ␤, p21, c-myc,
and c-jun) and to measure expression of genes not repre-
sented on the genechip (atrogin-1). Fluorophore-labeled
LUX primers (forward) and their unlabeled counterparts (re-
verse) were provided by Invitrogen (La Jolla, CA). LUX
primers were designed within Affymetrix probe sets se-
quences for each gene, and all primers were designed using
the software called LUX Designer (Invitrogen, www.invitro-
gen.com/lux). We performed multiplex PCR combining in
the same PCR mix each experimental gene with the house-
keeping gene GAPDH.
For each sample, 20␮l PCR contained 2␮l cDNA (first
diluted 1:10 after reverse transcription [RT]), 200nM of
each gene-specific primer (two pairs for multiplex PCR) and
1 ⫻ Platinum Quantitative PCR SuperMix-UDG (Invitro-
gen), and 1 ⫻ ROX reference dye (500nM in SuperMix).
PCR conditions were standard (Invitrogen, www.invitrogen-
.com/lux), and reactions were conducted in a 96-well spec-
trofluorometric thermal cycler (ABI PRISM 7700 Sequence
detector system; Applied Biosystems, Foster City, CA). Flu-
orescence was monitored during every PCR cycle at the an-
nealing or extension step and during the post-PCR temper-
ature ramp. Fold changes were measured according to
manufacturer instructions (Invitrogen), and ANOVA t test
was used for statistical analysis.
Immunoblotting
Protein extracts were recovered from the lower organic phase
of TRIZOL after supernatant isolation for RNA extraction
(GIBCO). Proteins then were quantified by the Bradford
method, and 20␮g of protein solubilized in Laemmli sample
buffer supplemented with protease inhibitors (10mg of apro-
tinin per ml, 1mg of leupeptin per ml, 1mM phenylmethl
sulfonyl fluoride). Immunoblotting was done using standard
methods with 4 to 12% and 10% SDS-PAGE gels. For im-
munoblotting, we used the following antibodies: goat poly-
clonal anti–TGF-␤ receptor I and II, goat polyclonal anti–
TAK-1 (diluted 1:200; Santa Cruz Biotechnology, La Jolla,
CA); rabbit polyclonal anti-junB (diluted: 1:1,000; Onco-
gene, Cambridge, MA); and rabbit polyclonal anti–R-Ras,
anti–p38 MAPK, anti–phospho-p38 MAPK, anti–c-jun,
anti–phospho-c-jun, anti–c-myc, anti–phospho-c-myc, anti–
phosho-SMAD 1, 5, 8,anti-MKK3, anti–phspho-MKK3,
anti-MKK4, ant-Ask1, anti–phospho-Ask1, mouse monoclo-
nal anti-JNK, and anti–phospho-JNK (Cell Signaling, Bev-
erly, MA). Immunocomplexes were visualized with ECL
chemiluminescence (Amersham, Arlington Heights, IL).
TUNEL and Immunocytochemistry
The TUNEL technique was used for detection of nuclear
DNA fragmentation in situ. Frozen muscle sections (10␮m)
from patients and control subjects were incubated under the
same coverslip with TUNEL reaction mixture, and incorpo-
rated fluorescein-dUTP was detected by using alkaline
phosphatase-conjugated antifluorescein antibodies according
to the manufacturer’s instruction (In Situ Cell Death Detec-
tion Kit; Boehringer, Mannheim, Germany). Unfixed muscle
sections adjacent to those analyzed by means of TUNEL
were processed for immunocytochemistry accordingly to
standard procedures. The same primary antibodies used for
immunoblotting were used also for immunocytochemistry,
and incubated overnight at 4°C. Sections were developed
with peroxidase staining. The count of fibers positive by
TUNEL and immunocytochemistry was performed in at
least 100 muscle fibers per section.
Results
Clinical and Pathological Features of Acute
Quadriplegic Myopathy and Controls
Muscle biopsies from five patients, each showing a
clinical and histological pattern consistent with the di-
agnosis of AQM, were studied (see Table 1). Severe
systemic illness was present in all patients, variably in-
cluding sepsis, intensive care unit treatment, surgery,
and corticosteroids administration. All patients had a
moderate/severe muscle weakness and atrophy. Electro-
myography showed myopathic changes compatible
with AQM. No clinical or electromyography evidence
of nerve involvement was seen, including lack of spon-
taneous activity (see Table 1). Apoptotic signs, as
shown by TUNEL and activated caspase 3 immunore-
activity were present in atrophic fibers in all patients
(see Table 1), as we have previously described.18
Both neurogenic and normal control muscles were
