Cell culture and treatment
Human skeletal muscle cells (HuSKMCs; Lonza AG, Basel, Switzerland) were cultured in growth medium (GM) consisting of skeletal muscle basal medium (skBM; Lonza) supplemented with 20% FCS (PAA Laboratories, Dartmouth, PA, USA). Differentiation was initiated 24 to 48 hours after seeding by changing to a serum-free differentiation medium (DM), skBM. For small interfering (si)RNA experiments, cells were transfected 24 hours after seeding in GM, and differentiation was initiated after another 24 hours.
To determine NFκB activity, HuSKMCs were infected 24 hours after seeding with human recombinant adenovirus-NFκB-luciferase (Vector Laboratories Inc., Burlingame, CA, USA) in GM for 48 hours, then the medium was removed and the cells stimulated for another 6 hours in serum-free skBM with the compounds under investigation To assess the effects on HuSKMC differentiation, the assessed compounds were added at the onset of differentiation, and cells were differentiated into myotubes for up to 120 hours. To measure TGF-β reporter-gene activity in supernatants (SNs) from differentiating HuSKMCs, a reporter-gene assay (RGA) was used. HEK293T cells stably transfected with pGL3-CAGA12-luc were seeded in serum-reduced medium (2% FCS) for 24 hours, then the medium was removed, and the cells stimulated with a 10:1 mixture of supernatant and serum-enriched medium (20% FCS) in the absence or presence of 500 ng/ml of a human Fc-TGF-β RIIb/Fc chimera (TGF-βRIIb) alone or in combination with neutralizing anti-Activin A antibody (αActA) for another 24 hours.
The following reagents were used: human IL-1α), IL-1β, TNF-α, TGF-βRIIb, andαActA (all R&D Systems Inc., Minneapolis, MN, USA), long-R3-iIGF-1, SB431542 (both Sigma-Aldrich, St Louis, MO, USA), SB203580, withaferin A (both Tocris Bioscience, Ellsville, MO, USA), and TAK-1 inhibitor (AnalytiCon Discovery AG, Potsdam, Germany). Stock solutions were prepared either in PBS supplemented with 0.1% BSA for Fc-TGF-βRIIBb, TNF-α and IL-1α, in 10 mmol/l HCl for IGF-1 or in dimethyl sulfoxide (DMSO) for TAK-1 inhibitor, SB431542, SB203580, and withaferin A. For immunostaining, a primary antibody against myosin heavy chain (MyHC) was used (clone A4.1025; Upstate Biotechnology, Lake Placid, NY, USA), and the secondary antibody was conjugated to a fluorescent dye (Alexa Fluor® 488 F (AB'); Invitrogen Corp., Carlsbad, CA, USA).
Primary antibodies against phospho-TAK-1 (Thr 184/187), phospho-SEK/MKK4 (Thr241), phospho-p38MAPK (Thr 180/Tyr182), phospho-c-Jun (Ser63), phospho-activating transcription factor (ATF)2 (Thr71), phospho-NFκB p65 (Ser536), phospho-SMAD2 (Ser465/467), phospho-AKT (Ser473) (all Cell Signaling Technology, Beverly, MA, USA), and phospho-SMAD3 (Ser423/425; Millipore Corp., Billerica, MA, USA) were used for western blotting. The loading control was α-tubulin (Sigma), and the secondary antibodies were labelled with horseradish peroxidase (HRP) (goat anti-rabbit IgG HRP and goat anti-mouse IgG HRP; Cell Signaling Technology).
Lysis buffer consisting of extraction reagent (Phosphosafe; Novagen Inc., Madison, WI, USA) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich) was added. Homogenates were separated by centrifugation for 10 minutes at 4°C (14,000 rpm). Supernatants were collected and protein contents measured a commercial kit for protein determination (BCA Kit; Sigma-Aldrich). Samples were diluted in SDS-PAGE sample buffer and denatured for 5 minutes at 95°C. Equal amounts of protein were loaded per lane of 4 to 12% polyacrylamide gel (NuPAGE Bis-Tris gel; Invitrogen Corp., Carlsbad, CA, USA), separated by electrophoresis, and then transferred onto nitrocellulose membranes. Membranes were blocked in TBS with 5% w/v non-fat milk powder. Primary antibodies were incubated in TBS with 0.1% Tween 20 and 5% BSA with the exception of phospho-SMAD2 (incubated with 5% non-fat milk), and secondary antibodies in TBS with 0.1% Tween 20, 0.05% SDS and 5% non-fat milk. Immunoreactivity was detected by SuperSignal West Femto Maximum Sensitivity Substrate and exposed to film.
