ATROPHY AND HYPERTROPHY SIGNALING IN THE DIAPHRAGM OF PATIENTS WITH COPD
DRIES TESTELMANS, TIM CRUL, KAREN MAES, ANOUK AGTEN, MARK CROMBACH, MARC
DECRAMER AND GHISLAINE GAYAN-RAMIREZ
Online data supplement
MATERIALS AND METHODS
Histochemistry
Serial sections of the diaphragm muscle were cut at 10 µm thickness with a
cryostat maintained at -20°C. Sections were stained with Hematoxylin and Eosin (H&E),
to determine histological changes in the muscles, and were blindly evaluated by an
independent pathologist. Sections were also stained for myofibrillar adenosine
triphosphatase (ATPase) after acid preincubation at pH 4.5 and 4.3. Fibers were identified
as slow-twitch type I and fast-twitch type II fibers. Cross-sectional area (CSA) was
determined from the number of pixels within the outlined borders using a Leitz Laborlux
X. microscope (Wetzlar, Germany) at x20 magnification, connected to a computerized
image system (Quantimet 500, Leica, Cambridge, UK). Around 150 fibers were used to
calculate CSA and proportion of all fiber types.
RNA extraction and real-time quantitative PCR
Total RNA was isolated using the Trizol method as advised by the supplier (Gibco
BRL, Life Technologies, Merelbeke, Belgium). In brief, approximately 50 mg of diaphragm
tissue was homogenized using an Ultra-Turrax homogenizer (Janke & Kunkel, Germany) in
1 ml Trizol Reagent. The homogenized samples were incubated at room temperature for five
minutes and 0.2 ml chloroform was added. After shaking for 15 seconds the samples were
incubated at room temperature for 2 minutes. Samples were centrifuged at 12,000 x g for 15
minutes at 4 ºC and the upper aqueous phase was transferred to a fresh tube. Isopropyl
alcohol (0.5 ml) was added to precipitate the RNA. Samples were incubated at room
temperature for 10 minutes and centrifuged at 12,000 x g for 10 minutes at 4 ºC. RNA pellet
was washed with 75% ethanol and centrifuged at 12,000 x g for 5 minutes at 4 ºC. RNA
pellets were briefly dried and dissolved in RNase free H2O. Quantity of the RNA was
determined by measurement of absorbance at 260 nm and 280 nm.
The reverse transcription (RT) was performed with the Superscript III First-Strand
Synthesis System (Invitrogen, Merelbeke, Belgium) according to the manufacturer’s
protocol, using 3 µg of total RNA and 150 ng of random hexamers. First-strand cDNA was
treated with RNase H.
Quantitative RT-PCR assay was performed on an ABI Prism 7700 Sequence
Detection System, (Applied Biosystems, Foster City, CA, USA) using the Platinum Sybr
Green qPCR Supermix UDG kit (Invitrogen, Merelbeke, Belgium), according to the
manufacturer’s protocol. Markers of the ubiquitin-proteasome pathway (MAFbx, MuRF1,
NEDD4), NF-B pathway (IB and , p50 and p65 subunits), muscle regulatory factors
(MyoD and myogenin), as well as myostatin were targeted in the present study. The primers
(Invitrogen) used for this study are shown in table 2. Expression of the 18S gene was used
to standardize the quantification of target cDNA (ABI Prism 7700 SDS software). Copy
numbers of unknown samples were calculated from plasmid cDNA standards. Data from
each gene was expressed as copy number of 18S.
Protein extraction
Approximately 40 mg of diaphragm were used for dual extractions of nuclear and
cytoplasmic proteins with NE-PER kit according to the company protocol (Pierce
Biotechnology, Erembodegem, Belgium)). Protease inhibitor cocktail (Pierce) was added to
the extraction reagents according to the company protocol. Protein concentration was
determined using the method of Bradford (Biorad, Nazareth, Belgium).
Gel electrophoresis and immunoblotting
Proteins from muscle lysates were denaturated in SDS loading buffer, boiled and
centrifuged briefly. Equal amount of muscle proteins were separated on SDSpolyacrylamide gels (12%) and transferred onto polyvinylidene fluoride (PVDF) membrane.
Membranes were blocked in non-fat dry milk for 1 h and incubated overnight with the
appropriate primary antibody. Histone H3 (Figure E2) (sc8654, Santa Cruz) and -tubulin
(T6074, Sigma) were investigated to confirm proper separation of the nuclear and
cytoplasmatic fractions, respectively. For the detection of the target proteins the following
antibodies were used: anti-human-MyoD polyclonal antibody (sc760, SantaCruz, Boechout,
Belgium), anti-human-myogenin monoclonal antibody (sc13137, Santa Cruz), anti-human
myostatin (ab996, Abcam, Cambridge, UK), anti-human-IBα monoclonal antibody (ab7547,
Abcam), anti-human-IBβ polyclonal antibody (ab32518, Abcam). The anti-human MAFbx
polyclonal antibody was a kind gift from Prof. F Maltais (Quebec, Canada). As secondary
antibodies anti-rabbit coupled with horseradish peroxidase (HRP) and anti-mouse-HRP
(Dako, Heverlee, Belgium) were used. Proteins were visualized with the use of an ECL Plus
detection kit (GE Healthcare, Buckinghamshire, UK), and quantified using Bio 1D (VilberLourmat, France). Equal loading was evaluated with Ponceau S staining for the nuclear
proteins (Figure E1 A, B and C) and data were expressed relative to controls. For the
cytoplasmic proteins, protein expression was normalized with -tubulin and expressed
relative to controls.
NF-B activity
Activation of NF-B p50 and p65 was detected using a microtiter plate assay by
Pierce Biotechnology. Nuclear proteins were incubated in wells coated with doublestranded NF-B consensus oligonucleotides. Bound p50 or p65 was identified with
antibodies for the respective protein and detected with a HRP-coupled secondary antibody
followed by ECL reagent, as recommended by the manufacturer. Luminescence signal was
read in a luminometer (kindly provided by F. Claessens, Labo Legendo).
Figure legends
Figure E1: Representative blots stained with PonceauS, illustrating equal loading for MyoD protein
(A), myogenin protein (B) and Myostatin nuclear protein (C) in the diaphragm of 6 different controls
and 6 different COPD patients.
Figure E2: Western blot illustrating Histone-H3 (16 kDa) expression in the diaphragm of 6 different
controls and 6 different COPD patients.
Figure E1
A
B
C
Figure E2