used to address the specificity of the changes in AQM
muscles. These age-matched controls included seven
biopsies from subjects with asymptomatic hyperCK-
emia (normal histopathology) and five biopsies from
patients from neurogenic disorders (motor neuron dis-
ease, lower motor neuron disease, spinal muscle atro-
phy type III, and chronic axonal neuropathy; all
showed neuropathic histopathology). A subset of neu-
rogenic atrophic fibers showed also apoptotic features
as previously reported.18
Unsupervised Hierarchical Clustering Accurately
Diagnoses Acute Quadriplegic Myopathy from
Neurogenic Atrophy
Each skeletal muscle biopsy was processed individually
for expression profiling (five AQM patients, five neuro-
genic disorders, and seven normal muscles). We expres-
sion profiled approximately 12,000 transcripts using Af-
fymetrix high-density oligonucleotide arrays Hu95 v2.
Data were processed according to bioinformatic methods
that we have previously shown provide good signal/noise
ratios for human muscle biopsies.20 Normalizations in-
cluded per chip (50th percentile) and per sample nor-
malization (to the median of each gene). We used sev-
eral types of data analysis: unsupervised and supervised
hierarchical clustering; statistical analysis using analysis
of variance (Welch ANOVA t test); nucleation of dys-
regulated transcripts using a 1.5-fold change cutoff be-
Di Giovanni et al: MAPK Cascade in AQM In Vivo 197
tween the different groups of diseases (probe sets differ-
ing in 50% expression level); functional clustering with
implementation of the GeneSpring gene query tool; and
functional ontology visualizations using both Gene-
Mapp,21 and David (http://apps1.niaid.nih.gov/david/
upload.jsp) software and databases.
Unsupervised hierarchical clustering based on stan-
dard distance metrics (GeneSpring) showed correct di-
agnosis of the three diagnostic groups (myogenic atro-
phy [AQM], neurogenic atrophy, normal controls) into
three specific branches on the dendrogram (Fig 1).
This analysis provides strong support for distinct mo-
lecular pathophysiological mechanisms for the two
types of atrophy and also indicates that the biological
variables were dominant over the sum of technical vari-
ables and interindividual noise.20
We then filtered data for dysregulated transcripts
based on a significant Welch ANOVA t test ( p ⬍
0.05) between AQM and neurogenic and AQM and
normal controls, combined with a 1.5-fold change
threshold. This resulted in 1,670 upregulated, and 709
downregulated transcripts (see http://microarray.
cnmcresearch.org/pgadatatable.asp for the complete
gene list). The AQM-specific transcripts then were fil-
tered for functional ontologies and further analyzed
and verified, as described below.
Strong Induction of Oxidative Stress Response
and Protease/Proteasome Clusters in Acute
Quadriplegic Myopathy
Visualization of known functional clusters showed con-
sistent activation of oxidative stress, protease (including
caspase 4 and 6), and ubiquitin pathways using “heat
map” methods (see Fig 1). Some of these pathway
members showed downregulation in neurogenic con-
trols, consistent with distinct underlying molecular
pathophysiologies in the two types of atrophy. The
ubiquitin-dependent proteolytic pathway was particu-
larly strongly induced in myogenic atrophy (see Fig 1),
including upregulation of several ubiquitin proteases
and ubiquitin activating enzymes. There was no evi-
dent upregulation of ubiquitin ligases; however, it is
known that many ubiquitin ligases are induced via ac-
tivation of tissue specific F-box proteins of the SCF
ligase complex.11 The muscle-specific F-box, atrogin-1
(FBx32),11 was not on the U95v2A microarrays used
for our expression profiling studies. Therefore, we
studied expression of the ubiquitin ligase atrogin-1, by
quantitative real-time RT-PCR. We found atrogin-1
mRNA 10-fold increased in both AQM and neuro-
genic muscle relative to controls (Table 2). To our
knowledge, this is the first data on atrogin-1 in hu-
mans in vivo, although our data are consistent with in
vitro and animal models experiments showing
atrogin-1 induced in muscle atrophy under diverse
conditions and stimuli.
