Experiments were performed in accordance with guidelines of the Canadian Council on Animal Care and Animal Ethics Committee (University Health Network, Toronto, Ontario, Canada). Mice were genotyped using DNA extracted from tail biopsies and the following primers: mgRb/Rblox (forward), 5′-ATTTCAGAAGGTCTGCCAAC-3′ and (reverse) 5′-AGAGCAGGCCAAAAGCCAGGA-3′; Rb mutant (forward), 5′-AATTGCGGCCGCATCTGCATCTTTATCGC-3′ and (reverse) 5′-GAAGAACGAGATCAGCAG-3′; Rb wild type (forward), 5′-AATTGCGGCCGCATCTGCATCTTTATCGC-3′ and (reverse) 5′-CCCATGTTCGGTCCCTAG-3′; Rbfloxed (Rb18 + Rb19E) (forward), 5′-GGCGTGTGCCATCAATG-3′ and (reverse) 5′-CTCAAGAGCTCAGACTCATGG-3′. For timed pregnancies, mice were mated overnight, and the day of vaginal plug observation was considered E0.5.
Primary myoblast cell cultures
Over 1,000 embryos from >200 timed pregnancies were used in this study to generate primary mouse embryonic myoblast cultures. To maintain consistency between experiments, primary myoblasts were induced to differentiate at passage 2. Myoblasts were isolated from skeletal muscle of limbs of E.16.5–17.5 embryos. Tissues were digested for 20 min at 37°C in 80 µl solution containing 1.5 U/ml collagenase IV (Sigma-Aldrich), 2.4 U/ml dispase (Roche), and 5 mM CaCl2
gently triturated and plated onto a 60-mm collagen I–coated culture dish. Myoblasts were maintained in growth medium (GM), HAM’s-F10 (Lonza) supplemented with 20% FBS (PAA), and 2.5 ng/ml basic FGF (Sigma-Aldrich), in a humidified incubator at 5% CO2
and 37°C. To induce differentiation, myoblasts were rinsed once in 1× PBS and shifted to DM, DME, high glucose, and sodium pyruvate (Sigma-Aldrich), supplemented with 3% horse serum (PAA; Ho et al., 2004
). Adenoviruses were amplified in 293T cells maintained in DME plus 10% FBS and penicillin/streptomycin (Sigma-Aldrich). For drug treatment, a single dose of 5 mM 3-MA was added upon differentiation. 500 µM bezafibrate and 200 µM minocycline were refreshed every other day. 40 µM chloroquine was added for 12 h before harvesting the cells. Aphidicolin was used at 2.5 µg/ml. For hypoxia experiments, confluent myoblast cultures were fed DM and transferred hypoxic conditions (1% O2
). 50 µM lonidamine (refreshed every other day; Sigma-Aldrich) and Ad.VHL-T7-HPACGV were added at DM-1.
BrdU DNA synthesis assay
DM-1 myotube cultures were restimulated in GM or maintained in DM supplemented with 20 µM BrdU for 16 h before fixation with 3.7% formaldehyde (10 min). Aphidicolin was added 5 h before addition of BrdU for 16 h. Cultures were permeabilized using 0.3% Triton X-100 for 10 min, treated with 2 N HCl for 25 min, and neutralized with two washes of 0.5 M sodium borate, pH 8.5, for 5 min. After blocking in 1.0% BSA for 20 min and anti-MHC antibody (1:50; Sigma-Aldrich) for 1 h, cells were washed three times for 3 min each with PBS. Secondary antibody fluorescein-conjugated Alexa Fluor 563 (red; Invitrogen) was used. BrdU was detected using anti-BrdU antibody conjugated to FITC (Alexa Fluor 488) and diluted as per manufacturer’s protocol (BD). Confocal images of 0.5-µm sections were captured at room temperature using a 40× or 63× 1.2 NA C Apochromat water objective lens using a confocal microscope (LSM510 META; Carl Zeiss, Inc.) and acquisition software (AIM 3.2; Carl Zeiss, Inc.). Photoshop (CS2; Adobe) was used to overlay images.
Immunofluorescence and EM
500,000 myoblasts were seeded on 22-mm round collagen I–coated coverslips (BD) and induced to differentiate. Cells were fixed in 3.7% formaldehyde, permeabilized in 0.3% Triton X-100, and blocked for 20 min in 1% BSA/PBS at room temperature. The following antibodies and dilutions were used: myogenin, 1:10 (F5D; Santa Cruz Biotechnology, Inc.); MHC, 1:50 (Sigma-Aldrich); MCK, 1:50 (Santa Cruz Biotechnology, Inc.), cytochrome c, 1:50 (clone A-8; Santa Cruz Biotechnology, Inc.). Secondary antibodies fluorescein-conjugated Alexa Fluor 563 and 488 (Invitrogen) were added for 45 min. Nuclei were counterstained with DAPI (Invitrogen) for 10 min and mounted in fluorescent mounting media (Dako). Mitochondrial LC3 (red/green) colocalization was conducted with the Colocalization plug-in for ImageJ (National Institutes of Health). Electron micrographs were captured at 80 kV using a transmission electron microscope (H-7000; Hitachi) equipped with an AMT digital camera (Hitachi) at the University of Toronto. Image contrast was enhanced using Photoshop.
