Extracellular Superoxide Dismutase Ameliorates Skeletal Muscle

Transcription

Extracellular Superoxide Dismutase Ameliorates Skeletal Muscle Abnormalities,
Cachexia and Exercise Intolerance in Mice with Congestive Heart Failure
Okutsu et al: EcSOD-mediated Protection Against Cardiac Cachexia
Mitsuharu Okutsu, PhD1,4; Jarrod A. Call, PhD1,4; Vitor A. Lira, PhD1,4;
Mei Zhang, B.S.Med.1,4; Jean A. Donet, MD1,4; Brent A. French, PhD5;
Kyle S. Martin, BS5; Shayn M. Peirce-Cottler, PhD5; Christopher M. Rembold, MD1;
Brian H. Annex, MD1; Zhen Yan, PhD1,2,3,4
Downloaded from http://circheartfailure.ahajournals.org/ by guest on November 18, 2016
Departments of Medicine1, Pharmacology2, and Molecular Physiology and Biological
Physics3; Center for Skeletal Muscle Research4 at Robert M. Berne Cardiovascular Research
g
Charlottesville, VA
Center, Department of Biomedical Engineering5, University of Virginia,
Correspondence to
Zhen Yan, PhD
R4-6041A
A, P
.O
O. B
ox 801394
801
139
94
409 Lane Road, M
MR4-6041A,
P.O.
Box
Charlottesville, VA
V 22908
Telephone: 434-982-4477
982
82-4
447
477
7
39
3
9
Fax: 434-982-3139
Email: [email protected]
DOI: 10.1161/CIRCHEARTFAILURE.113.000841
Journal Subject Code: [110] Congestive heart failure
1
Abstract
Background—Congestive heart failure (CHF) is a leading cause of morbidity and mortality,
and oxidative stress has been implicated in the pathogenesis of cachexia (muscle wasting)
and the hallmark symptom, exercise intolerance. We have previously shown that a nitric
oxide (NO)-dependent antioxidant defense renders oxidative skeletal muscle resistant to
catabolic wasting. Here, we aimed to identify and determine the functional role of the
NO-inducible antioxidant enzyme(s) in protection against cardiac cachexia and exercise
intolerance in CHF.
Methods and Results—We demonstrated that systemic administration of endogenous nitric
oxide donor S-Nitrosoglutathione in mice blocked the reduction of extracellular superoxide
dismutase (EcSOD) protein expression, the induction of MAFbx/Atrogin-1 mRNA expression
wed
d tthat
hatt en
ha
endo
doge
do
geno
ge
no EcSOD,
and muscle atrophy induced by glucocorticoid. We further showed
endogenous
rily
y by type
typ
y e II
IIId/x
d/x and IIa myofibers
myofiber
f er
ers and enriched
ed at endothelial ccells, is
expressed primarily
cise training
ci
ng. Mu
ng
Musc
scle
sc
le-spe
le
peccific ooverexpression
pe
ver
erex
ex
xpr
p es
essi
siion
n ooff Ec
cSO
SOD
D by ssomatic
omaa gene
om
induced by exercise
training.
Muscle-specific
EcSOD
g nes
gen
esis
is [muscle
[mu
usscl
c e creatine
c eaati
cr
tine
ne kkinase
inas
in
asee (M
as
MCK)CK
K))-Ec
EcSO
Ec
SOD]
SO
D] iin
n mi
mice
ce ssignificantly
ig
gnifi
niifi
ficc
transfer or transgenesis
(MCK)-EcSOD]
le atrophy. Importantly,
Importa
taanttly
ly, when
when
en crossbred
cro
osssbr
bred
ed iinto
noam
nt
mouse
ouse genetic mo
o of CHF
attenuated muscle
model
y ch
chain
chai
ain
ai
n (M
(MHC)
(MHC
HC)) ca
HC
calsequestrin]
cals
lseq
ls
eque
uest
stri
rin]
ri
n] MC
MCK
K EcSOD
EcSO
Ec
SOD
SO
D ttransgenic
rans
ra
nsge
geni
nicc mice
ni
mice h
had
ad significant
[D-myosin heavy
(MHC)-calsequestrin]
MCK-EcSOD
attenuation of cachexia with preserved whole body muscle strength and endurance capacity
in the absence of reduced heart failure. Enhanced EcSOD expression significantly
ameliorated CHF-induced oxidative stress, MAFbx/Atrogin-1 mRNA expression, loss of
mitochondria and vascular rarefaction in skeletal muscle.
Conclusions—EcSOD plays an important antioxidant defense function in skeletal muscle
against cardiac cachexia and exercise intolerance in CHF.
Key Words: oxidative stress, catabolic muscle wasting, exercise intolerance, mitochondrial
dysfunction, vascular rarefaction
2
Congestive heart failure (CHF) is a leading cause of morbidity and mortality in industrialized
countries. Cachexia, characterized by severe loss of muscle mass and exercise intolerance,
manifested as fatigue and dyspnea during minimal physical activities, are important
predictors of mortality in CHF. The progressive decline of exercise capacity results from
multiple skeletal muscle abnormalities, including atrophy, impaired excitation-contraction
coupling, mitochondrial dysfunction and vascular rarefaction1-3. Oxidative stress due to
educ
ed
uced
uc
ed aantioxidant
ntio
nt
ioxi
io
xida
xi
da defenses
excessive production of reactive oxygen species (ROS) and/or reduced
has emerged as a central
cen
en l player
entral
playe
y r in cardiac cachexia,
cachexiia, and is mechanistically
m ch
me
hanistically
y linked
link d to
intracellular signaling,
contractile
n
naling,
contractil
ile protein
p ottein function
pr
f nction and
fu
d ddegradation
egrad
dation
i 3-6. Although
Alth
houghh animal
studies have shown
treatment,
clinical
wn eeffectiveness
ffec
ff
ecti
tive
ti
vene
ness
ss o
off an
antioxidant
anti
tiox
ti
oxid
idan
id
antt ssupplements
uppl
up
plem
pl
emen
ents
ts iin
n tr
treatment
trea
eatm
tmen
entt cclinica
lini
li
nica
ni
ca trials have
limited successes7, 8, possibly due to lack of specificity to the target cells and subcellular
environment leading to either poor efficacy or impairment of physiological signaling. It is
critical to identify a viable approach to augment the endogenous antioxidant defense system
to cope with this complicated clinical syndrome.
Our body has very sophisticated, functional antioxidant defense systems for maintaining a
delicate redox homeostasis. The first line of antioxidant defense is comprised of superoxide
dismutases (SODs), which scavenge superoxide (O2-) to produce the less reactive hydrogen
3
peroxide (H2O2). Skeletal muscle expresses all three isoforms of SOD: the cytosolic
copper/zinc-containing SOD (CuZnSOD or SOD1), the mitochondrial manganese-containing
SOD (MnSOD or SOD2) and the extracellular SOD (EcSOD or SOD3). EcSOD is a
glycoprotein that is secreted and has high affinity for sulfated polysaccharides, such as
heparin and heparin sulfate, through its haprin-binding domain on the C-terminus. EcSOD
may also be internalized by the cells that it binds through endocytosis9. Therefore, EcSOD
rodu
ro
duci
du
cin
ci
ng ccells
ng
e and target
may exert antioxidant function intracellularly and extracellularlyy in pproducing
cells.
Expression of this
antioxidant
pathological
i ant
is
tioxida
d nt enzyme
enzym
y e appears to be
be prone
p one to
pr
t suppression
suppressiion bby
y path
h
conditions, potentially
diseases.
example,
reduced
EcSOD
ntially
ntia
nt
iall
ia
lly
ll
y un
underlying
unde
derl
de
rlyi
rl
ying
yi
ng tthe
he eetiology
tiol
ti
olog
ol
ogy
y of tthe
he ddiseases
isea
is
ease
sess F
For
or eexample
xamp
xa
mple
le re
redu
du
expression has been reported in many chronic diseases, including CHF10, 11. In terms of
functionality, genetic deletion of the EcSOD gene exacerbates12-14 whereas transgenic
overexpression attenuates the pathologies in mouse models of chronic diseases14, 15. These
findings have clearly established the functional importance of EcSOD in protection against
diseases caused by oxidative stress; however, its importance of skeletal muscle mass and
function is unknown.
We have recently identified a nitric oxide (NO)-dependent antioxidant defense mechanism
4
that underlies the resistance of oxidative muscles to catabolic wasting3, 16, 17. To ascertain the
protective function of NO and to identify the antioxidant gene(s) that plays a major role in
this protection, we first administer systemically an endogenous NO donor,
S-nitrosoglutathione (GSNO), in a mouse model of catabolic muscle wasting induced by
glucocorticoid, a hormone that is elevated in CHF and involved in muscle catabolism18. Our
findings underscore the importance of NO-mediated induction of EcSOD expression in
protection against catabolic muscle wasting. We then employed two
two independent
inde
in
depe
de
pend
pe
nden
nd
e t genetic
en
approaches and showed
muscle ameliorates
sho
sho
how
wed that
thatt enhanced EcSOD expression
th
ex
xpression in
in skeletal
skel
eletal
l
amee
cardiac cachexiaa andd exercise
with
atrophy
gene
exerciise in
iintolerance
tolerance along
allong wi
ith
h bblunted
lunted
t d oxidative
oxid
idatiive stress, aatr
r
expression, mitochondrial
model
chondrial
chon
ch
ondr
dria
dr
iall loss
ia
loss aand
nd m
microvascular
icro
ic
rova
vasc
scul
ular
ul
ar aabnormalities
bnor
bn
orma
mali
liti
li
ties
ti
es iin
n a mo
mouse
mous
usee mo
mode
de of CHF.