198 Annals of Neurology Vol 55 No 2 February 2004
The Transforming Growth Factor–␤/MAPK
Signaling Cascade Is Specifically Activated in Acute
Quadriplegic Myopathy Myogenic Atrophy
The GeneMapp and David databases were used for
functional classification of dysregulated transcripts to
identify potential signaling pathways specifically associ-
ated with myogenic atrophy. Both databases showed the
induction of several members of the MAPK signaling
cascade in atrophic AQM muscles. MAPK induction
plays an important role in the regulation of cell growth,
atrophy, mitosis, and apoptosis.22 This prompted us to
further evaluate with both the expression profiling data
set and RT-PCR the expression levels of all known
members of this pathway and of possible related path-
ways, including TGF-␤ and RAS signaling cascades. We
found the specific upregulation of TGF-␤ receptor II,
TAK-1, ASK-1, RAS family members, SMADs (Smad
1, 3, and 4), JNKK2, JNK, MKK3/6, p38 MAPK,
c-jun, junB, c-fos, c-myc, gadd-45␤, and p21 cell cycle
inhibitor. Fold changes and p values for each transcript
are reported (Table 2). Transcription of the extracellular
signal-repulsated kinase (ERK) branch of the large
MAPK pathway was inhibited or not differentially reg-
ulated in AQM muscles (data not shown).
Real time RT-PCR confirmation was performed for
a subset of the TGF-␤/MAPK pathway members (p21,
GADD45 ␤, c-myc, c-jun; see Table 2). In all tran-
scripts tested, the RT-PCR data were consistent with
the microarray data, and all genes showed significant p
values by both independent assay methods.
Activation of the Transforming Growth Factor–␤/
MAPK Pathway Is Seen at the Protein Level
The majority of activation of the TGF-␤/MAPK path-
way occurs at the protein level (phosphorylation, local-
ization, stability), and mRNA studies are a relatively in-
sensitive means of assessing the status of the entire
pathway. For this reason, we performed immunoblotting
for many members of the TGF-␤/MAPK cascade in
protein extracts from all AQM muscle biopsies and
compared them with the neurogenic and normal con-
trols. We measured both total and phosphorylated (p)
forms for the following proteins: ASK-1, SMADs,
JNKKs, JNKs, MKK3/6, P38-MAPK, c-jun, c-fos,
c-myc. Protein levels also were assessed for r-RAS,
TGF-␤ receptors I and II, TAK-1, junB (Fig 2A). Our
immunoblot data were consistent with a wide activation
of the TGF-␤/MAPK pathway, with increases of both
total and phosphorylated protein forms in AQM atro-
phic muscles. These data were also in agreement with
the transcriptional upregulation seen by both microarray
and RT-PCR. Fold changes and p values from quanti-
tated immunoblots were calculated for TGF-␤ receptor
II, ASK-1, p38-MAPK, phosphorylated p38-MAPK,
c-myc, phosphorylated c-myc, SMADs, and junB (see
Fig 2B); all demonstrated highly significant activation
 Fig 1. Diagnostic and func-
tional ontology analysis of
microarray data. (A) Unsu-
pervised hierarchical cluster-
ing shows correct diagnosis of
acute quadriplegic myopathy
(AQM) subjects. Shown is
unsupervised hierarchical
clustering based on average
distance metrics (Gene-
Spring), with correct diagno-
sis of the three groups studied
(myogenic atrophy [AQM],
neurogenic atrophy, normal
controls) into three specific
branches on the dendrogram.
Specifically, each group of
patients is clustered into a
specific branch of the gene
tree, corresponding to genes
with similar expression levels.
Bar graph on the right shows
the color code for gene expres-
sion level (red: high; blue:
low). AQM patient numbers
refer to Table 1. Neurogenic
atrophy and controls are
numbered based on order in
the figure. (B) AQM shows
specific induction of stress
response and proteolysis path-
ways. Shown are supervised
hierarchical clusters obtained
after filtering for more than
one “present” call across the
17 profiles, p value less than
0.05 (Welch ANOVA t test),
and 1.5-fold changes in ex-
pression between AQM and
controls; these filters then
were combined with func-
tional clustering of oxidative
stress and proteases and
ubiquitin-dependent pathway.
Bar graph on the right shows
the color code for gene expres-
sion level (red: high; blue:
low).

