healthmedicinet

Animals

Transgenic C57BL/6J mice overexpressing adiponectin (ApN mice), which had been generated
in our lab, were used; native full-length ApN was placed under the control of the
adipocyte aP2 promoter, thereby targeting ApN expression to white adipose tissue.
As ApN is secreted, these mice also showed a moderate elevation of circulating ApN
levels 12]. C57BL/10ScSn mdx mice were purchased from Jackson Laboratory (Maine, USA) and crossed
with ApN mice in order to obtain mdx mice overexpressing ApN (mdx-ApN mice). Three
groups of F
₁
mice were studied in all experiments: the mdx-ApN mice, their mdx littermates (true
controls), and wild-type (WT) mice; the three groups of mice were maintained on the
same C57BL/6J-10 ScSn genetic background. Only male mice were studied.

These animals were maintained under a standard laboratory chow and housed at a constant
temperature with a fixed light-dark cycle. At the end of the experiments, mice were
sacrificed by cervical dislocation (between 09.00 and 11.00 h). Blood samples were
saved. Pairs of tibialis anterior (TA) muscles or inguinal fat pads were weighed, frozen in liquid nitrogen, and stored
at ?80 °C for subsequent analyses. For some experiments dealing with evaluation of
muscle injury, additional muscles were sampled.

All procedures were approved by the Ethical Committee for Animal Experimentation from
the Medical Sector at the Catholic University of Louvain.

Light microscopy, immunohistochemistry, and morphometry

Muscle samples were fixed in 10 % formalin for 24 h and embedded in paraffin. Sections
5 ?m thick were stained with hematoxylin-erythrosin-safran (HES). The percentage of
muscle fibers with central nuclei (a characteristic feature of DMD) was determined
on more than 1000 fibers per muscle section. For immunohistochemistry, sections were
processed as previously described 6] using rabbit polyclonal antibodies directed against peroxiredoxin 3 (PRDX3, dilution
1:700, incubation 2 h), peroxiredoxin 5 (PRDX5, 1:300, 2 h) [gifts from B. Knoops,
University of Louvain, Brussels, Belgium 13]], 4-hydroxy-2-nonenal (HNE, 1:100, 2 h), tumor necrosis factor alpha (TNF?, 1:200,
2 h), interleukin-1? (IL-1?, 1:300, overnight), and nuclear factor kappa B (NF-?B,
1:50, overnight) (all from Abcam, Cambridge, UK). Rat monoclonal antibodies directed
against markers of macrophages (CD68) or of T lymphocytes (CD3) (1:50, overnight,
Abcam) were also used. Before immunostaining, sections were submitted to heat-mediated
antigen retrieval using a microwave oven and Tris-citrate buffer (pH 6.5). Binding
of antibodies was detected by applying for 30 min at room temperature a second antibody,
which was a biotinylated goat anti-rabbit IgG (H+L) or a biotinylated rabbit anti-rat
IgG (H+L) (Labconsult, Brussels, Belgium). Peroxidase activity was revealed with 3,3?-diaminobenzidine
(DAB) (Life Technologies, Gent, Belgium), which produces a brown staining. For each
marker, all slides from the three groups of mice were treated simultaneously for immunohistochemistry
analysis and DAB revelation, and then analyzed together. Immunohistochemical controls
were performed by omission of the first antibody or of the first and second antibodies
or by using pre-immune serum. For quantification of NF-?B expression, the percentage
of immunolabeled nuclei was determined after counting an average of 1000 fiber nuclei
per slide for each mouse. For PRDX3/5, HNE, TNF?, IL-1?, CD3, and CD68, whole muscle
section were scanned using the Leica SCN400 slide scanner (Leica Microsystems, Diegem,
Belgium), and then the percentage of DAB surface area within muscle fibers was quantified
using the Tissue Image Analysis 2.0 (Leica).

In vivo studies of global force or resistance

Mice were submitted to three main tests:

Wire test. Animals were suspended by their forelimbs from a 1.5 mm-thick, 60 cm-long metallic
wire at 45 cm above soft ground. The time (seconds) until the mouse completely released
its grasp and fell down was recorded. Three trials were performed per session, with
a 30-s recovery period between trials. The maximum time per trial was set to 180 s.
For each mouse, the scores of the three trials were averaged 14].

Grip test. The grip strength test measures the muscle strength of forelimb or of combined fore-
and hindlimb muscles. Limb strength was recorded using a grid connected to a sensor
(Panlab-Bioseb, Vitrolles, France). The mice were gently laid on the top of the grid
so that their front paws (forelimb test) or both fore and hind paws (combined test)
can grip the grid. Then, mice were pulled back steadily until the grip was released
down the complete length of the grid. Each test was repeated three times at an interval
of 20 min. Results are presented as the mean of the two highest values of force recorded,
related to body weight 15].

A third test of eccentric exercise was also performed mainly to evaluate muscle injury (see below).

Quantification of muscle damage markers in plasma

Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) activities were quantified
to evaluate skeletal muscle damage as injured muscles release CK and LDH into the
bloodstream at high levels. Kits were based on colorimetric methods (Gentaur, Kampenhout,
Belgium). CK and LDH activities were expressed as IU/L.