We have, therefore, provided convincing evidence to support the effectiveness of enhancing
EcSOD expression and antioxidant activity in protecting skeletal muscle structural and
functional integrity under the condition of CHF.
Methods
Animals
Wild type mice (male, 8 weeks old, C57BL/6J) were obtained commercially (Jackson
5
Laboratory, Bar Harbor) and housed in temperature-controlled (22 °C) quarters with a
12:12-h light-dark cycle and free access to water and normal chow (Harlan). A
muscle-specific EcSOD transgenic mouse line was generated at the University of Virginia
Gene Targeting and Transgenic Facility. Briefly, RT-PCR was performed using mouse
skeletal muscle RNA with forward primer
5’-TGGATCCACCATGTTGGCCTTCTTGTTC-3’ and reverse primer
EcS
cSOD
OD cDNA
cDN
DNA
A (NM_011435).
(N
5’-AGGTACCAGTGGTCTTGCACTCGCTCTC-3’ to obtain EcSOD
ragmen
ra
ent (7
en
(762 bp)
p of the codingg region
reg
gion was
wa then
th
hen fused to a FLAG
FLA
A sequence
A Bam H1-Kpn I ffragment
o codon
op
codon
d in
in pTarge
T g t (P
Promega
g , Madison)
M dison)
Ma
dii n)) to create pEcSOD.
pE
EcSOD
OD. A 1.3-kb
1
followed by a stop
pTarget
(Promega,
Hind
ment
me
nt o
off FL
FLAG
AG ttagged
agge
ag
ged
d EcSOD
EcSO
Ec
SOD
SO
D was
was then
the
hen
n inserted
inse
in
sert
rted
ed iinto
nto
nt
o pMCKhGHpolyA
pMCKhGHp
pMCK
pM
CKhG
CK
hGHp
hG
Hp
III-Eco RI fragment
FLAG-tagged
between a 4.8-kb mouse muscle creatine kinase (MCK) promoter and the human growth
hormone polyadenylation site19. A linear Not I-Xho I fragment (6.7 kb) was isolated and used
for pro-nuclear injection to generate transgenic mice (C57BL/6.SJL F1 background). We
backcrossed the transgene onto pure C57BL/6J background (7 generations). To determine if
enhanced EcSOD expression prevents CHF-induced muscle atrophy, MCK-EcSOD mice
were crossbred with D-myosin heavy chain (Į-MHC)-calsequestrin mice (CSQ; DBA
background)20 to generate double transgenic MCK-EcSOD:Į-MHC-CSQ mice (DTG). The
6
wild type (WT) littermates or littermates with only MCK-EcSOD allele (TG) were used as
controls. Therefore, the mice used were of the same genetic background. To prevent negative
impact of heart failure on embryonic development, the Į-MHC-CSQ breeder mice were
treated with E-blocker metoprolol in drinking water before and during breeding6, and the
E-blocker was removed at birth such that none of the mice used in the studies received
E-blocker after birth. Voluntary wheel running protocol is as described21. All animal
Car
aree and
and Use
Use Committee.
Com
Co
m
protocols were approved by the University of Virginia Animal Care
R of
o tail
taiil DNA
DN
NA was performed
p rformed usin
pe
ing MCK
K transgenic
tra
r nsge
g nic forward pr
r
Genotyping--PCR
using
primer,
5’G CAG
CA
AG GCT
GCT
GC
T GTA
GTA
GT
A AC-3’;
AC-3’; EcSOD
AC
E S
Ec
SO
OD intron
inttron forward
forward
d primer,
prim
i er, 5’-GGG
5’-G
GGG AGG
GAC TGA GGG
TGG GAA GAG
primer,
G TTA
TTA GG-3’;
GG 3’;
3’;; aand
nd Ec
EcSOD
EcSO
SOD
SO
D ccoding
odin
od
ing
in
g re
reverse
reve
vers
rsee pr
primer
prim
imer
im
er 5’
55’-CAC
C
CAC
AC CTC
CTC CAT CGG
GTT GTA GT-3’. Exogenous EcSOD expression in adult skeletal muscle was confirmed by
immunoblot as described6.
Dexamethasone and GSNO injections
Mice received daily injection of dexamethasone (25 mg/kg, i.p.) (Sigma, St. Louis) or normal
saline with/without GSNO (2 mg/kg) twice a day for 7 days22. At 12 and 24 hrs after the last
injection of GSNO and dexamethasone, respectively, soleus, plantaris, tibialis anterior,
gastrocnemius, extensor digitorum longus (EDL) and white vastus lateralis muscles were
7
harvested and processed for further analyses.
Semi-quantitative RT-PCR and real-time PCR
These analyses were performed for MAFbx/Atrogin-1, MuRF1 and Gapdh mRNAs in
plantaris muscles as described23. Real-time PCR was performed using primers for
MAFbox/Atrogin 1 and 18S rRNA from Applied Biosystems (Foster City, CA).
Immunoblot analysis—Immunoblot analysis was performed as described6 with the following
SOD
SO
D (R
(R&D
&D,, Mi
&D
Min
n
primary antibodies: CuZnSOD (Abcam), MnSOD (Abcam), EcSOD
(R&D,
Minneapolis),
peroxisome proliferator-activated
receptor
1-D
ife
ife
ferrator-ac
a tiivated recept
p or J coactivator
coacttiv
vator 1D (PGC(PG
GC- 1D))
i e)), cytochrome
ipor
cytoch
hrome ooxidase
xida
id se IIV
V (C
COX
X IIV)
V) (In
I vitrogen)
i
), cytochrome
cytochr
h om
m C (Cyt C)
(Chemicon/Millipore),
(COX
(Invitrogen),
(Cell Signaling, D
Danvers),
Inc.,
Danvers)
anve
an
vers
rs)) m
malondialdehyde
alon
al
ondi
dial
di
alde
al
dehy
de
hyde
hy
de (MDA)
(MD
MDA)
A) (Academy
(Ac
Acad
adem
ad
emy
y Biomedical
Biom
Bi
omed
edic
ed
ical
ic
al Company,
C
Com
om
Houston) and 4-hydroxynonenal (4-HNE) (ab48506, Abcam). Hybridoma for antibodies
against MHC I (BA-F8), MHC IIa (SC-71) and MHC IIb (BF-F3) were purchased from
German Collection of Microorganisms and Cell Cultures. All the bands for analysis have
been validated previously3, 6, 24. OxyBlot Protein Oxidative Detection Kit (Millipore) was
used for immunblot detection of carbonylated proteins. Immunoblots were analyzed by
Odyssey Infrared Imaging System (LI-COR Biosciences, NE) and quantified by Scion Image
software.
8
Immunohistochemistry
Fresh frozen muscle sections (5 Pm) were stained with rabbit anti-EcSOD (Sigma), and
images acquisition and analysis of capillary density were performed as described3.
Somatic gene transfer and measurement of myofiber size
We performed electric pulse-mediated gene transfer by injecting DNA (15 Pg of pGFP3 + 50
Pg of pEcSOD or pCI-neo) in tibialis anterior muscles as described24. Confocal microscopy
iti
tive
ve ffibers
iber
ib
erss fiber
er
fibe
fi
berr size (>100
be
(Fluoview 1000; Olympus) was performed to measure GFP-positive
m
ous
use) using
usi
s ng
si
g Image
g J6.
fibers for each mo
mouse)
ing test
testt
Treadmill running
Blood
Bloo
ood
d lactate
lact
la
ctat
atee level
leve
le
vell was
was measured
meas
me
asur
ured
ed before,
bbefore
efor
ef
oree at
a 40 min
formed
form
fo
rmed
ed as
as de
described
desc
scri
ribe
ri
bedd3. Bl
be
The test was performed
and immediately after the test by Lactate Scout (SensLab, Leipzig)
Whole body tension test (WBT)
WBT was performed according to an established method25 in a custom-designed device. Ten
trials were performed with 10-sec intervals for each mouse, and the mean value of the five
peak readings was used for statistical analysis.
Magnetic resonance imaging (MRI)
MRI was used to assess cardiac function as described26.
9
Electrocardiography
Electrocardiography was performed on conscious mice using the ECGenie (Mouse Specifics,
Boston). Mice were placed on the platform for 10 min to acclimate. Data with continuous
recordings of over 40 heart beats were used for analyses by using eMOUSE, a physiologic
waveform analysis platform (Mouse Specifics, Boston).