specific for AQM myogenic atrophy, including the ratio at both at the transcriptional and protein level, including
of phosphorylated versus total p38-MAPK. We conclude activated phosphorylated states of specific signaling pro-
that the TGF-␤/MAPK cascade is coordinately activated teins, in myogenic atrophy in AQM.

Di Giovanni et al: MAPK Cascade in AQM In Vivo 199

Table 2. TGF-␤ MAPK Signaling Cascade mRNA Expression Data Show Strong Upregulation Specific to Myogenic Atrophy
AQM vs Control AQM vs Neurogenic Atrophy

Fold Percentage of Positive Fibers* Fold

Genbank No. Gene Name Change p (⫾ SD) Change p

D50683 TGFbeta type II receptor

mRNA Affymetrix 3 ⬍0.01 2.4
Protein in atrophic fibers 90 ⴞ 5(AQM) 20 ⴞ 5(Neur) <0.01
V00568 MYC
mRNA Affymetrix 9 ⬍0.01 12.6 ⬍0.01
mRNA real-time PCR 8.8 ⬍0.01 5.1 ⬍0.05
Protein in atrophic fibers 82 ⴞ 3(AQM) 12 ⴞ 4(Neur) <0.01
(p-c-MYC)
AF078077 GADD45beta
mRNA Affymetrix 4.3 ⬍0.05 5.1 ⬍0.05
mRNA real-time PCR 7.1 ⬍0.01 4.6 ⬍0.05
Protein in atrophic fibers 86 ⴞ 5(AQM) 8 ⴞ 2(Neur) <0.01
U59423 SMAD1
mRNA Affymetrix 2.1 ⬍0.01 1.8 ⬍0.01
U59912 SMAD1
mRNA Affymetrix 1.9 ⬍0.01 2.2 ⬍0.01
X89750 SMAD2
mRNA Affymetrix 4.1 ⬍0.01 2.1 ⬍0.05
U59913 SMAD5
mRNA Affymetrix 2.8 ⬍0.01 7.4 ⬍0.01
SMAD 1, 5, 8
Protein in atrophic fibers 88 ⴞ 4(AQM) 3 ⴞ 1(Neur) <0.01
L35263 P38 MAPK
mRNA Affymetrix 1.9 ⬍0.01 1.8 ⬍0.05
Protein in atrophic fibers 65 ⴞ 10(AQM) 3 ⴞ 1(Neur) <0.01
M14949 R-RAS
mRNA Affymetrix 3 ⬍0.05 5.5 ⬍0.01
AF043938 M-RAS R-RAS3
mRNA Affymetrix 1.6 ⬍0.01 1.4 ⬍0.05
RAS
Protein in atrophic fibers 95 ⴞ 5(AQM) 4 ⴞ 1(Neur) <0.01
J04111 JUN
mRNA Affymetrix 3.3 ⬍0.01 2.3 ⬍0.05
mRNA real-time PCR 6 ⬍0.01 4.5 ⬍0.05
U03106 CDKN1 P21
mRNA Affymetrix 14.5 ⬍0.01 7.2 ⬍0.01
mRNA real-time PCR 8 ⬍0.01 5 ⬍0.05
p-ASK
Protein in atrophic fibers 72 ⴞ 4(AQM) 3 ⴞ 1(Neur) <0.01
X14885 TGFbeta 3
mRNA Affymetrix 3.1 ⬍0.05 2.6 ⬍0.05
L07594 TGFbeta type III receptor
mRNA Affymetrix 2.6 ⬍0.01 3.4 ⬍0.01
L41690 TRADD
mRNA Affymetrix 2.1 ⬍0.05 2.8 ns
AB009356 TAK1
mRNA Affymetrix 4.4 ⬍0.01 1.8 ⬍0.05
U71087 MEK5 PRKMK5
mRNA Affymetrix 1.8 ⬍0.01 3 ⬍0.01
AF002715 MAPKKK4 MEKK4
mRNA Affymetrix 1.7 ⬍0.01 1.4 ns
U67156 MAPKKK5
mRNA Affymetrix 1.6 ⬍0.05 1.3 ns
U09759 JNK2
mRNA Affymetrix 2.3 ⬍0.01 2.3 ⬍0.05
M12174 RHOH6
mRNA Affymetrix 10.8 ⬍0.01 3.1 ⬍0.01
L24564 RAD1
mRNA Affymetrix 5.4 ⬍0.05 11.5 ⬍0.01
L25080 RhoA
mRNA Affymetrix 2.9 ⬍0.01 4.1 ⬍0.01
U02570 CDC42GAP
mRNA Affymetrix 2.7 ⬍0.01 4.3 ⬍0.01
M23379 GAP RASA
mRNA Affymetrix 2.5 ⬍0.01 2.1 ⬍0.05
U52522 POR1
mRNA Affymetrix 5.4 ⬍0.01 6.1 ⬍0.01
Atrogin-1
mRNA real time PCR 10.8 ⬍0.01 No NS
change

Refers to immunohistochemistry experiments.

TGF ⫽ transforming growth factor; AQM ⫽ acute quadriplegic myopathy; PCR ⫽ polymerase chain reaction; NS ⫽ not significant.

200 Annals of Neurology Vol 55 No 2 February 2004

Fig 2. Immunoblotting and quantitation of members of transforming growth factor (TGF)–␤/MAPK cascade. (A) Shown is protein
quantitation for several members of TGF-␤ MAPK cascade in acute quadriplegic myopathy (AQM), neurogenic atrophy, and nor-
mal control muscle. Both total and phosphorylated proteins were measured. Total proteins were measured using Bradford method,
and an equal amount of proteins (20␮g) was loaded for each sample. Both total and phosphorylated forms of TGF-␤/MAPK path-
way members were markedly elevated in AQM. (B) Shown is quantitation of immunoblots for some representative members of
TGF-␤/MAPK cascade. Fold changes and p values from quantitated immunoblots are provided for TGF-␤ receptor II, Ask-1,
SMADs, p38-MAPK, phosphorylated p38-MAPK, c-myc, phosphorylated c-myc, and junB. All show highly significant activation in
AQM myogenic atrophy, including the ratio of phosphorylated versus total p38-MAPK.

