ONLINE ONLY MATERIAL
Title:
Atrophy and hypertrophy signalling of the quadriceps and diaphragm in
chronic obstructive pulmonary disease
Authors:
Mariève Doucet1, Annie Dubé1, Denis R. Joanisse1,2, Richard Debigaré1,
Annie Michaud1, Marie-Ève Paré1, Rosaire Vaillancourt1, Éric Fréchette1,
François Maltais1.
From:
1
Centre de recherche, Institut universitaire de cardiologie et de pneumologie
de Québec, Université Laval, Québec, Canada, 2Division de Kinésiologie,
Université Laval, Québec, Canada.
Correspondence to:
Dr François Maltais
Centre de Pneumologie
Institut Universitaire de cardiologie et de pneumologie
de Québec
2725 Chemin Ste-Foy
Québec, QC
G1V 4G5
Tel: 418-656-4747
Fax: 418-656-4762
E-mail:francois.maltais@med.ulaval.ca
Subjects
The diagnosis of COPD was based on a current or past smoking history (> 10 packyear) and pulmonary function testing showing irreversible airflow obstruction (postbronchodilator forced expiratory volume in 1 s [FEV1] < 80% of predicted value and
FEV1/forced vital capacity [FVC] < 70%). Patients with comorbid conditions that could be
associated with muscle wasting such as active inflammatory illnesses, heart failure,
diabetes, systemic corticosteroids, unstable nutritional status and treatment with
chemotherapy or radiotherapy were excluded.
Skeletal Muscle and Data Analysis
Quantitative PCR
All protocols consisted of one denaturing cycle at 90C for 10 minutes, followed by
40 cycles of denaturing at 90C for 30 seconds, annealing at 60C for 60 seconds and
elongation at 72C for 60 seconds followed by final elongation at 72C for 5 minutes. At
the end of the PCR amplifications the samples were subjected to a melting curve analysis.
To control for any variations due to efficiencies of reverse transcription and PCR, acidic
ribosomal phosphoprotein PO (RPLPO or 36B4) was used as an internal control. The
comparative threshold cycles (Ct) values for Atrogin-1, MuRF1 and FoxO-1 study were
normalized for RPLPO reference genes and analyzed using the 2-∆∆Ct method.[1] All qPCR
runs were performed in triplicate to ensure quantitative accuracy. PCR primer sequences
are provided in table 1.
Protein extraction and Western blotting
Cytoplasmic protein extraction was performed with  30 mg of muscle using a
commercial kit according to the manufacturer protocol (NE-PER; Pierce Biotechnology,
Rockford, USA). Muscle samples were homogenised using a Polytron PowerGen 125
(Omni International, Marietta, GA, USA). Protease inhibitor cocktails for mammalian cell
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and tissue extracts (Sigma, Oakville, ON, Canada) and phosphatase inhibitor cocktails I and
II (Sigma, Oakville, ON, Canada) was added to the extraction buffer. Protein content was
determined by a DC protein essay (BioRad, Mississauga, ON, Canada) based on a modified
Lowry method (Lowry and al., 1972). Electrophoresis was performed using 10-15 % SDS
PAGE gels in buffer containing 25 mM Tris pH 8.8, 200 mM glycine and 1 % SDS. After a
3-hour protein transfer in cold (4C) buffer containing 15 mM Tris pH 8.8, 120 mM glycine
and 10 % methanol, nitrocellulose membranes were blocked for 1 hour or overnight with
5% non-fat dry milk in TBS containing 0.1 % Tween-20. The membranes were then
incubated either 1 hour at room temperature or overnight at 4C using the primary
antibody. The membranes were then washed 3 x 20 minutes with TBS containing 0.1 %
Tween-20 and incubated for 1 hour at room temperature with the secondary antibody and
again washed as above. Secondary antibodies used were donkey anti-rabbit IgG (GE
Healthcare, Little Chalfont, Buckinghamshire, UK) and sheep anti-mouse IgG (GE
Healthcare, Little Chalfont, Buckinghamshire, UK) conjugated to horseradish peroxidase.
Finally, the membranes were treated for 1 minute (ECL) or 5 minutes (ECL plus) with
chemiluminescence substrates (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and
an X-ray film (Clonex Corporation, Markham, ON, Canada) was exposed over the
nitrocellulose membranes.
Antibodies and the phosphorylation site recognized along with their dilutions are
shown in table 2.
The antibodies against phosphorylated-p70S6K, total AKT,
phosphorylated-AKT, phosphorylated-GSK3β, phosphorylated-4E-BP1 (Cell Signalling
technology Inc., Danvers, MA, USA), were used as previously described.[2] The antibodies
against Atrogin-1 was developed in our centre as previously reported.[2] Protein content
were normalized to α-tubulin (Sigma, Oakville, ON Canada) as previously described by our
team.[2]
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REFERENCES
1.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative pcr and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-408.
2.
Doucet M, Russell AP, Léger B, et al. Muscle atrophy and hypertrophy signaling in
patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med
2007;176:261-269.
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TABLE E1. PRIMER SEQUENCES USED FOR PCR ANALYSES
Atrogin-1
Forward
Reverse
GCA GCT GAA CAA CAT TCA GAT CAC
CAG CCT CTG CAT GAT GTT CAG T
MuRF1
Forward
Reverse
CCT GAG AGC CAT TGA CTT TGG
CTT CCC TTC TGT GGA CTC TTC CT
FoxO-1
Forward
Reverse
AAG AGC GTG CCC TAC TTC AA
CTG TTG TTG TCC ATG GAT GC
RPLPO
Forward
Reverse
TCT ACA ACC CTG AAG TGC TTG ATA TC
GCA GAC AGA CAC TGG CAA CAT T
Definition of abbreviations : MuRF1 = Muscle ring finger-1, FoxO-1 =
Forkhead transcription factor-1, RPLPO = acidic ribosomal phosphoprotein PO.
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TABLE 2. ANTIBODIES AND THEIR DILUTIONS
Cytoplasmic proteins
Atrogin-1
Total AKT
p-AKT
p-p70S6K
p-GSK3β
p-4E-BP1
Site of
phosphorylation
Ser 473
Thr 389
Ser 21/9
Thr 37/46
Dilution
1:2000
1:5000
1:1000
1:2000
1:1000
1:1000
Control for protein content
α-tubulin
1:20000
Definition of abbreviations : p-AKT = phosphorylated-AKT, p-p70S6K =
phospholylated-70kDa ribosomal S6 Kinase, p-GSK3β = phospholylatedglycogen synthase kinase 3β, p-4E-BP1 = phospholylated-eukaryotic
translation initiation factor 4E binding protein-1.
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