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On line data depository
Effect of pulmonary rehabilitation on muscle remodelling in cachectic
patients with COPD
Authors: Ioannis Vogiatzis, Davina C. M. Simoes, Grigoris Stratakos, Evangelia Kourepini,
Gerasimos Terzis, Panagiota Manta, Dimitrios Athanasopoulos, Charis Roussos, Peter D. Wagner,
and Spyros Zakynthinos.
METHODS
Pulmonary rehabilitation programme
The rehabilitation program was multidisciplinary and included supervised exercise training,
breathing control and relaxation techniques, disease education, dietary advice, and psychological
support on issues relating to chronic disability. All patients performed interval exercise at intervals
of 30-s work alternating with 30-s rest as previously described [1-4]. Exercise was performed on
electromagnetically braked ergometers (CatEye-Ergociser, EC-1600; Osaka, Japan) for 45 min/day,
3 days/week for 10 weeks. The workload for both non-cachectic and cachectic patients was initially
set at 90% of the individual patient’s peak work rate (assessed at baseline during an incremental
cycle ergometer test) and it was subsequently increased on a weekly basis so as to present both noncachectic and cachectic patients with a similar overall training load throughout rehabilitation.
Muscle biopsy analysis
Previous work [5] has demonstrated that the mRNA content of both IGF-I and MyoD 24 h after an
exercise bout is not different compared to that before the exercise bout. As such, vastus lateralis
muscle percutaneous biopsies were obtained 24 h after the first (baseline) and 24 h after the last
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(Post) training sessions. Muscle percutaneous biopsies of the right vastus lateralis muscle were
performed at mid-thigh (15 cm above the patella). Muscle samples were cut in two pieces. One was
placed immediately into liquid nitrogen and the other was aligned under a stereoscope in order to
have most of the fibres in parallel. The muscle sample was placed in embedding compound and
frozen in isopentane pre-cooled to its freezing point. All biopsies were kept at -80oC until the day of
analysis. Cryostat transverse sections of 10 m thick from the embedded samples were cut at -20oC
and were stained for myofibrillar ATPase after pre-incubation at pH 4.3, 4.6 and 10.3 [6]. A mean
of 426 ± 43 muscle fibers were classified as type I, IIa or IIb from each sample. The cross sectional
area of at least 300 fibers from each sample was measured with an image analysis system
(ImagePro, Media Cybernetics Inc, Silver Spring, MD, USA) at a known and calibrated
magnification. The mean cross-sectional area of all fiber-types was calculated taking into account
the relative contribution of each fibre type to total surface [7]. A-amylase-periodic acid shift was
used to visualize capillaries. The number of capillaries identified in a certain area was divided by
the number of fibres found in the corresponding muscle section [8].
RNA isolation and cDNA synthesis
Total RNA was extracted from 30 mg of muscle biopsies using an RNeasy Fibrous Tissue (Qiagen,
West Sussex, UK). To eliminate residual genomic DNA, the RNA samples were treated with
DNAse I. Subsequently, total RNA was quantified (260 nm) and adjusted to a concentration of
1μg/μl. Reverse transcription was performed using 1 μg of total RNA from each sample, using a
SuperScript First–Strand Synthesis System (Invitrogen, Carlsbad, CA) for cDNA synthesis
according to the manufacturer’s instructions.
Quantitative real-time PCR
Two μl of each cDNA sample were used as template for the amplification reaction. Each PCR
reaction included 2.5 μl of 10x PCR buffer with 2.5 mM MgCl2, 80 nM dNTPs, 0.3 μM of each
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primer, 2.5 units of Platinum Taq Polymerase and SYBR Green I at final concentration of 0.1x.
Primer sequences for TNF-α, Myostatin, IGF-I, MGF, MyoD, and Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) are given in Table 1. Primers were designed using the
http://www.genome.wi.mit.edu/cgibin/primer/primer3_www.cgi_http://biotools.idtdna.com/biotools
/ mfold/mfold.asp web sites.
Quantification utilized standard curves of plasmids constructs containing partial sequences of genes
amplified with primers as described in Table 2. Transcripts were cloned into pCR-II-TOPO vector
(TOPO TA Cloning, Invitrogen, Carlsbad, CA). Their sequences were confirmed against database
(GeneBank Accession No: NM_004862.2, NM_005259.1, NM_000618.3, U40870, NM_002478.4,
NM_002046). Absolute number of copies was calculated using a standard curve subjected to 10fold dilutions, containing from 108 to 100 copies/ μl. PCR amplifications of each gene and GAPDH
were performed in triplicates alongside with their respective standard curves in a Chromo4 Detector
and PTC-200 Peltier Thermal Cycler and analysed with Opticon software 2.03 (MJ Research,
Massachusetts, USA). Expression of GAPDH mRNA was used for normalization.