Specific Key Members of Transforming Growth
Factor–␤/MAPK Cascade Are Expressed in Atrophic
Myofibers with Apoptotic Features in Acute
Quadriplegic Myopathy
We have shown previously the presence of apoptotic fea-
tures (activated caspases 3 and TUNEL-positive fibers) in
atrophic myofibers in a subset of patients in AQM.18 To
extend these previous findings, we localized several mem-
bers of the TGF-␤/MAPK cascade in cryosections of pa-
tient muscle biopsies by using immunohistochemistry.
We found TGF-␤, TGF-␤ receptor, p-ASK1, p38-
MAPK, GADD45 ␤, c-jun, and c-mycall strongly ex-
pressed specifically in atrophic myofibers with apoptotic
features (TUNEL-positive) and rare normotrophic fibers
in AQM muscle (Fig 3, 4). TGF-␤ receptor showed both
membrane and cytoplasmic staining, and p38-MAPK
showed diffuse cytoplasm localization, whereas c-jun
showed characteristic nuclear localization (see Fig 3). On
serial sections, TUNEL-positive picnotic nuclei in atro-
phic fibers strongly immunoreacted for this transcription
factor (see Fig 4). No or very faint staining was observed
in control muscles and in neurogenic muscle fibers.
Quantitation of the percentage of atrophic fibers (myo-
genic and neurogenic biopsies), immunoreactive for
TGF-␤ II receptor, phosphorylated c-myc, GADD45 ␤,
SMAD (1, 5, 8), P38, RAS, and phosphorylated ASK-1

Di Giovanni et al: MAPK Cascade in AQM In Vivo 201

Fig 3. Immunohistochemistry shows localization of transforming growth factor (TGF)–␤ MAPK cascade in atrophic fibers in acute
quadriplegic myopathy (AQM). Shown is specific localization in atrophic fibers in AQM (A, C, E, G, I, M) and neurogenic mus-
cles (B, D, F, H, L, N) of TGF-␤ receptor (A, B), SMADs (C, D), p38 (E, F), Ras (G, H), p-c-Myc (I, L), and gadd45 beta
(M, N). Control muscles immunostained with TGF-␤ receptor (P), SMADs (Q), Ras (R), and p-c-Myc (S), show no signal. In-
tense immunostaining is present only in AQM atrophic muscles for all proteins. Several myofibers are positive for TGF-␤ receptor
also in neurogenic atrophy (B). Original magnification, ⫻250

was also performed. In all cases, the percentage of atrophic
fibers immunopositive for TGF-␤/MAPK members was
significantly higher in myogenic (AQM) compared with
neurogenic atrophy (see Table 2, boldface).
Discussion
Comparative Genomics of Myogenic and Neurogenic
Atrophy
We present protein and mRNA data in AQM myogenic
atrophy, neurogenic atrophy, and normal controls that
shows activation of TGF-␤/MAPK signaling cascade spe-
cifically in muscle in myogenic atrophy in AQM patients.
Neurogenic atrophy and myogenic atrophy shared similar
strong upregulation of the muscle-specific ubiquitin ligase
atrogin-1 (FBx32), consistent with the myofiber atrophy
seen in both conditions. The AQM-specific induction of
the TGF-␤/MAPK pathway was demonstrated at both
the transcriptional and protein level, including protein
phosphorylation consistent with constitutive activation of
this signaling cascade. We also showed colocalization of
TGF-␤/MAPK pathway members with apoptotic atro-
phic myofibers in patient muscle, including TGF-␤ recep-
tor II, ASK1, p38 MAPK, c-myc, and c-jun.
Transforming Growth Factor–␤/MAPK Signaling
Cascade: Mechanisms of Activation and Effectors
Atrophic myofibers in AQM patients showed strong
coordinated upregulation of TGF-␤ MAPK signaling
cascade, Ras family members, and cell cycle inhibitors
(Table 2, Fig 5). Oxidative and osmolar stress, pH im-
balance, and cytokine release all are documented trig-
gers for the TGF-␤/MAPK signaling cascade in diverse

202 Annals of Neurology Vol 55 No 2 February 2004

Fig 4. TUNEL shows colocalization with members of MAPK cascade in atrophic fibers in acute quadriplegic myopathy (AQM).
Shown are TUNEL-positive fibers (B, F) that, respectively, co-localize with p38 (A, arrows) and c-jun (F, arrows show some rep-
resentative fibers) in atrophic fibers in AQM. Shown are also control sections processed for p38 (C), c-jun (G), and TUNEL (D,
H) that do not show TUNEL or p38 positivity and express faint nuclear staining for c-jun. Original magnification, ⫻250.