Whole mount immunofluorescence
d ffor
or smooth
smo
moot
oth
ot
h mu
Flexor digitorum brevis (FDB) muscles were immunolabeled
muscle D-actin
27
.
a) and
a)
and isolectin
iso
solect
so
c in (IB4-Alexa647
(IB4-Alexa647 orr Alexa488,
Alexa488,
8 Invitrogen)
8,
Invitroge
g n) as descr
r
(IA4-Cy3, Sigma)
described
Statistics
rresented
esen
es
ente
ted
d as mean
mea
ean
n ± SE w
with
ith
it
h each
each of
of the
the in
individual
indi
divi
di
vidu
vi
dual
du
al ddata
ataa po
at
points
poin
ints
in
ts p
pl
l
The results are presented
plotted.
Comparisons between two groups were analyzed by the Wilcoxon rank sum test. A two-way
ANOVA was used for comparisons across groups, and when appropriate a post-hoc test was
used for detection of differences. When the data failed to meet the normality assumption,
logarithmic transformation (base 10) was used prior to ANOVA. When the data failed to
meet the equal variance assumption, a non-parametric test (Kruskal-Wallis) was used, and
when appropriate, the Dunn’s post-hoc analysis to detect difference across specific means. A
p-value <0.05 was required to report significance.
10
Results
Systemic administration of GSNO induces EcSOD expression and prevents
dexamethasone-induced muscle wasting
We have recently shown that an NO-dependent antioxidant defense system may underlie the
resistance of oxidative muscles to catabolic muscle wasting3, 6, 16. To determine whether NO
donor is sufficient to provide protection in vivo and to identify the antioxidant gene(s) that is
functionally involved, we performed daily injections of dexamethasone
thas
th
ason
as
onee (25
on
(25 mg/kg,
mg/k
mg
/k i.p.)
with/without endogenous
for
dogenous
dog
ogeenouss NO
og
O donor GSNO (2
( mg/kg,
mg/
g/kg, i.p.
p twice
twi
w ce a day)
day
y) in mice fo
o 7 days.
Dexamethasone inje
muscle
injection
cttion le
lled
d to a significant
sig
gniificantt increase in
in lipid
lipid peroxidation
peroxid
id
dation
t
iin
n sk
sskeletal
ke
as assessed by 4-HNE
S1).
injection
HNE
H
NE immunoblot
imm
mmun
unob
oblo
ob
lott (S
lo
(Supplemental
(Sup
uppl
plem
pl
emen
enta
tall Fi
Figu
Figure
gure
re S
S1)
1) Dexamethasone
Dexa
De
xame
meth
thas
th
ason
onee in
caused a significant reduction of EcSOD protein expression (-29.7%, p<0.001), but not for
CuZnSOD, MnSOD or catalase (Figure 1A). GSNO alone induced a moderate increase of
EcSOD (p<0.05) and completely prevented the reduction of EcSOD protein expression
induced by dexamethasone (Figure 1A). Dexamethasone induced a significant increase of
ubiquitin E3 ligase MAFbx/Atrogin-1 mRNA (1.9-fold, p<0.05), which was completely
blocked by GSNO (Figure 1B). No significant differences were detected for MuRF1 mRNA.
Importantly, dexamethasone injection induced significant atrophy in fast-twitch, glycolytic
11
EDL (-14.5%, p<0.001), tibialis anterior (-4.6%, p<0.05), gastrocnemius (-10.8%, p<0.001)
and plantaris muscles (-9.5%, p<0.001), but not in slow-twitch, oxidative soleus muscle.
Therefore, endogenous NO donor administration significantly attenuated muscle atrophy in
all the fast-twitch, glycolytic muscles (Figure 1C).
EcSOD is highly expressed in oxidative muscle, induced by endurance exercise training
and accumulates at capillary endothelial cells
empl
em
ploy
pl
oyed
oy
ed
To determine which type(s) of muscle fibers expresses EcSOD, w
wee employed
ence.
en
nce
ce. We found that EcSOD was expressed
expressed
d at
a a moderate level in
n most of the
immunofluorescence.
t glycolytic
tch,
gllycoly
l tic
i plantaris
planta
l
riis muscle
musclle (Figure
(Fi
Figure 2A).
2A)). In
I contrast,
contr
t ast, EcSOD
EcS
SOD
D was highly
fibers in fast-twitch,
sstt of tthe
he fibers
fib
iber
erss in sslow
low
lo
w tw
twitch
twit
itch
it
ch o
oxidative
xida
xi
dati
da
tive
ti
ve ssoleus
oleu
ol
euss muscle
musc
mu
scle
le with
wit
ith
h clear
clea
cl
earr evidence of
expressed in most
slow-twitch,
enrichment at capillary endothelial cells (Figure 2A, indicated by arrows). The specificity of
the immunofluorescence was confirmed both by negative staining without primary antibody
(not shown) and by the positive staining of aortic smooth muscle and bronchioles of the lung
(Figure 2A). Fiber type analysis revealed that EcSOD was primarily expressed by type IIa
and IId/x myofibers (Figure 2B) while neither type IIb nor type I fibers expressed detectable
levels of EcSOD. Furthermore, immunoblot analysis provided clear evidence that EcSOD is
expressed more abundantly in slow-twitch, oxidative soleus muscle than fast-twitch,
12
glycolytic white vastus lateralis muscle (p<0.05; Figure 2C). Finally, voluntary running (4
wks) induced EcSOD protein expression in plantaris muscle (+79.9%, p<0.01; Figure 2D).
Therefore, EcSOD expression in skeletal muscle is enhanced by contractile activities, which
may underlie the protective effects of exercise training and oxidative phenotype3, 6
Somatic gene transfer-mediated EcSOD overexpression in adult skeletal muscle protects
myofibers from atrophy
pre
ress
ssio
ss
ion
io
n in atrophying
atr
trop
oph
op
h
Our finding that dexamethasone injection decreases EcSOD expression
muscle
duced
du
uce
ced
d EcSOD
EccSO
SOD
D expression
expr
p ession pplays
lays
y a rrole
ole in cat
catabolic
tab
a ollic wasting. Block
suggests that reduced
Blocking the
SOD
D expression
expressiion along
allong withh the
the prevention
preventtion off muscle atrophy
atroph
t hy by
by G
reduction of EcSOD
GSNO
administration further
notion.
expression
is
urther
urth
ur
ther
th
er ssupports
uppo
up
port
rtss th
this
is n
notion
otio
ot
ion
io
n T
To
o de
determine
dete
term
rmin
inee if eenhanced
in
nhan
nh
ance
ced
d EcSOD
EcSO
Ec
SOD
SO
D ex
exp
p
sufficient to protect myofibers from catabolic wasting, we performed electric pulse-mediated
gene transfer in fast-twitch, glycolytic tibialis anterior muscle. High levels of exogenous
EcSOD expression were confirmed when compared to the contralateral control muscle
(pCI-neo) (Figure 3A). Systemic administration of dexamethasone resulted in a leftward shift
(reduction) of the fiber size histogram with a significant reduction of the mean
cross-sectional area (CSA) in pCI-neo transfected myofibers (-14.5%, p<0.01), whereas
myofibers transfected with EcSOD were protected from atrophy (Figure 3B and 3C).
13
Muscle-specific EcSOD expression in transgenic mice significantly attenuates muscle
wasting
To further ascertain the protective role of EcSOD in catabolic muscle wasting, we generated
a transgenic mouse line with muscle-specific overexpression of EcSOD under the control of
the MCK promoter (Figure 4A, top left panel). We detected significantly greater EcSOD
protein expression in skeletal muscles in MCK-EcSOD mice than in the wild-type littermates
prof
pr
ofou
of
ound
ou
nd in
in plantaris
plan
pl
an
(Figure 4A, top right panel). The increases appeared to be more profound
and
e al
eral
alis
is mus
usclles than in soleus muscl
us
cle. Immun
un
nof
o luorescence of EcS
S
white vastus lateralis
muscles
muscle.
Immunofluorescence
EcSOD
along
g analysis
anallysiis showed
sh
howed
d thatt exogenous EcSOD
EcS
SOD
D was highly
highly expressed
d iin type IIb
with fiber typing
fibers (Figure 4A,
panel).
expression
are
A bottom
bot
otto
tom
m pa
panel)
pane
nel)
l) These
Thes
Th
esee fiber
fibe
fi
berr type-specific
be
type
ty
pe sspecific
peci
pe
cifi
ci
ficc ex
fi
expr
pres
essi
sion
si
on patterns
patt
tter
erns
ns aar
r consistent
with the notion that the MCK promoter is more active in fast-twitch, glycolytic fibers.
EcSOD also appeared to be enriched at endothelial cells (Figure 4A, bottom panel by arrows).