Muscle protein immunoblotting
Vastus lateralis muscle biopsies stored at -80ºC were homogenized in 10 volumes (wt/vol) of a lysis
buffer containing protease inhibitor cocktail (Complete Mini, Roche Diagnostics, Mannheim
Germany) and phosphatase inhibitor cocktail (Complete PhosSTOP, Roche Diagnostics, Mannheim
Germany). Protein concentration was determined by using DC protein assay (BioRad, Hercules,
CA). Samples were subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE), followed
by transfer to a polyvinylidene fluoride membrane (PVDF) (Millipore Corp. Bedford, MA).
Immunoblotting was carried out by using primary antibodies raised against human TNF-a (1:1000),
IGF-I (1:200) and MyoD (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA); Myostatin (1:2500)
(GeneTex Inc, San Antonio, USA); Akt (1:1000), phospho -Akt (Ser473) (1:1000), IκB-α (1:1000),
and phospho -IκB-α (Ser32) (1:1000) (Cell Signaling Technology Inc, Danvers, MA.); Atrogin-1
(1:200) (Santa Cruz Biotechnology, Santa Cruz, CA); MuRF-1 (1:200) (Santa Cruz Biotechnology,
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Santa Cruz, CA); Nitrotyrosin (1:1000) (Cayman Chemical). Membranes were incubated with
horseradish peroxidase (HRP) -linked anti- rabbit IgG (1:5000) or anti-goat (1:5000, Santa Cruz
Biotechnology, Santa Cruz, CA) secondary antibodies. Since it was impossible to load samples of
pre- and post-rehabilitation from all patients in one single gel, several precautions were taken to
allow us to compare the gels. The precautions were as follows: protein extraction and quantification
from all biopsies were carried out at the same day, followed by denaturation. Aliquots were made
and kept at -80°C for few days until all gels and membranes were prepared following standardized
protocols. A control protein was analysed in all the membranes studied to allow for loading
normalization. In addition, a control skeletal muscle sample from a healthy aged-matched subject
was placed in all the gels for allowing normalisation among gels.
To validate protein content, immunoblots were normalized with a monoclonal anti-sarcomeric αactinin antibody (1:1000, Sigma-Aldrich, St. Louis, MO) and with a secondary HRP-linked antimouse Ab (1:10000, Cell Signalling Technology Inc, Danvers, MA.). Bands were visualized using
Chemiluminescent Substrate or SuperSignal West Femto, Maximum Sensitivity Substrate (Pierce,
Rockford, IL). Data was digitalised and quantified by densitometric analysis.
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Table 1: Sequences of PCR primers used for amplification and sequencing.
Target
Forward primer (5’/3’)
Reverse primer (5’/3’)
mRNA
TNF-α
Myostatin
IGF-I
MGF
MyoD
GAPDH
GeneBank
Ref.
Accession No.
TGTGTTGTCGTCCT
CTTGTAGGTGCCCA
TCCTGCAAC
GGAGAG
ACCATGCCTACAG
GATTCAGGTTGTTT
AGTCTGA
GAGCCAA
ATCGTGATGAGTGC GAGGGTCTTCCTAC
TGCTTC
ATCCTG
AGCTCGCTCTGTCC
GAGACTTCGTGTTC
GTGC
TTGTT
TGCCACAACGGAC
CGGTCCAGGTCTTC
GACTTC
GAA
GAAGGTGAAGGTC
CATGGGTGGAATC
GGAGT
ATATTGGA
NM_004862.2
4
NM_005259.1
*
NM_000618.3
*
U40870
*
NM_002478.4
5
NM_002046
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Definition of abbreviations: TNF-α: tumor necrosis factor-alpha; IGF-I: insulin-like growth factor-I;
MGF: mechano growth factor; MyoD: myogenic differentiation factor D and GAPDH:
glyceraldehyde-3-phosphate dehydrogenase. * see section of quantitative real-time PCR for
information on primer design.
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Table 2: Sequences of PCR primers used for cloning partial sequences of each gene in pCR IITOPO vector.
Gene
TNF-α
Myostatin
IGF-I
MGF
MyoD
GAPDH
Forward primer (5’/3’)
Reverse primer (5’/3)
GeneBank
for TOPO cloning
for TOPO cloning
Accession No.
TGGACCGCCCTATCC
ACGTCTGGCTGAGT
AAATGT
CCTACA
AGTGGATGGAAAAC
TCAATGCTCTGCCA
CCAAAT
AATACC
ATCGTGATGAGTGCT
GAGGGTCTTCCTAC
GCTTC
ATCCTG
AGCTCGCTCTGTCCG
GAGACTTCGTGTTC
TGC
TTGTT
GACGGCTCTCTCTGC
GGAAGTGCGAGTG
TCCTT
CTCTT
GAAGGTGAAGGTCG
CATGGGTGGAATC
GAGT
ATATTGGA
Ref.
NM_004862.2
*
NM_005259.1
*
NM_000618.3
*
U40870
*
NM_002478.4
*
NM_002046
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Definition of abbreviations: TNF-α: tumor necrosis factor-alpha; IGF-I: insulin-like growth factor-I
(IGF-I); MGF: mechano growth factor; MyoD: myogenic differentiation factor D and GAPDH:
glyceraldehyde-3-phosphate dehydrogenase. * see section of quantitative real-time PCR for
information on primer design.
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