Di Giovanni et al: MAPK Cascade in AQM In Vivo 203

Fig 5. Induction of transforming growth factor (TGF)–␤/Mapk signaling cascade in myogenic atrophy. Observed modulation of the
TGF-␤ MAPK cascade in acute quadriplegic myopathy (AQM).
cell types.22–28 Both the TGF-␤ and Ras pathways
converge on the MAPK pathway, likely leading to con-
stitutive activation, and the resulting proteolysis and
apoptosis that are histological features of AQM. The
association of AQM with oxidative and osmolar stress
has been seen clinically; however, our data provide mo-
lecular confirmation of this via our observed upregula-
tion of anti–oxidative stress enzymes HO-1, MnSOD,
and GPX (see Fig 2), and these changes were not
shared with neurogenic atrophy.
The upstream triggers, downstream regulation, and
cell-specific regulation of the TGF-␤/MAPK pathway
is complex; yet, it is recognized that this regulation is
critical in determining the biological response of many
cell types, toward either proliferation or atrophy/apo-
ptosis.21–23,25,26 A few members of this cascade appear
to be key in influencing this biological outcome,
namely, p38 MAPK, JNKs, c-myc, and c-jun. These
specific pathway members have been shown to pro-
mote cell atrophy and death rather than mitosis, par-
ticularly in postmitotic tissues, including skeletal mus-
204 Annals of Neurology Vol 55 No 2 February 2004
cle.29 –38 Importantly, the MAPK and Ras pathways
have been shown to be necessary for muscle proteolysis
in Caenorhabditis elegans,39 and TGF-␤–mediated
MAPK activation was shown to antagonize muscle hy-
pertrophy and the insulin-like growth factor–1 path-
way, through activation of proapoptotic insulin-like
growth factor receptor binding proteins.40
Taken together, our data and data from the litera-
ture allow us to propose a model for muscle cell atro-
phy in AQM in which activation of TGF-␤ receptors,
exacerbated by stress response and corticosteroid/Ras
pathways,28,41 begins constitutive intracellular signaling
of the MAPK cascade in specific myofibers leading to
muscle atrophy with apoptotic features (see Fig 3, 4).
Consistent with this model, we show that many of the
pathway members are in the hyperphosphorylated, ac-
tivated state, and that these pathway components colo-
calize with apoptotic cells in patient muscle biopsies.
To briefly describe the roles of some of the proteins
we found specifically activated in AQM, the most im-
portant TGF-␤ signal transducers are SMADs and
TAK-1 that after phosphorylation translocate to the
nucleus and promote transcription of downstream tar-
gets (c-jun, junD, and c-myc), and also phosphorylate
downstream MAPK members (see Fig 5). Transcrip-
tion factors c-jun, junD, and c-myc can both promote
apoptosis and induce TGF-␤ transcription providing a
positive feedback to the entire pathway. They can be
activated at the transcriptional (activated by SMADs)
and protein level (phosphorylated by p38MAPK,
JNKs). Ras and Rho family members are typically trig-
gered by TGF-␤ signaling, whereupon they mediate
actin reorganization, possibly participating in cytoskel-
eton reshaping. SMADs activation can also promote
transcription of p21 and gadd45 ␤, both of which in-
duce cell growth arrest and interact with ASK-1.
ASK-1 and other MAPK pathway members promote
apoptosis and proteolysis.42– 45
In summary, this signaling cascade likely leads to
muscle actin-cytoskeleton reorganization, cell atrophy,
apoptosis, and proteolysis, all distinguishing features of
AQM muscle. Importantly, our data prove that neuro-
genic atrophy and myogenic atrophy (AQM) share the
ubiqutin ligase pathway, but only AQM activates the
TGF-␤/MAPK pathway. Note also that the TGF-␤/
MAPK cascade is not dysregulated in forms of inflam-
matory myopathies associated with necrotic features,
including dermatomyositis, polymyositis, and inclusion
body myopathy.46,47
Transforming Growth Factor–␤/MAPK Cascade:
Possible Therapeutic Targets
The novel pathophysiological cascades defined here for
AQM suggests new targets for potential therapies in-
hibiting muscle atrophy. As shown in Figure 5, expres-
sion of the ERK branch of MAPK cascade is either
inhibited or unchanged in AQM. ERKs family mem-