Consistent with findings presented in Figure 1, dexamethasone injection induced muscle
mass loss in wild-type mice by -27.5% for EDL (p<0.001), -19.5% for tibialis anterior
(p<0.01), 19.0% for gastrocnemius (p<0.001) and 14.7% for plantaris (p<0.001) muscles, but
not in soleus muscle (Figure 4B). More importantly, dexamethasone injection did not cause
significant reduction in muscle mass in any of these muscles in MCK-EcSOD mice,
14
suggesting that enhanced EcSOD expression is sufficient to protect fast-twitch, glycolytic
fibers from catabolic wasting (Figure 4B). Protein expression of antioxidant enzymes
(CuZnSOD, MnSOD and Catalase), mitochondrial (COX IV and Cyt C) and contractile
proteins (MHC I, IIa and IIb) were not significantly different between MCK-EcSOD mice
and the wild-type littermates in these muscles (Figure 4C), suggesting that the protection in
MCK-EcSOD mice was most likely due to enhanced antioxidant function, not adaptive
responses induced by the transgene overexpression.
EcS
Ec
SOD ttransgenic
ransggenic mice are resi
istant to ccachexia
a hex
ac
xia and exercise
exerci e intolerance
Muscle-specific EcSOD
resistant
F
induced by CHF
nd tthe
he ccurrent
urre
ur
rent
nt ffindings
indi
in
ding
di
ngss (Figure
(Fig
(F
igur
ig
uree 1,
1 3 aand
nd 4)
4) su
suggest
sugg
gges
estt th
that
at enhanced
udies
u
dies
di
es6,, 166 aand
Our previous studies
EcSOD expression is sufficient to preserve skeletal muscle mass and function under
conditions of cachexia. We expected that MCK-EcSOD mice would be resistant to CHF in
developing cachexia and exercise intolerance. To this end, we generated
MCK-EcSOD:Į-MHC-CSQ double transgenic mice by crossbreeding. Although Į-MHC-CSQ
and MCK-EcSOD:Į-MHC-CSQ double transgenic mice had similar mortality probably due to
severe dilated cardiomyopathy (Supplemental Figure S2), a significant attenuation of
cachexia (assessed by body weight) was observed in MCK-EcSOD:Į-MHC-CSQ double
15
transgenic mice at 7 wks of age compared with Į-MHC-CSQ mice (p<0.01; Figure 5A).
Importantly, CHF-induced exercise intolerance as indicated by a dramatic reduction in
treadmill running distance (p<0.001) was blunted in MCK-EcSOD:Į-MHC-CSQ mice
(p<0.001 vs. CSQ; Figure 5B). This functional protection was confirmed by blood lactate
levels; all four groups of mice displayed increases in blood lactate, but the highest levels
independent of time points were observed in Į-MHC-CSQ mice (p<0.05 vs. WT, TG, DTG;
0m
in ooff th
thee ru
rrunning
unn
Figure 5C). The area under curve of blood lactate for the first 40
min
test was
her iin
n Į-MHC-CSQ
Į-M
-MHC
-M
H -CSQ
Q mice comp
parred with wild-type
wild
wi
l -typ
y e mice (p<0.01),
(p<
p 0.011 which was
significantly higher
compared
- SOD
-EcSO
SOD:Į-MHC
SO
MHC-CSQ
SQ mice (p<0.05
SQ
( <0
(p
0.05
05 vs. CSQ).
CSQ). Non-invasive
Non-iinvasiive whole
wh
h body
blunted in MCK-EcSOD:Į-MHC-CSQ
wed
we
d th
that
at Į-MHC-CSQ
ĮM
MHC
HC CSQ
CSQ mice
mic
icee have
have a significantly
sig
igni
nifi
ni
fica
fi
cant
ntly
ly rreduced
educ
ed
uced
ed fforce
orce
or
ce p
pr
r
tension test showed
production
(39.9%, p<0.01) compared with wild-type mice (Figure 5D), which was also blunted in
MCK-EcSOD:Į-MHC-CSQ mice (p<0.05 vs. CSQ).
Improved skeletal muscle function in MCK-EcSOD:Į-MHC-CSQ mice could theoretically be
a result of improved cardiac function by protection from overexpressed EcSOD redistributing
to the heart. To test this possibility, we first performed immunoblot and semi-quantitative
RT-PCR and found significantly increased EcSOD protein in the heart without increased
EcSOD mRNA (Supplemental Figure S3) along with elevated EcSOD in the serum in
16
MCK-EcSOD mice, suggesting that muscle EcSOD redistributed to the heart through the
circulation. MCK-EcSOD:Į-MHC-CSQ mice, however, developed the same degree of
cardiac hypertrophy (Figure 5E) and reduction of ejection fraction as Į-MHC-CSQ mice
(Figure 5F). Electrocardiography showed significantly increased QRS intervals (Table) and
decreased heart rate (p<0.01) in these mice to the same degree (Figure 5G). Therefore,
reduced cachexia and improved muscle function in MCK-EcSOD:Į-MHC-CSQ mice did not
sbre
sb
red
re
d MC
MCKK-Ec
KEcSO
Ec
SO mice
appear to be a result of improved cardiac function. We also crossbred
MCK-EcSOD
m
odel
od
e of amyotrophic
el
amyo
am
y trop
phic lateral sclerosis
sclero
osis (A
((ALS),
LS
S), SOD1-G93A
SO
OD1-G93A mice. SOD1-G93A
with a genetic mod
model
sgene expressing
expressing
i aut
autosomal
tosomall dominant
domiinantt fashion
f shio
fa
i n off a mutant
mutant
t form
form of
mice harbor transgene
esults
esul
es
ults
ul
ts iin
n AL
ALS
S li
like
ke m
motor
otor
ot
or n
neuron
euro
eu
ron
n di
disease
dise
seas
asee SOD1-G93A
SOD1
SO
D1 G93A
G93
93A
A and
and
CuZnSOD that results
ALS-like
disease.
SOD1-93A:MCK-EcSOD mice had similar survival curve (Supplemental Figure S5) with
significant reduction of body weight at 14 wks of age and reduced muscle force production at
8 weeks of age (Supplemental Figure S5). Therefore, EcSOD did not provide protection for
skeletal muscle when the insults were of motor neuron origin.
Muscle-specific EcSOD transgenic mice are resistant to CHF in developing cachexia and
multiple abnormalities
To obtain structural and biochemical evidence of EcSOD-mediated protection, we measured
17
muscle mass and normalized by tibia length to minimize the effect of body weight reduction.
CHF led to a significant loss of muscle mass in EDL (-38.4%; p<0.01), tibialis anterior
(-37.3%; p<0.01), gastrocnemius (-42.3%; p<0.001), plantaris (-43.3%; p<0.01) but to a less
degree in soleus (-28.7%; p<0.001) muscles in Į-MHC-CSQ mice compared to the wild-type
littermates, which were significantly attenuated in MCK-EcSOD:Į-MHC-CSQ mice (Figure
6A). Fiber size analyses showed that Į-MHC-CSQ mice had significantly reduced IId/x/IIb
EcSO
SOD
SO
D ov
over
erex
er
expr
ex
pree
pr
fiber size (-42.8%; p<0.05) (Figure 6B), which was blunted by Ec
EcSOD
overexpression
Į-MHC-CSQ
-MHC
-M
HC-CSQ
HC
Q mice had
d significantly
Q). C
Q)
onsis
istent
is
n with our previous
p evious finding
pr
fiin
nding
g3, Į-M
(p<0.05 vs. CSQ).
Consistent
decreased capillary
in
glycolytic
type
IId/x/IIb
fibers
a contacts
ary
contacts
t
in glyc
ly ollytic
t typ
pe II
Id/x//IIIb fi
fib
bers (p<0.01),
(p<0.001),
1)) bu
bbutt not inn oxidative
type IIa fibers. T
The
he lloss
osss of ccapillarity
os
apil
ap
illa
il
lari
la
rity
ri
ty w
was
as ssignificantly
igni
ig
nifi
ni
fica
fi
cant
ntly
ly aattenuated
tten
tt
enua
uate
ted
d in
MCK-EcSOD:Į-MHC-CSQ mice (p<0.05 vs. CSQ; Figure 6C). We also performed whole
mount immunofluorescence staining for CD31 and smooth muscle D-actin in FDB muscle
and measured the fractal dimension of microvessels (arterioles and venules), a measure of
number of branches and microvessels. There were no significant differences in fractal
dimension among different groups (Figure 6D), suggesting that vascular rarefaction did not
occur at the arteriole and venule levels in Į-MHC-CSQ mice. Į-MHC-CSQ mice are known
to have reduced mitochondrial (Cyt C, COX IV) and PGC-1D protein expression indicative
18
of mitochondrial dysfunction in fast-twitch, glycolytic muscles3, 6. Here, we showed that a
preferentially reduced expression of these proteins in fast-twitch, glycolytic white vastus
lateralis muscle was absent in MCK-EcSOD:Į-MHC-CSQ mice (Figure 6D), suggesting that
overexpression of EcSOD is sufficient to block CHF-induced mitochondrial dysfunction in
skeletal muscle. Consistently, CHF condition in Į-MHC-CSQ mice did not cause any
significant changes in myosin heavy chain protein isoforms, and EcSOD overexpression
ithe
it
her.
he
r. R
ealea
l-ti
ltim
ti
m PCR
alone in MCK-EcSOD mice did not cause compensatory changess eeither.