Evaluation of muscle injury after in vivo staining with Evans blue dye

Evans blue dye (EBD) irreversibly binds to plasma albumin in vivo, and upon injury, the complex
leaks into the surrounding tissues 16], 17]. EBD (Sigma-Aldrich, Bornem, Belgium) was dissolved in PBS buffer, sterilized by
filtration through membrane with pore size of 0.2 ?m, and kept at 4 °C. The dye solution
was injected ip into the mice (1 mg dye/0.1 ml/10 g body weight). The mice were sacrificed 24 h later
either in basal (sedentary) conditions or after bouts of eccentric exercise 18].

Eccentric exercise. The mice were subjected to a downhill running exercise on a treadmill with a downward
inclination of 15°, and at a speed of 10 m/min for 10 min. This training was repeated
daily for 3 days, and the mice were sacrificed 1 h after the 3rd session and 24 h
after the EBD injection 18]. Six different muscles were studied: biceps brachii (BB) and triceps brachii (TB) from the forelimbs; TA, gastrocnemius (G), extensor digitorium longus (EDL), and soleus (S) from the hindlimbs.

Spectrophotometric detection of EBD. EBD was extracted from powdered muscle using dimethylformamide, and the absorbance
was read spectrophotometrically at 620 nm. The extravasated EBD concentration was
calculated against a standard curve and was expressed as ng of EBD/mg muscle weight
16].

Fluorescence microscopy detection of EBD. Muscles were embedded in Tissue-Tek (Sakura, Alphen-aan-den-Rijn, Netherlands) using
iso-pentane frozen in liquid nitrogen. Three different transverse cryostat sections
(10 ?m thick) were obtained for each muscle and observed by fluorescence microscopy
17].

Western blotting and immunoprecipitation

Skeletal muscle was homogenized in a lysis buffer (Cell Signaling Technology, BIOKE,
Leiden, The Netherlands) supplemented with 100 mM NaF and 1 % protease inhibitor cocktail
(Active Motif, Rixensart, Belgium). Immunoblotting was performed as reported 19] by using rabbit polyclonal antibodies directed against phospho-NF-?B p65 (phosphor
Ser536) and myogenic factor 5 (Myf5) (both from Abcam); AMPK, P-AMPK, and Sirtuin
1 (SIRT1) (all form Cell Signaling); peroxisome proliferator-activated receptor-?
coactivator-1? (PGC-1?) and Utrophin (both from Santa Cruz, Heidelberg, Germany);
or a mouse monoclonal antibody directed against myogenic differentiation (MyoD) (Abcam).
Signals were revealed by enhanced chemiluminescence, then quantified and normalized
to those of actin, Ponceau stain or the studied protein without post-translational
modifications (when these modifications were studied) using ImageJ program (National
Institutes of Health, Maryland, USA).

To obtain protein extracts for immunoprecipitation, skeletal muscle was homogenized
in the lysis buffer indicated above, supplemented with 1 % protease/phosphatase inhibitor
cocktail (Cell Signaling), 5 mM nicotinamide, and 1 mM sodium butyrate. A total of
1000 mg of proteins from muscle samples were used. The immunoprecipitation was carried
out using magnetic beads (Thermo Fisher Scientific, Lafayette, CO) and anti-PGC-1?
antibody (2 ?l antibody/1 g total protein; Santa Cruz) following the manufacturer’s
instructions. The beads were collected using neodymium magnets and washed with TBS-Tween
0.05 %. After the final wash, the samples were dissolved in 100 ?l of elution solution
(0.1 M glycine, pH 2.5); 20 ?l were then separated by SDS-PAGE and immunoblotted,
as described 20], using an acetyl-lysine antibody (1:1.000; Cell Signaling) and anti-PGC-1? antibody
(1:1.000; Santa Cruz), respectively.

Culture of human myotubes

Primary cultures of human skeletal muscle cells were initiated from satellite cells
of quadriceps samples obtained from male organ donors (?=?5, 40?±?9 years) or from latissimus dorsi flap samples obtained from women undergoing reconstructive surgery after breast cancer
(?=?6, 48?±?13 years). The last series of biopsy samples were used for siRNA transfection
experiments. For each patient, the culture experiments were done in duplicate and
the data from a given individual were then averaged. Patients provided written informed
consent, and the study protocol had the approval of the local Ethical Committee of
Saint-Luc University Hospital.

Cultures were performed as described 21] with minor modifications. Myoblasts were grown in 35-mm (or 6 wells; siRNA transfection)
plates at 37 °C in the presence of 5 % CO
₂
in F-12 (Ham) supplemented with 12 % fetal bovin serum (FBS), 1 %?L-glutamine, and 100 ?g/ml Primocin™ (Invivogen, Toulouse, France) (all other products
from Life Technologies, Inc., Merelbeke, Belgium). After 3–5 days of proliferation,
at the end of which the seeding density has reached 70–80 %, the growth medium was
replaced by the fusion medium which consists of 1 part DMEM, 1 part F-12 (Ham), 2 %
horse serum (HS), 1 %?L-glutamine, and 1 % penicillin-streptomycin (Life Technologies). This fusion medium
was then changed every 2 days, and differentiation was allowed to continue for 10 days
(time required to obtain mature myotubes with characteristic elongated and multinucleated
morphology) before the experimentation period.