bers are known for their progrowth and proregenera-
tion potential in diverse conditions and cell types.
Therefore, upregulation of these members might shift
the cascade toward growth as opposed to atrophy. An-
other therapeutic target resides in the inhibition of key
players of the proatrophic pathway, including SMADs,
p38 MAPK, JNKs, ASK-1, and cell cycle inhibitor
p21. JNK inhibitors will soon be in clinical trials in
cancer and rheumatoid arthritis.22 Importantly, we
would anticipate that modulation of this signaling cas-
cade might also show efficacy in diverse conditions
showing muscle atrophy, including cancer cachexia and
acquired immune deficiency syndrome.
This work was supported by grants from the National Institutes of
Health (National Heart Lung and Blood Institute U01
HL66614-01 “Programs in Genomic Applications” HOPGENE;
NINDS N01-NS-1-2339, NINDS 3R01 NS29525-09, E.P.H.) and
Telethon Italy (GGP02253, S.S.).
References
1. Hirano M, Ott BR, Raps EC, et al. Acute quadriplegic
myopathy: a complication of treatment with steroids, nondepo-
larizing blocking agents, or both. Neurology 1992;42:
2082–2087.
2. Larsson L, Li X, Edstrom L, et al. Acute quadriplegia and loss
of muscle myosin in patients treated with nondepolarizing neu-
romuscular blocking agents and corticosteroids: mechanisms at
the cellular and molecular levels. Crit Care Med 2000;28:
34 – 45.
3. Argov Z. Drug induced myopathy. Curr Opin Neurol 2000;
13:541–545.
4. Rich MM, Bird SJ, Raps EC, et al. Direct muscle stimulation
in acute quadriplegic myopathy. Muscle Nerve 1997;20:
665– 673.
5. Sander HW, Golden M, Danon MJ. Quadriplegic areflexic
ICU illness: selective thick filament loss and normal nerve his-
tology. Muscle Nerve 2002;26:499 –505.
6. Lacomis D. Critical illness myopathy. Curr Rheumatol Rep
2002;4:403– 408.
7. Mitch WE, Goldberg AL. Mechanisms of muscle wasting. The
role of the ubiquitin proteosome pathway. N Engl J Med 1996;
335:1897–1905.
8. Helliwell TR, Wilkinson A, Griffiths RD, et al. Muscle fiber
atrophy in critically ill patients is associated with loss of myosin
filaments and the presence of lysosomal enzymes and ubiquitin.
Neuropathol Appl Neurobiol 1998;24:507–515
9. Showalter CJ, Engel AG. Acute quadriplegic myopathy: analysis
of myosin isoforms and evidence for calpain-mediated proteol-
ysis. Muscle Nerve 1997;20:316 –322.
10. Matsumoto N, Nakamura T, Yasui Y, Torii J. Analysis of mus-
cle proteins in acute quadriplegic myopathy. Muscle Nerve
2000;23:1270 –1276.
11. Bodine SC, Latres E, Baumhueter S, et al. Identification of
ubiquitin ligases required for skeletal muscle atrophy. Science
2001;294:1704 –1708.
12. Glass DJ. Signalling pathways that mediate skeletal muscle hy-
pertrophy and atrophy. Nat Cell Biol 2003;5:87–90
13. Rich MM, Pinter MJ. Sodium channel inactivation in an ani-
mal model of acute quadriplegic myopathy. Ann Neurol 2001;
50:26 –33.
14. Rich MM, Pinter MJ, Kraner SD, Barchi RL. Loss of electrical
excitability in an animal model of acute quadriplegic myopathy.
Ann Neurol 1998;43:171–175.
15. Rich MM, Kraner SD, Barchi RL. Altered gene expression in
steroid-treated denervated muscle. Neurobiol Dis 1999;6:
515–522.
16. Jagoe RT, Lecker SH, Gomes M, Goldberg AL. Patterns of
gene expression in atrophying skeletal muscles: response to food
deprivation. FASEB J 2002;16:1697–1712.
17. Childs TE, Spangenburg EE, Vyas DR, Booth FW. Temporal
alterations in protein signaling cascades during recovery from
muscle atrophy. Am J Physiol Cell Physiol 2003;285:
C391–C398.
18. Di Giovanni S, Mirabella M, D’Amico A, et al. Apoptotic fea-
tures accompany acute quadriplegic myopathy. Neurology
2000;55:854 – 858.
19. Di Giovanni S, Knoblach SM, Brandoli C, et al. Gene profiling
in spinal cord injury shows role of cell cycle in neuronal death.
Ann Neurol 2003;53:454 – 468.
20. Seo J, Bakay M, Zhao P, et al. Building a coherent data pipe-
line in micorarray data analysis: visual optimization of signal/
noise ratios. IEEE ICME 2003;III:461– 465.
21. Church DLM, Lash AE, Leipe DD, et al. Database resources of
the National Center for Biotechnology Information. Nucleic
Acids Res 2001;29:11–16.