Real-time
that
a Į-M
at
MHC
HC-CSQ
Q mice had significantly
sign
g iifficantly
y elevated
elev
el
e atted
e MAFbx/Atrog
g
analysis showed th
Į-MHC-CSQ
MAFbx/Atrogin-1
mRNA
siigniifi
ficantly
ly red
duced
d in
in MCK-EcSOD:Į-MHC-CSQ
MCK
MC
K-Ec
E SO
SOD:
D Į-MHC
MHC
C-CSQ
C Q mice
miice (p<0.05 vs.
(p<0.001), whichh was significantly
reduced
F) We did
did n
not
ot d
detect
etec
et
ectt si
significantly
sign
gnif
ifiica
if
cant
ntly
ly differences
dif
iffe
fere
fe
renc
nces
es of
of ap
apoptosis
apop
opto
tosi
siss as
si
assess
asse
sess
ss by
CSQ) (Figure 6F).
assessed
TUNEL staining (Supplemental Figure S5). Most importantly, we have performed three
different analyses to determine if EcSOD overexpression had any significant impact on
oxidative stress induced by CHF. We found that CHF (in Į-MHC-CSQ mice) led to
significantly elevated levels of oxidative stress markers, MDA and protein carbonyls, in
fast-twitch, glycolytic white vastus lateralis muscle, which were significantly reduced in
MCK-EcSOD:Į-MHC-CSQ mice (Figure 6G and Supplemental Figure 6S). A similar trend,
although not statistically significant, was found for another oxidative stress marker of lipid
19
peroxidation, 4-HNE (Supplemental Figure 6S). These findings provide evidence for EcSOD
function in protecting skeletal muscle from cardiac cachexia at molecular, biochemical,
structural and functional levels likely through its ectopic antioxidant function.
Discussion
Studies with genetic and pharmacological interventions in cultured myocytes and intact
ant
ntio
ioxi
io
xida
xi
dant
da
n ddefenses
nt
efee
ef
skeletal muscles have provided ample evidence that intracellularr antioxidant
are
w provide
ainin
ain
in
nin
ing normal
no
ormal
a redox state,, musclee m
ass and
d fu
ffunction
nction28, 29. Here, we
critical in maintaining
mass
clear in vivo evidence
under
dence th
that E
EcSOD
cSOD
SOD
D expressio
expression
i n iin
n sk
skeletal
kelettall muscl
muscle
le iiss red
reduced
duced
d und
un
de
glucocorticoidism,
associated
m a condition
con
ondi
diti
di
tion
ti
on of
of many
many chronic
chr
hron
oniic di
diseases
dise
seas
ases
es including
inc
nclu
ludi
lu
ding
di
ng CHF
CHF tthat
hatt is aas
ha
s
with increased oxidative stress30, muscle wasting and loss of muscle function18. We showed
that systemic administration of endogenous NO donor could attenuate skeletal muscle
atrophy induced by dexamethasone in mice concurrent with a restoration of EcSOD
expression. These findings suggest that NO-dependent EcSOD expression is important for
muscle maintenance in vivo. We then employed two independent genetic approaches and
showed that myofibers with acquired EcSOD expression do not undergo significant atrophy
upon glucocorticoidism. Muscle-specific EcSOD transgenic mice are resistant to CHF,
20
displaying significant protections against cachexia, exercise intolerance, and loss of
mitochondria, rarefaction of microvasculature, and increased atrophy gene expression in
skeletal muscle. Most importantly, we have shown evidence of reduced oxidative stress when
EcSOD is ectopic overexpressed in a mouse model of cardiac cachexia. These findings
provide for the first time unequivocal evidence that induced EcSOD expression protects
myofibers from catabolic muscle wasting in vivo, suggesting that promoting EcSOD
cou
ould
ld bbee an effective
eff
ffee
expression by augmenting NO bioavailability in skeletal muscle could
CHF patients.
pattie
i ntts.
intervention for CHF
tant si
signaling
ignali
lliing rrole
ole
l in ph
physiological
hysiiologic
l
all and
d pathological
patho
h logical
i processes. W
NO plays important
We have
eection
ctio
ct
ion
io
n of lipopolysaccharide
lip
ipop
opol
olys
ol
ysac
acch
char
ch
arid
idee (LPS),
id
(LPS)
(LPS
(L
PS)) a m
PS
major
ajor
aj
or mediator
med
edia
iato
ia
torr of ssepsis
epsi
ep
siss iin
si
n
reported that injection
sepsis,
induces
greater NO production in slow-twitch, oxidative muscle than in fast-twitch, glycolytic muscle
in mice3, 16. Since these oxidative muscles are resistant to catabolic muscle wasting under
many disease conditions, the findings suggest that elevated NO production is protective. Our
previous studies in cultured myocytes provided further evidence for a functional link between
NO and antioxidant gene expression6, 16. Consistently, the data presented in this and previous
studies16, 31 showed robust induction of EcSOD expression when there is increased NO
bioavailability, suggesting that NO-induced EcSOD expression in skeletal muscle may
21
contribute significantly to the protection associated with oxidative phenotype.
The functional importance of EcSOD has been shown by its reduced expression associated
with various chronic diseases10, 11, and by the fact that genetic deletion and overexpression of
EcSOD leading to exacerbation and protection against pathological changes in animal models
of diseases, respectively12-15. Glucocorticoids are used as therapeutic agents due to their
potent anti- inflammatory and immunosuppressive functions; however, high dose and
long-term use of glucocorticoids causes muscle atrophy22, 32. Previous
evio
ev
ious
io
us sstudies
tudi
tu
diees
di
es and
and
nd our current
ncreased
n
creased
cr
d oxidative
oxiidative stress induced
d bby
y dexame
dexamethasone
m th
hasone in skeletal muscle with
data support an increased
2 333
.O
Oxidative
xid
idatiive stress may promotes cat
id
catabolic
t
direct evidence of
radicals
o increased
d ffree
ree radi
icals22,
wasting and loss of
signaling,
contractile
protein
of muscle
musc
mu
scle
le function
fun
unct
ctio
ionn through
io
thro
th
roug
ugh
h ce
cell
ll ssignaling
igna
ig
nali
ling
li
ng im
impairment
impa
pair
irme
ir
ment
nt o
off co
contra
cont
ntra
ra
and promotion of protein degradation4, 5, 34, 35. Here, we provide new evidence for reduced
EcSOD protein expression in catabolic muscle wasting and the functional importance of the
NO-mediated EcSOD expression in protection against catabolic wasting.
A key question is how EcSOD protects skeletal muscle from atrophy. Since EcSOD is the
only isoform of superoxide dismutase that is secreted to the extracellular space, one
straightforward explanation is that elevated extracellular ROS, which could be scavenged by
EcSOD, plays an importantly role in cachexia and exercise intolerance. Several pathological
22
steps might be relevant here. First, extracellular ROS could cause endothelial cell apoptosis36,
leading to vascular rarefaction, an important feature of cardiac cachexia. Vascular rarefaction
not only impairs microcirculation and tissue perfusion but also exacerbates muscle wasting
due to hypoxia. Second, superoxide reacts readily with NO to reduce NO bioavailability37,
impairing exercise-induced increase of blood flow and contributing to exercise intolerance.
Finally, extracellular ROS-mediated intracellular ROS production and signaling38 may play
D ex
expr
pres
pr
essi
es
sion
si
on ccould
oull provide
ou
an important role in catabolic muscle wasting. Enhanced EcSOD
expression
protection through
mechanisms.
Studies
protectivee effects of
ghh oone
ne oorr more of these mechanis
sms. Studie
ies showing
ie
shhowing
g protecti
antioxidants when
applied
EcSOD
en ap
pllied directly
di ly to cultured
cultured
d cells
cellls appear
app
ppear to
pp
t bbee consistent
consiistent with
wiith
hE
function in scavenging
enging
engi
en
ging
gi
ng eextracellular
xtra
xt
race
cell
llul
ll
ular
ul
ar ssuperoxide;
uper
up
erox
oxid
ide;
id
e; however,
however
h
owev
ow
ever
er in llight
ight
ig
ht o
off the
the fi
findings
find
ndin
nd
ings
in
gs that EcSOD
can be internalized by cells that it binds9, elevated intracellular EcSOD in myofibers and/or
other cells in skeletal muscle may also contribute to the protection.
The protective role of EcSOD is likely mediated by its antioxidant properties. Based on the
evidence of reduced oxidative stress markers and atrogene expression in glycolytic muscles
(Figure 6F and 6G), we believe that transgenic overexpression of EcSOD in skeletal muscle
exerts its protective function by counteracting oxidative stress induced by CHF. The findings
of muscle mass, mitochondrial protein and vascular rarefaction are all extremely consistent
23
with this notion. Future studies should focus on elucidating the necessity of EcSOD
expression in skeletal muscle in maintain muscle mass and function under catabolic wasting
conditions.