Western blot analysis
Cells were lysed on ice in K4IP buffer (50 mM Hepes, pH 7.5, 0.1% Tween-20, 1 mM EDTA, 2.5 mM EGTA, 150 mM NaCl, 1.0 mM DTT, and 10% glycerol) containing protease inhibitors (Sigma-Aldrich). The following antibodies and dilutions were used for 3 h at room temperature or overnight at 4°C: α/β-tubulin, 1:4,000 (Cell Signaling Technology); MHC, 1:200 (Sigma-Aldrich); LC3b, 1:500 (Cell Signaling Technology); MCK, 1:200 (Santa Cruz Biotechnology, Inc.); pRb, 1:1,000 (BD); troponin T-FS, 1:200 (clone c-18; Santa Cruz Biotechnology, Inc.); poly(ADP-ribose) polymerase, 1:500 (Cell Signaling Technology); and caspase-3, 1:500 (Cell Signaling Technology). Secondary antibodies used were HRP-linked anti-IgG at 1:2,000 (Cell Signaling Technology) for 1.5 h in blocking buffer, and HRP activity was detected using SuperSignal West Dura chemiluminescent substrate (Thermo Fisher Scientific) and captured by x-ray film. Films were digitized using a scanner (Canon). Quantification of Western blots was conducted using Photoshop.
Mitochondrial and nuclear DNA quantitative PCR
Mitochondrial DNA copy number was quantified for an 80-bp fragment of mouse mitochondrial DNA (primers: forward, 5′-GCAAATCCATATTCATCCTTCTCAAC-3′; reverse, 5′-GAGAGATTTTATGGGTGTAATGCGGTG-3′) relative to the NADH dehydrogenase (ubiquinone) flavoprotein 1 single-copy nuclear gene (GenBank/EMBL/DDBJ accession no. NM_133666) (primers: forward, 5′-CTTCCCCACTGGCCTCAAG-3′; reverse, 5′-CCAAAACCCAGTGATCCAGC-3′) using quantitative real-time PCR (7900T lightcycler; Applied Biosystems).
ATP, MitoTracker red CMXRos, and TUNEL assays
ATP quantification was performed using a luminescence detection system according to the manufacturer’s protocol (ATPlite; PerkinElmer). Mitochondria membrane potential was detected using MitoTracker red CMXRos according to the manufacturer’s protocol (Molecular Probes). For TUNEL, differentiating myoblasts on collagen I–coated coverslips were fixed in 3.7% formaldehyde for 10 min, washed three times in PBS, permeabilized with 0.3% Triton X-100 solution, and washed three times in PBS. Subsequently, 30 U TdT (Fermentas) was added to 50 µl TUNEL label solution (Roche).
Adenovirus Cre infections
For adenovirus Cre (Ad.cre; Vector Biolabs) infection of Rbf/f myoblasts, cells were transduced at a multiplicity of infection of 1,300. At a lower multiplicity of infection (<1,000), some pRb-positive nuclei within myotubes were still detected. 48 h after transduction, myoblasts were induced to differentiate. For all other recombinant adenoviruses, transduction was performed when cultures were induced to differentiate. The following adenovirus vectors were provided by the indicated sources: Ad.Bcl-2 (M. Crescenzi, Higher Institute of Health, Rome, Italy), Ad.GFP-LC3 and Ad.RFP-LC3 (A.M. Tolkovsky, University of Cambridge, Cambridge, England, UK), Ad.HIF-1α/NF-κB and Ad.EV (K.A. Vincent, Genzyme Corporation, Framingham, MA), Ad.VHL-T7-HPACGV (M. Ohh, University of Toronto, Toronto, Ontario, Canada), Ad.Vps34DN (D. Murphy, University of Bristol, Bristol, England, UK), Ad.mTOR, Ad.KD.mTOR, and Ad.ca.mTOR (C.J. Rhodes, The University of Chicago, Chicago, IL), Ad.FOXO3a and Ad.DN.FOXO3a (K. Walsh, Boston University School of Medicine, Boston, MA), Ad.RbΔK11 and Ad.p27 (D.S. Park, University of Ottawa, Ottawa, Ontario, Canada), and Ad.XIAP and anti-XIAP antibody (R.G. Korneluk, University of Ottawa, Ottawa, Ontario, Canada).
Brightfield images and videos
Brightfield images and videos were captured at room temperature using 20× or 40× air objective lenses on a microscope (TE200; Nikon) fitted with a charge-coupled device digital camera (Hamamatsu Photonics). Images were acquired using imaging software (SimplePCI; Hamamatsu Photonics). Photoshop was used to enhance clarity and contrast using same parameters for control and experimental samples.
Online supplemental material
Fig. S1 shows characterization of myogenic defects during abortive differentiation of Rb−/−
myoblasts. Fig. S2 shows that cell cycle inhibitors aphidicolin and p27Kip1
accelerate the degeneration of Rb−/−
myotubes. Fig. S3 shows differential effects of mTOR, Foxo3, minocycline, and XIAP on differentiation of Rb−/−
myoblasts. Video 1 shows twitching control myotubes (Ad.Bcl-2 transduced), Video 2 shows twitching Rb−/−
myotubes (Ad.Bcl-2 transduced), Video 3 shows twitching control myotubes (3-MA treated), Video 4 shows twitching Rb−/−
myotubes (3-MA treated), Video 5 shows twitching control Ad.EV-transduced Rbf/f
myotubes (hypoxia), and Video 6 shows twitching RbΔf
myotubes (hypoxia). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201005067/DC1