Di Giovanni et al: MAPK Cascade in AQM In Vivo 205
22. Johnson GL, Lapadat R. Mitogen-activated protein kinase path-
ways mediated by ERK, JNK, and p38 protein kinases. Science
2002;298:1911–1912.
23. Massague J, Chen YG. Controlling TGF-beta signaling. Genes
Dev 2000;15;14:627– 644.
24. Thannickal VJ, Day RM, Klinz SG, et al. Ras-dependent and
-independent regulation of reactive oxygen species by mitogenic
growth factors and TGF-beta1. FASEB J 2000;14:1741–1748.
25. Chang L, Karin M. Mammalian MAP kinase signalling cas-
cades. Nature 2001;410:37– 40.
26. Hazzalin CA, Mahadevan LC. MAPK-regulated transcription: a
continuously variable gene switch? Nat Rev Mol Cell Biol
2002;3:30 – 40.
27. Obata T, Brown GE, Yaffe MB. MAP kinase pathways acti-
vated by stress: the p38 MAPK pathway. Crit Care Med 2000;
28:N67–N77.
28. Tu Y, Wu C. Cloning, expression and characterization of a
novel human Ras-related protein that is regulated by glucocor-
ticoid hormone. Biochim Biophys Acta 1999;1489:452– 456.
29. Noguchi K, Yamana H, Kitanaka C, et al. Differential role of
the JNK and p38 MAPK pathway in c-Myc- and s-Myc-
mediated apoptosis. Biochem Biophys Res Commun 2000;267:
221–227.
30. Kang HJ, Soh Y, Kim MS, et al. Roles of JNK-1 and p38 in
selective induction of apoptosis by capsaicin in ras-transformed
human breast epithelial cells. Int J Cancer 2003;103:475– 482.
31. Deschesnes RG, Huot J, Valerie K, Landry J. Involvement of
p38 in apoptosis-associated membrane blebbing and nuclear
condensation. Mol Biol Cell 2001;12:1569 –1582.
32. Ning W, Song R, Li C, et al. TGF-beta1 stimulates HO-1 via
the p38 mitogen-activated protein kinase in A549 pulmonary
epithelial cells. Am J Physiol 2002;283:L1094 –L1102.
33. Hyman KM, Seghezzi G, Pintucci G, et al. Transforming
growth factor-beta1 induces apoptosis in vascular endothelial
cells by activation of mitogen-activated protein kinase. Surgery
2002;132:173–179.
34. Singleton JR, Baker BL, Thorburn A. Dexamethasone inhibits
insulin-like growth factor signaling and potentiates myoblast
apoptosis. Endocrinology 2000;141:2945–2950.
206 Annals of Neurology Vol 55 No 2 February 2004
35. Shaulian E, Karin M. AP-1 as a regulator of cell life and death.
Nat Cell Biol 2002;4:E131–E136.
36. Pelaia G, Cuda G, Vatrella A, et al. Effects of TGF-␤ and
budesonide on MAPK activation and apoptosis in airway epi-
thelial cells. Am J Respir Cell Mol Biol 2003;29:12–18.
37. Yoshida K, Kuwano K, Hagimoto N, et al. MAP kinase acti-
vation and apoptosis in lung tissues from patients with idio-
pathic pulmonary fibrosis. J Pathol 2002;198:388 –396.
38. Agusti AG, Sauleda J, Miralles C, et al. Skeletal muscle apopto-
sis and weight loss in chronic obstructive pulmonary disease.
Am J Respir Crit Care Med 2002;166:485– 489.
39. Szewczyk NJ, Peterson BK, Jacobson LA. Activation of Ras and
the mitogen-activated protein kinase pathway promotes protein
degradation in muscle cells of Caenorhabditis elegans. Mol Cell
Biol 2002;22:4181– 4188.
40. Rajah R, Valentinis B, Cohen P. Insulin-like growth factor
(IGF)-binding protein-3 induces apoptosis and mediates the ef-
fects of transforming growth factor-beta1 on programmed cell
death through a p53- and IGF-independent mechanism. J Biol
Chem 1997;272:12181–12188.
41. Mulder KM. Role of Ras and Mapks in TGFbeta signaling.
Cytokine Growth Factor Rev 2000;11:23–35.
42. Yue J, Frey RS, Mulder KM. Cross-talk between the Smad1
and Ras/MEK signaling pathways for TGFbeta. Oncogene
1999;18:2033–2037.
43. Zhang Y, Feng XH, Derynck R. Smad3 and Smad4 cooperate
with c-Jun/c-Fos to mediate TGF-beta-induced transcription.
Nature 1998;394:909 –913.
44. Shibuya H, Yamaguchi K, Shirakabe K, et al. TAB1: an acti-
vator of the TAK1 MAPKKK in TGF-beta signal transduction.
Science 1996;272:1179 –1182.
45. Derynck R, Zhang Y, Feng XH. Smads: transcriptional activa-
tors of TGF-beta responses. Cell 1998;95:737–740.
46. Tezak Z, Hoffman EP, Lutz JL, et al. Gene expression profiling
in DQA1*0501⫹ children with untreated dermatomyositis:
a novel model of pathogenesis. J Immunol 2002;168:
4154 – 4163.
47. Greenberg SA, Sanoudou D, Haslett JN, et al. Molecular pro-
files of inflammatory myopathies. Neurology 2002;59:
1170 –1182.