It has been shown that EcSOD expression by smooth muscle can be enhanced by exercise
training in an NO-dependent manner31, and EcSOD mRNA is increased by acute exercise in
skeletal muscle39. It has also been shown recently that exercise training protects oxidative
duc
uced
ed skeletal
ske
kele
leta
le
taal muscle
m
stress and proteasome-dependent protein degradation in CHF-induced
nans
na
nswereed qu
qquestion
estion is which effec
effecter
cteer ge
ggene(s)
ne(s
((ss) is responsible for th
the
h enhanced
atrophy40. An unanswered
antioxidant function
induced
exercise
training.
be such
t
tion
indduced
d by
by exer
cise
i trai
ining
g. Our
Our findings
fiind
dings suggest
suggestt EcSOD
EcSOD
SOD
O could
c
an important player.
expression
in
yerr W
ye
Wee sh
show
ow h
here
eree th
er
that
at eexercise
xerc
xe
rcis
isee tr
is
training
trai
aini
ai
ning
ni
ng p
promotes
romo
ro
mote
tess Ec
EcSOD
EcSO
SOD
SO
D ex
expre
expr
pree
skeletal muscle (Figure 2), and that enhanced EcSOD expression is sufficient to provide
protection against cardiac cachexia and exercise intolerance.
Another issue is whether enhanced expression of EcSOD from skeletal muscle redistributes
to the extracellular space of the heart leading to improved cardiac function and preventing
cachexia. EcSOD-mediated protection against cardiac toxicity has been shown by Kliment et
al where EcSOD global knockout mice showed exacerbated cardiac dysfunction, myocardial
apoptosis and fibrosis following doxorubicin injection41. We have previously shown that
24
adenovirus-mediated gene transfer of EcSOD in the heart elicits protective effects on cardiac
function following myocardial infarction in rabbits42. In this study, although forced
expression of EcSOD from skeletal muscle redistributed to the heart (Supplemental Figure
S3), it did not improve cardiac function and prevent premature death. These findings allowed
us to focus on the impact of ectopic EcSOD expression in skeletal muscle on catabolic
muscle wasting without confounding factors of EcSOD overexpression in impeding heart
ng is
is caused
caus
ca
used
us
ed bby
y ca
a
failure. Sicne abnormal calcium and G-protein coupling signaling
calsequestrin
the
he nega
negative
g tive
ve findings
find
ding
gs of EcSOD effects in
ffrom
om with
thin cardiac myocytes
th
myo
y cy
ytes43, th
overexpression fro
within
the heart suggestt thatt el
elevated
levated
d EcSOD
EcS
SOD
D in
in the
h hheart
eart off Į-MHC-CSQ
Į-M
MHC-C
HC CSQ
HC
Q mouse is
is not enough to
deal with such a ssevere
origin.
ever
ev
eree CHF
CHF condition
cond
co
ndit
nd
itio
it
ion
io
n or with
wit
ith
h pa
pathogenesis
path
thog
th
ogen
enes
esis
is of
of CHF
CHF of an
an intracellular
intrace
intr
in
trac
acee
Previous findings support the view that exercise intolerance in CHF is a result of skeletal
muscle abnormalities although the failing heart is the primary cause44-46. Consistent with this
scenario, our findings suggest that the improved skeletal muscle outcomes in
MKC-EcSOD:Į-MHC-CSQ mice were not due to improved cardiac function. Our
measurements of baseline heart function, heart weight, skeletal muscle weight, and survival
curve collectively suggest that EcSOD overexpression had a negligible impact on the
progression of heart failure induced by cardiac overexpression of calsequestrin in our model.
25
On the contrary, skeletal muscle abnormalities are significantly blocked/attenuated along
with improved contractile function and exercise capacity. We, therefore, believe that the
effects of elevated EcSOD in the heart on cardiac function, if present, likely has a minor
impact on exercise capacity. It would be interesting to ascertain whether muscle
overexpression of EcSOD is protective against other types of CHF.
The concept that exercise intolerance in CHF results from multiple skeletal muscle
vent
ve
ntio
nt
ions
io
ns.. We hhave
ns
aav
ve previously
abnormalities will be instrumental to developing effective interventions.
lea
ead
ds too preferential
pref
eferential vascular rare
ef
efaction in
n fastt twitch, glycolytic
tglyc
y olytt muscle
shown that CHF leads
rarefaction
fast-twitch,
a th
that
hatt skeletal
ske
k letall muscles
musclles with
wiith
h overexpression
overexppression off PGCPGC
C- 1D have
have ssignificantly
fibers in CHF3, and
xpre
xp
ress
ssio
ion
io
n less
les
esss muscle
musc
mu
scle
le atrophy
atr
tro
oph
phy
y and
and vascular
vasc
va
scul
ular
ul
ar rarefaction
rar
aref
efaact
ef
ctio
ion
io
n under
unde
un
derr tthe condition
de
elevated EcSOD eexpression
expression,
of CHF6. Here, we have again detected vascular rarefaction in fast-twitch, glycolytic fibers in
Į-MHC-CSQ mice, which was significantly attenuated by EcSOD overexpression. Since
EcSOD accumulates at the endothelium, EcSOD may also protect endothelial cells against
oxidative stress-induced apoptosis. We have also previously shown that skeletal muscles
undergoing catabolic wasting lose mitochondria with reduced expression of PGC-1D and
increased oxidative stress3, 6, which may contribute directly to exercise intolerance. The fact
that EcSOD overexpression prevents the reduction of mitochondrial and PGC-1D proteins
26
strongly suggests that ROS induced by CHF induces pathological events in muscle. This
could explain why enhanced EcSOD expression in skeletal muscle can provide potent
protection and lead to preserved exercise capacity in MCK-EcSOD:Į-MHC-CSQ mice.
Previous studies have also linked oxidative stress to catabolic muscle wasting with evidence
for enhanced ubiquitin-proteasome16, 47-49 and the autophagy-lysosome systems22, 50. The
link of oxidative stress to autophagy, particularly mitochondrial autophagy (mitophagy), may
underlie the loss of mitochondria in CHF. A vicious cycle between
oxidative
stress
een
ee
n ox
oxid
idat
id
ativ
at
ivee st
iv
stre
ree and
mitochondrial dysfunction
development
cachexia
ysf
ysf
sfu
unctio
on could be critical for thee developm
pmentt of cardiac cache
pm
e and
exercise intolerance.
n
nce.
Finally, we have p
muscle
leads to
previously
revi
re
viou
vi
ousl
sly
sl
y shown
show
sh
own
n that
that overexpression
ove
vere
rexp
xpre
ress
ssio
ion
io
n of PGC-1Į
PGC
PGC 1Į
1Į iin
n sk
skeletal
skel
elet
el
etal
al m
mu
u
increased iNOS, eNOS, EcSOD, MnSOD, CuZnSOD and CAT expression in skeletal muscle
along with increased FOXO and Akt phosphorylation6. We postulated that PGC-1Į enhances
FOXO and Akt phosphorylation in parallel with enhanced NO-antioxidant defense axis. Our
current findings, consistent with this posulation, suggest that increased NO can directly
stimulate EcSOD in skeletal muscle providing protection. It is of note that increased NO
alone without overexpression of PGC-1Į only stimulates EcSOD, but not the other
antioxidant enzymes. This NO inducibility of EcSOD expression helped us to focus on the
27
function of this important, yet understudied, antioxidant enzyme in protection against muscle
wasting.
In summary, we have obtained novel findings to support that NO-mediated protection against
catabolic muscle wasting is associated with enhanced EcSOD protein expression. Genetic
interventions with forced expression of EcSOD in skeletal muscles significantly attenuates
muscle atrophy. Importantly, muscle-specific EcSOD transgenic mice are resistant to CHF in
rot
otec
ecti
ec
tion
ti
on aagainst
gain
ga
insst
in
st oxidative
developing cachexia and exercise intolerance with significant protection
gene
enee expression,
en
expr
prressi
sion,
i , and loss of mitochondria
mitoch
chondria aand
n microvasculature. We now
nd
stress, atrophic gen
here enhanced
enha
h nced
d skeletal
skelletal muscle EcSOD
EcS
SOD
D expression prevents en
n
propose a model wh
where
endothelial
normalities
n
orma
or
mali
liti
li
ties
ti
es in
in cachexia
cach
ca
chex
ch
exia
ia aand
nd exercise
exe
xerc
rcis
isee intolerance
is
into
in
tole
lera
le
ranc
ncee li
likely
like
kely
ke
ly ddue
ue tto
o th
thee
and muscular abnormalities
maintenance of redox homeostasis (Figure 7). The findings pave the way for targeting
NO-dependent EcSOD expression for the prevention and treatment of cardiac cachexia.
Acknowledgements
We thank Mrs. John Sanders and R. Jack Roy for their excellent technical supports in
immunofluorescence and MRI, respectively.
28
Sources of Funding
This project was supported by grant AR050429 (Z.Y.). The research was also supported by
Nakatomi Foundation (M.O.), Postdoctoral Fellowship in Physiological Genomics by the
American Physiological Society (V.A.L.), National Health Institutes T32-HL007284
(J.A.C.), and American Heart Association 12POST12030231 (J.A.C.). This research was
supported in part by grant to S.M.P. (HL082838).