RNA was isolated using commercial kits (RNEasy Mini Kit, RNEasy MinElute Kit and RNase-Free DNase Set; Qiagen Inc., Valencia, CA, USA), following the manufacturer's protocol. Samples were prepared for reverse transcription PCR (QuantiFAST Multiplex RT-PCR+R Kit; Qiagen Inc.) and run in an automated PCR machine (7500 FAST Real-time PCR machine; Applied Biosystems, Foster City, CA, USA). A TaqMan probe set for the Activin A β-chain was used (Inhibin β A; Rn00567500_m1: rat; Hs01081598_m1: human; Applied Biosystems, Foster City, CA, USA).
Small interfering RNA
The siRNA pools used were designed against SMAD2, SMAD3, the Activin A β-chain (Inhibin β A), and a non-targeting control (NTC) targeting an unknown mammalian sequence together with transfection reagent (DharmaFECT 1; Dharmacon/Thermo Fisher Scientific Inc., Rockford, IL, USA) were used, to give a final siRNA concentration of 100 nmol/L.
After washing with cytoskeleton stabilizing buffer (CSB; 80 mmol/l PIPES, 5 mmol/l EGTA, 1 mmol/l MgCl2, 40 g/l PEG3500 in distilled water at pH7.4), cells were fixed with 4% paraformaldehyde in CSB. Cells were then permeabilized with 0.2% Triton in CSB, and non-specific binding blocked with normal goat serum (Zymed Laboratories Inc., South San Francisco, CA, USA) followed by incubation with MyHC diluted in PBS and subsequently with fluorescent dye (Alexa Fluor® 488 F (AB'); Invitrogen Corp.) diluted in PBS. Cells wre mounted with in an antifade reagent with DAPI (ProLong Gold; Invitrogen Corp.).
HEK293T cells stably transfected with the TGF-β responsive construct CAGA12-luc cloned into the reporter construct pGL3 (Promega Corp., Madison, WI, USA) were kindly provided by C. Lu (Developmental and Molecular Pathways, Novartis Pharma AG, Basel, Switzerland). Infection of HuSKMCs was performed using the human recombinant adenovirus-NFκB-luciferase reporter (Vector Laboratories Inc., Burlingame, CA, USA). Reporter-gene activity was measured using Britelite Plus (Perkin Elmer, Waltham, MA, USA), and chemiluminescence was read using a spectrophotometer (Spectramax M5; Molecular Devices Inc., Sunnyvale, CA, USA).
Creatine kinase activity assay
Cells were washed three times with PBS and then lysed with reporter lysis buffer (Promega Corp.) and stored at -80°C until measurement. Creatinine kinase (CK) activity was measured using a commercial reagent (CK (IFCC) Reagent; Thermo Electron, Waltham, MA, USA), prepared according to the manufacturer's instructions. Lysates were adjusted to room temperature, CK reagent was added, and absorbance was immediately read at 340 nm for 20 minutes with a reading interval of 1 minute. CK standard curves were freshly prepared using CK from rabbit muscle (Roche Diagnostics, Basel, Switzerland). Protein content was determined using a commercial (BCA Kit; Sigma-Aldrich), as before.
Activin A ELISA
ELISA for Activin A (Human Activin A DuoSet; R&D Systems Inc.) was performed according to the manufacturer's instructions, using a modified chemiluminescent measurement. Briefly, plates were coated with capture antibodies overnight at 4°C, and non-specific binding was blocked with PBS plus 1% BSA for at least 1 hour. Supernatants and 6M Urea (1:1) were added and incubated for 2 hours at room temperature. Detection antibody was added for 2 hours at room temperature followed by incubation with HRP-labeled streptavidin for 20 minutes at room temperature. After addition of a chemiluminescent substrate (SuperSignal ELISA Femto Maximum Sensitivity substrate (PierceBiotechnology Inc., Rockford, Illinois, USA) chemiluminescence was read using a spectrophotometer (Spectramax M5; Molecular Devices Inc.).
In vivo experiments
All animal procedures were approved by the cantonal veterinarian office of Basel (license number 2127).
Male Wistar rats (Janvier, Le Genest Saint Isle, France) of various ages (4 per group) were housed for 2 weeks on a 12 hour light/dark cycle with unrestricted access to food and water. Rats were asphyxiated using CO2 at 6, 18, 21 and 24 months (29, 81, 93 and 106 weeks) of age, and the gastrocnemius muscle was immediately dissected, weighed and snap-frozen in liquid nitrogen before processing for RNA extraction. Compared with 6-month-old rats, the gastrocnemius weight was unchanged for 18-month-old rats and reduced by 41% and 49% in 21- and 24-month-old rats, respectively.
Differences between groups were analyzed using one-way ANOVA for all experiments. P < 0.05 was considered significant.