Ten days after differentiation, cells were treated with human recombinant TNF? (10 ng/ml)?+?interferon
gamma (IFN?) (10 ng/ml) and/or ApN (5 ?g/ml), at the indicated concentrations, for
24 h (TNF? from Tebu-Bio, Boechout, Belgium, IFN? from RD systems, Abington, UK,
and ApN from Biovendor, Heidelberg, Germany). In some experiments, cells were first
transfected before inflammatory challenge and/or ApN treatment. Briefly, 3.10
⁵
cells/well were transfected with either the On-Targetplus non-targeting pool siRNAs
(negative control, NT siRNAs), or a specific On-Targetplus siRNA SMARTpool against
human AdipoR1 (50 nM) or human SIRT1 (50 nM) or human PGC-1? (70 nM) (all from Dharmacon,
Thermo Fisher Scientific) using 7 ?l Lipofectamine RNAiMAX reagent (Life Technologies)
for 24 h. siRNA silencing was effective, ranging from 70 to 95 % in all experiments.
Next, the medium was renewed and cells were treated with TNF??+?IFN? with or without
ApN for an additional 24 h. At the end of the experiments, cells were collected and
rinsed twice in PBS before RNA extraction.

RNA extraction and real-time quantitative PCR (RT-qPCR)

RNA was isolated from cultured cells and from mouse adipose tissue with TriPure reagent
(Roche Diagnostics, Vilvoorde, Belgium). Of the total RNA, 1–2 ?g were reverse transcribed,
as described previously 21]. RT-qPCR primers were designed for mouse cyclophilin, ApN as reported 21], for interleukin 10 (IL-10; sense, 5?-TGA TGC CCC AGG CAG AGA AGC A-3?; antisense,
3?-GGG GAG AAA TCG ATG ACA GCG CCT C-5?), myosin heavy chain 7 (Myh7; sense, 5?-GGT
GCC AAG GGC CTG AAT GAG GAG-3?; antisense, 3?-GGT CTG AGG GCT TCA CGG GCA C-5?), myosin
heavy chain 1 (Myh1; sense, 5?-AGC TTC AAG TTT GGA CCC ACG GTC G-3?; antisense, 3?-GCA
GCC TCC CCG AAA ACG GC-5?), myosin heavy chain 6 (Myh6/Mrf4; sense, 5?-TGC GGA TTT
CCT GCG CAC CT-3?; antisense, 3?-GCA TCC ACG TTT GCT CCT CCT TCC-5?), myosin heavy
chain 3 (Myh3/eMyHC; sense, 5?-CAG AAA TGG AGA CAC GGA TCA GA-3?; antisense, 3?-AGA
GGT GAA GTC ACG GGT CTT TGC C-5?), myogenin (MyoG; sense, 5?-CCA GCC CAT GGT GCC CAG
TGA A-3?; antisense, 3?-TGT AGG GTC AGC CGC GAG CAA-5?), and utrophin A (sense, 5?-GCC
AGA CAC TAT GAC CCC TCC CA-3?; antisense, 3?-GGG CAT GCT CAT CCT CCA CGC T-5?). RT-qPCR
primers for human TATA box-binding protein (TBP), TNF?, interleukin-6 (IL-6) were
similar to those previously reported 22], for I?B (sense, 5?-TGT GGC AAC TGG AAG CAA GCC C-3?; antisense, 3?-ACC ATG GCC GCA
TCA ATG TGC T-5?), and utrophin A (sense, 5?-AGT TCC ACC AGG CAG CGG GAG-3?; antisense,
3?-ACC TCC GTG AGA TCC GTG CTG G-5?). Cyclophilin (mouse) and TBP (human) were used
as reporter genes. Of the total RNA equivalents, 40–80 ng were amplified using an
iCycler iQ real-time PCR detection system (Bio-Rad Laboratories, Nazareth, Belgium)
21]. The threshold cycles (Ct) were measured in separate tubes and in duplicate. The
identity and purity of the amplified product were checked by electrophoresis on agarose
minigels, and analysis of the melting curve was carried out at the end of the amplification.
To ensure the quality of the measurements, each plate included a negative control
for each set of primers.

Result presentation and statistical analysis

Results are means?±?SD for the indicated number of mice or experiments. Comparisons
between the three groups of mice (WT, mdx, and mdx-ApN) were carried out by one-way
ANOVA followed by Tukey’s test (Prism 5; GraphPad Software, California, USA). When more than three experimental conditions were compared in vitro, the influence
of ApN and that of TNF?/IFN? were assessed by two-way ANOVA with F test, followed
by post hoc two by two comparisons with Bonferroni correction for multiple comparisons (Prism
5). Comparisons between two myotubes conditions from a given subject were made using
two-tailed paired Student’s t test. Differences were considered statistically significant at P??0.05.