Disclosures
None.
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34
Table. Muscle-specific EcSOD expression does not provide significant protection against
CHF in Į-MHC-CSQ mice as assessed by electrocardiography.
WT
TG
CSQ
DTG
(n = 11)
(n = 16)
(n = 8)
(n = 6)
PR (ms)
25.1±1.3
26.1±0.7
29.9±3.1**
38.9±6.8†
QRS (ms)
10.9±0.2
10.8±0.2
16.5±1.8***
17.4±2.0§§§
QT (ms)
39.6±0.8
40.4±0.7
57.6±4.1***
64.4±8.6§§§
Data
were
obtained
from
MCK-EcSOD
(TG),
Į-MHC-CSQ
Į-M
-MHC
HC-C
HC
-CSQ
-C
SQ
((CSQ)
and
MCK-EcSOD:Į-MHC-CSQ double transgenic mice (DTG) and
nd their
th
hei
eirr wild-type
wild
wi
ld-t
ld
-ttyp littermates
(WT) and presented
mean
vs.
n ed as m
nted
ean ± SE. ** and *** denote
deenote p<0.01
d
p<0
0.0
. 1 aand
nd p<0.001, respectively,
respp
§§
WT; † denotes p<0.05
<0.05 vs. CS
C
CSQ;
SQ;; §§§
denotes
denot
otes
t p<0
p<0.001
<0.001 vs.
<0
vs. TG.
TG.
35
Figure Legends
Figure 1. Systemic administration of endogenous NO donor GSNO induces EcSOD
expression and prevents dexamethasone-induced muscle atrophy. Wild type mice were
subjected to daily injections of dexamethasone (Dex) or saline as control (Con) with/without
endogenous NO donor GSNO for 7 days. Various muscles were harvested for muscle weight
measurements, and plantaris muscles were processed for immunoblot and semi-quantitative
PCR analyses. A) Immunoblot images of antioxidant enzymes in plantaris muscle (40 Pg
total protein) with samples from a MCK-Pgc-1D transgenic mouse (Pgc-1D TG) and a
wild-type littermate (WT) as positive and negative controls (20 Pg), respectively. D-tubulin
was probed as loading control. Quantitative and statistical analyses are presented on the right
(n=9-11/group); B) Images of semi-quantitative RT-PCR analysis for MAFbx/Atrogin-1 and
MuRF1 mRNA with quantitative and statistical analyses presented
the
ted on
on th
he right
righ
ri
ghtt
gh
(n=9-11/group); and C) Quantification of muscle weight of EDL,
anterior
L, tibialis
tib
bia
iali
lis an
li
nte
teri
rior
ri
o (TA),
gastrocnemius (GA),
plantaris
(SO)
GA)
GA)
A), pl
plan
antaari
an
ris (PL) and soleus (S
SO)
O muscless from
fro
om mice. Data aree presented
as normalized muscle
weight
weight
(n=9-11/group).
***
denote
usccle weigh
gh
ht by
b body
bod
ody we
weig
ig
ght (n
n=9-111/g
/gro
roup
ro
upp). *,
*, **
* and
d **
** de
deno
o p<0.05,
p<0.01 and p<0.001,
001
001
01,, respectively.
r sp
re
spec
ecctiive
vely
ly
y.
Figure 2. EcSOD
endurance
D is hhighly
ighl
ig
h y ex
hl
expr
expressed
p es
pr
esse
sed
se
d in ooxidative
xida
xi
dati
da
tive
ti
ve m
muscle
usscl
clee an
and
d is iinduced
nduc
nd
uced
uc
ed bby
y en
endu
duu
exercise training. Various tissues were harvested from wild type mice and from sedentary
mice and exercise trained mice after 4 weeks of voluntary running and processed for
immunoblot analysis and immunohistochemistry. A) Immunofluorescence staining showing
EcSOD expression in plantaris (PL) and soleus (SO) muscle sections in comparison with the
aorta and lung sections. High magnification images were shown in the brackets with arrows
pointing to the capillary endothelial cells. Scale bar = 100 Pm; B) Immunofluorescence
staining of PL and SO muscle sections showing EcSOD (blue) is mainly expressed by type
IIa (pink) and IId/x (fibers with no staining) myofibers; C) Immunoblot images of EcSOD
protein in SO and white vastus lateralis (WV) muscles (40 Pg total protein) with D-tubulin as
loading control. Quantitative and statistical analyses are presented on the right (n=6/group);
and D) Immunoblot images of EcSOD protein in plantaris muscle (40 Pg total protein) from
sedentary (Sed) and exercise trained (Ex) mice after 4 weeks of voluntary running with
D-tubulin as loading control. Quantitative and statistical analyses are presented on the right
(n=10/group). * and ** denote p<0.05 and p<0.01, respectively.
36
Figure 3. Somatic gene transfer-mediated EcSOD overexpression in adult skeletal muscle is
sufficient to prevent dexamethasone-induced catabolic muscle wasting. Ten days after
electric pulse-mediated gene transfer in tibialis anterior muscles (see Methods), mice
received daily injections of dexamethasone (Dex) or saline (Sal) as control for 7 days, and the
tibialis anterior muscles were harvested and processed for protein and muscle fiber size
analyses by immunoblot and fluorescent microscopy. A) Immunoblot images of EcSOD
protein expression in left tibialis anterior muscle transfected with pEcSOD and the
contralateral control muscle transfected with empty vector pCI-neo (pCI) with D-tubulin as
loading control (40 Pg total protein); B) Fluorescent microscopy images of muscle sections
with co-transfection of pGFP3; and C) Histograms of fiber size distribution according to
cross-sectional area (CSA) (left, top and bottom panels) and comparison of the mean CSA
across treatment conditions (right panel, n=5-9/group). ** and *** denote p<0.01 and
p<0.001, respectively.
Figure 4. Muscle-specific EcSOD transgenic mice are resistant to dexamethasone-induced
deexa
xame
meth
me
thas
th
aso
as
one
on
n
catabolic musclee w
wasting.
Skeletal
astingg. Sk
Skelet
etal
et
a muscles
al
mus
uscl
us
c es were
wer
ere ha
hharvested
arv
ves
e ted
ted fr
te
ffrom
rom
m MCK-EcSOD
MC
CKK EcSO
SOD
SO
D mice
m (TG)
and wild type littermates
immunofluorescence
immunoblot
termates
ter
e mates (W
er
((WT)
T) aand
nd
d pprocessed
roceesssed
d ffor
or imm
mm
mun
u of
oflu
lu
uores
esceence an
and
d im
mmu
analyses. Muscles
weight
e were also harvested
es
harves
essteed for muscle
mu
usclee w
eiigh
g t from TG and WT micee following 7
days of dexamethasone
A) EcSOD
hasone
h
ason
as
onee injections
on
inje
in
ject
ctio
ct
ions
ns (Dex)
(De
D x) w
with
ith
it
h saline
sali
sa
l ne ((Sal)
li
Sal)
Sa
l)) iinjections
njjec
ecti
tion
onss as ccontrol.
on
ontr
on
trol
tr
ol.. A
transgene construct
illustrated
top.
Immunoblot
in
ctt iiss iill
ll st
strated
tratted
d att th
the ttop
op IImm
mm n
noblot
obl
blott iimages
mages off E
EcSOD
cSO
SOD
SO
D eexpression
pre
soleus (SO), plantaris (PL) and white vastus lateralis (WV) muscles from TG and WT mice
(40 Pg total protein) with D-tubulin as loading control are presented on the right.
Immunofluorescence images are presented at the bottom showing EcSOD overexpression
mainly in fast-twitch, glycolytic type IIb fibers (green) with enrichment around endothelial
cells (indicated by arrows); B) Quantification of muscle weight in EDL, tibialis anterior (TA),
gastrocnemius (GA), PL and SO muscles as normalized muscle weight by body weight
(n=9-12/group); and C) Immunoblot images of other antioxidant enzymes, mitochondrial and
muscle contractile proteins in SO, PL and WV muscle in TG and WT mice. *, ** and ***
denote p<0.05, p<0.01 and p<0.001, respectively.
Figure 5. Muscle-specific EcSOD transgenic mice are resistant to cardiac cachexia and
exercise intolerance. MCK-EcSOD mice (TG) were crossbred with Į-MHC-CSQ mice (CSQ)
to generate MCK-EcSOD:Į-MHC-CSQ double transgenic mice (DTG). These mice were
analyzed for body weight, running capacity, whole body tension, electrocardiography and
MRI with wild-type littermates (WT) as control. A) Body weight at 7 wks of age
37
(n=8-14/group); B) Treadmill running test at 5 wks of age; C) Blood lactate levels
(n=11-17/group) during treadmill running test (left) and the area under the curve (AUC)
during the first 40 min of the test (right); D) Representative force production records of WBT
at 7 wks of age (left) with quantitative and statistical analyses presented on the right
(n=8-13/group); E) Representative heart images (left; bar = 1 cm) with quantitative and
statistical analyses presented on the right (n=10-12/group); F) Representative MRI (left; scale
bar = 2 cm) of the thoracic chambers with quantification of end-systolic volume (ESV),
end-diastolic volume (EDV) and left ventricle ejection fraction (LVEF) of the heart at 6 wks
of age (n=4-6/group); and G) Representative CKG recordings (left, scale bar = 0.5 sec) and
quantification of the heart rate (right) at 7 wks of age (n=6-16/group). *, ** and *** denote
p<0.05, p<0.01 and p<0.001, respective
Figure 6. Muscle-specific overexpression of EcSOD prevents CHF-induced catabolic muscle
wasting, loss of mitochondria, vascular rarefaction and oxidativee stress.
str
tres
ess.
es
s. Skeletal
s.
Ske
kele
leta
leta
le
tall muscles
were harvested from MCK-EcSOD (TG), Į-MHC-CSQ (CSQ) and
nd
MCK-EcSOD:Į-MHC-CSQ
wild-type
littermates
-MHC-C
-M
CSQ ddouble
ou
ub
blle
le transgenic
trran
ansg
sggen
nic mice
mice (DTG)
((D
DTG
TG) and
a d th
an
ttheir
eirr wi
wild-t
typ
ypee li
i
(WT) at 7 wks off aage
processed
measurements
muscle
weight,
ge and pro
roceessed
d for m
easuurem
men
nts ooff mu
uscle
le w
eigh
ht,, muscle
muscl
clle fiber size,
contractile and mitochondrial
protein
expression,
mitoch
hondrial protei
ein
ei
n expr
p essi
sion,
i
aatrogene
at
ro
og
geene mRNA expression and
a d oxidative
stress. A) Quantification
anterior
ification
ific
if
icat
atio
at
ion
n of muscle
mus
uscl
clee weight
weig
we
i h
ig
htt nnormalized
orma
or
mali
ma
lize
zed
ze
d by tibia
tib
ibia
ia length
len
engt
gth
gt
h in EDL,
EDL
DL,, tibialis
tib
ti
b
(TA), gastrocnemius
B)
mii s (GA),
(GA)
(G
A) plantaris
plant
l taris
i (PL)
(PL) andd soleus
sole
sol
le s (SO)
(SO
SO)) muscles
m scl
scles
les (n=9-12/group);
((n
n 9 12/gro
12/
2/gro
Immunofluorescence images of type IIa (green) and type IId/x/IIb fibers (negative staining)
with quantification of fiber size in plantaris muscle (n=4-5/group); C) Immunofluorescence
staining for CD31 (red) and MHC IIa (green) with quantification of fiber type specific
capillary contacts in plantaris muscle (n=4-5/group); D) Whole mount immunofluorescence
staining of CD31 (green) and smooth muscle D-actin (red) in FDB muscle (left) with
quantification of the fractional dimension of microvessels (right) (n=5-12/group); E)
Representative immuoblot images of PGC-1D, COX IV, Cyt c, EcSOD, MHC I, MHC IIa,
and MHC IIb proteins in SO and white vastus lateralis muscles (WV) with E-actin as loading
control; F) Real-time PCR analysis for MAFbx/Atrogin-1 mRNA expression in PL muscle
(n=7-9/group); and G) Representative immunoblot images of protein carbonylation in WV
muscle with GAPDH as loading control. *, ** and *** denote p<0.05, p<0.01 and p<0.001,
respectively.
Figure 7. Schematic presentation of the working hypothesis for EcSOD-mediated protection
against CHF-induced catabolic muscle wasting and exercise intolerance. CHF and the
38
resulting systemic factors exacerbates the production of extracellular free radical superoxide
ion O2.-, which in turn cause abnormalities in muscle vasculature (endothelial dysfunction
and apoptosis) and muscle fibers (atrophy and mitochondrial dysfunction). These
abnormalities in skeletal muscle underscore the development of exercise intolerance
presumably by impairing blood supply and muscle contractile and metabolic functions.
EcSOD in extracellular space in skeletal muscle may provide the first line of defense against
the excessive production of extracellular free radicals under the condition of CHF, ultimately
preserving muscle function.
39
Fig. 1
A
C
B
Fig. 2
A
C
B
D
Fig. 3
B
C
A
Fig. 4
B
C
A
Fig. 5
F
G
E
D
C
B
A
Fig. 6
A
B
C
Fig. 6 (continued)
G
F
E
D
Fig. 7
Extracellular Superoxide Dismutase Ameliorates Skeletal Muscle Abnormalities, Cachexia and
Exercise Intolerance in Mice with Congestive Heart Failure
Mitsuharu Okutsu, Jarrod A. Call, Vitor A. Lira, Mei Zhang, Jean A. Donet, Brent A. French, Kyle S.
Martin, Shayn M. Peirce-Cottler, Christopher M. Rembold, Brian H. Annex and Zhen Yan
Circ Heart Fail. published online February 12, 2014;
Circulation: Heart Failure is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2014 American Heart Association, Inc. All rights reserved.
Print ISSN: 1941-3289. Online ISSN: 1941-3297
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circheartfailure.ahajournals.org/content/early/2014/02/12/CIRCHEARTFAILURE.113.000841
Data Supplement (unedited) at:
http://circheartfailure.ahajournals.org/content/suppl/2014/02/12/CIRCHEARTFAILURE.113.000841.DC1.html
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Circulation: Heart Failure can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about this
process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation: Heart Failure is online at:
http://circheartfailure.ahajournals.org//subscriptions/
Supplemental data
Fig. S1. Dexamethasone injection induces oxidative stress in white vastus muscles in mice. Western
blot was performed for 4-HNE in muscle homogenates in mice i.p. injection of Dex (25 mg/kg) for 7
days. A sample from a mouse with lipopolysaccharide (LPS: 1 mg/kg, 12 hrs) injection were used as
positive control.
Fig. S2. Muscle-specific overexpression of EcSOD does not improve survival in mice with CHF.
Kaplan–Meier survival curves for MCK-EcSOD (TG), α-MHC-CSQ (CSQ) and
MCK-EcSOD:α-MHC-CSQ double transgenic mice (DTG) and their wild-type littermates (WT).
Death occurred between 5-7 wks of age and showed no significant differences between CSQ and
DTG mice (n=12-25/group).
1
Fig. S3. Elevated EcSOD levels in the heart of muscle-specific EcSOD transgenic mice due to
redistribution through circulation. A) Western blot image showing significantly increased EcSOD
protein expression in the hearts of MCK-EcSOD mice (TG) compared with wild-type littermates
(WT). WT and TG plantaris skeletal muscle (SKM) were used as controls; B) Semi-quantitative
RT-PCR showing similar EcSOD mRNA expression in both TG and WT mice; and C) Western blot
image showing significantly elevated EcSOD levels in the serum of TG mice compared with WT
mice.
Fig. S4. Measurement of apoptosis in skeletal muscle. Fresh frozen plantaris sections were used to
detect apoptosis by TUNEL staining with DAPI staining for nuclei staining. The number of
TUNEL-positive nuclei was counted from at least 1000 nuclei. Representative TUNEL staining (left,
scale bar = 50 µm) and quantification of the apoptosis (right) at 7 wks of age (n=4-6/group).
Fig. S5. Muscle-specific overexpression of EcSOD does not reduce mortality, loss of body weight and
muscle function in ALS mice. MCK-EcSOD mice (TG) were crossbred with SOD1-G93A mice
2
(G93A) to generate G93A:TG double transgenic mice and measured for life span, body weight (BW)
and whole body tension test (WBT). A) Kaplan–Meier survival curves for TG, G93A, G93A:TG and
WT mice. Death occurred between 20 wks of age and showed no significant differences between
G93A and G93A:TG mice (n=4-5/group); B) Body weight in G93A and G93A:TG mice were
significantly decreased starting at 14 wks of age. * denotes p<0.05; C) Muscle force production
measured by WBT (n=4-5/group). ** and *** denote p<0.01 and p<0.001, respectively, between
G93A and WT at either 8 or 16 wks. †† and ††† denote p<0.01 and p<0.001, respectively, between
G93A:TG and TG at either 8 or 16 wks.
Fig. S6. Muscle-specific overexpression of EcSOD prevents CHF-induced oxidative stress in
skeletal muscle. Glycolytic white vastus lateralis muscles (WV) were harvested from
MCK-EcSOD (TG), α-MHC-CSQ (CSQ) and MCK-EcSOD:α-MHC-CSQ double transgenic
mice (DTG) and their wild-type littermates (WT) at 7 wks of age and processed for
measurements of oxidative stress. A) Representative Western blot images of protein
carbonylation in WV muscle with GAPDH as loading control. Quantification with statistical
analysis were shown on the right; and B) Representative Western blot images of 4-HNE in
WV muscle with Ponceau S staining as loading control. Quantification with statistical
analysis were shown on the right. ** denotes p<0.01.
3

Extracellular Superoxide Dismutase Ameliorates Skeletal Muscle

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