Animals and in vivo treatments
Normal wild-type C57/BL6, C57Bl6 mdx mice and Nod/SCID mice were purchased from the Jackson Laboratories. Ubiquitin-GFP mice were a kind gift from G. Cossu; EGFP transgenic mice (a kind gift from Dr. Bill Stallcup) and a L2G85 (FLuc) strain ubiquitously expressing luciferase from the ACTB promoter were used to generate the double-transgenic animals. Animals were used at the specified age and treated for the indicated periods with daily i.p. injections of Trichostatin A, TSA (0.6 mg/kg/day; Sigma), dissolved in saline solution, or saline alone as control (Ctrl). Mice were bred and maintained according to the standard animal facility procedures, and all experimental protocols were approved by the internal Animal Research Ethical Committee according to the Italian Ministry of Health and complied with the NIH Guide for the Care and Use of Laboratory Animals.
Cell preparation and FACS isolation
For fluorescence-activated cell sorting, hind limb muscles were minced and digested in HBSS (Gibco) containing 2 µg/ml Collagenase A (Roche), 2.4 U/ml Dispase I (Roche), 10 ng/ml DNase I (Roche), 0.4 mM CaCl2 and 5 mM MgCl2 for 90 min at 37°C. Cells were stained with primary antibodies (10 ng/ml) CD31-PacificBlue (Invitrogen), CD45-eFluor450 (eBioscience), Ter119-eFluor450 (eBioscience), CD11b-Pacific blue, Sca-1-FITC (BD Pharmingen), CD34-Biotin (eBioscience) and α7integrin-APC (kindly provided by Dr. Fabio Rossi) for 30 min on ice. A subsequent incubation, 30 min on ice, with Streptavidin-PE-Cy7 (1/500; BD Pharmingen) was performed. Cells were finally washed and resuspended in HBSS containing 0.2% w/v BSA and 1% v/v Penicillin–Streptomycin. Flow cytometry analysis and cell sorting were performed on a DAKO-Cytomation MoFlo High Speed Sorter.
Muscle satellite cells (MuSCs) were isolated as Ter119−/CD45−/CD31−/CD34+/α7-integrin+/Sca-1− cells; FAPs cells were isolated as Ter119−/CD45−/CD31−/CD34+/α7-integrin−/Sca-1+ cells. FACS procedure in which CD11b was used in place of Ter119 gave rise to an identical population, with indistinguishable phenotype, biological activity and response to TSA.
Culture conditions of FAPs and MuSCs
Freshly sorted cells were plated on 0.1% gelatin-coated dishes in BIOAMF-2 complete medium (ATGC) as a growth medium (GM). For adipogenic differentiation, after 7 days of GM, cells were exposed for 3 days to adipogenic induction medium consisting of DMEM with 10% FBS (Gibco), 0.5 mM IBMX (Sigma), 0.25 µM dexamethasone (Sigma) and 10 µg/ml insulin (Sigma), followed by further 3 days in adipogenic maintenance medium, consisting of DMEM with 10% FBS and 10 µg/ml insulin. For TSA in vitro
treatment, cells were treated for 24 h with 50 nM TSA in GM and then switched in adipogenic DM without the drug. Cell culture inserts with 1.0-µm pore and 6-well culture plates (BD bioscience) were used for transwell co-culture. Inserts and plates were coated with 0.1% gelatin. 1 × 104
freshly sorted MuSCs were plated in the bottom of the plate, while 1 × 104
FAPs cells were plated on the upper insert. Transwell co-cultures were maintained in GM for 7 days and then harvested for analyses. Where specified, FAPs were exposed to a neutralizing antibody against Follistatin (R&D) at the final concentration of 4 µg/ml (Iezzi et al, 2004
Downregulation of Follistatin expression in FAPs was performed by transfection with lipofecatamin 2000 (Invitrogen) of a shRNAi against follistatin (shFollistatin; Iezzi et al, 2004
), pSuper empty vector was used as ctrl.
Muscle injury and transplantation experiments
Muscle injury was performed by intramuscular injection of 10 µg/ml notexin (NTX, Sigma). For transplantation experiments in Nod/SCID mice, recipient animals were anaesthetized via isoflurane inhalation and received 10 µl intramuscular injections into the tibialis anterior of either 10 µg/ml notexin for local tissue injury, or freshly isolated muscle stem cells (MuSC) and FAPs cells resuspended in PBS.
For transplantation experiments in mdx mice, recipient animals were anaesthetized via isoflurane inhalation and received intramuscular injections into the tibialis anterior of 10 µL NTX (10-5M) for local tissue injury. Twenty-four hours post injury freshly FACS isolated cells (MuSCs and/or FAPs) resuspended in PBS were then injected intramuscularly.
The paper explained
This manuscript elucidates the role of distinct cellular components of the skeletal muscle repair machinery in the pathogenesis of Duchenne Muscular Dystrophy (DMD), and indicates the biological rationale for the pharmacological correction by HDAC inhibitors (HDACi) of a key pathogenic event in the disease progression – i.e., the functional exhaustion of the regeneration potential of muscle satellite cells and the parallel fibroadipogenic degeneration.
The data demonstrate that a population of muscle interstitial cells that is distinct from satellite cells and has previously been defined to as FibroAdipogenic Progenitors (FAPs), contribute to DMD pathogenesis by progressively failing to support satellite cell-mediated regeneration of dystrophic muscles in mdx mice.
The functional interactions between FAPs and satellite cells are mediated by soluble factors, such as follistatin, a previously identified as a target of HDACi. The functional status of FAPs appears to determine the ability of HDACi to promote regeneration and prevent fibro-adipogenic degeneration in young, but not old, dystrophic muscles.
The information derived by this study will have an impact on the immediate translation of HDACi in the pharmacological treatment of DMD patients, as it indicates cellular targets and mechanism of action that will help to the identification of new biomarkers for patient selection in clinical trials and for monitoring the efficacy of the intervention.
Histology and immunofluorescence
Tibialis anterior muscles were snap frozen in liquid nitrogen-cooled isopentane and then cutted transversally. Cryosections (8 µm) and cultured cells were fixed in 4% PFA for 20 min and permeabilized with 100% methanol for 6 min at −20°C. To avoid unspecific binding, muscle sections were first blocked with a solution containing 4% BSA in PBS and then with anti-mouse AffiniPure Fab fragment (Jackson, 1:100). Immunostaining with primary antibodies was performed overnight at 4°C. Antibody binding was revealed using species-specific secondary antibodies coupled to Alexa Fluor 488 or 543 (Molecular Probes). Nuclei were visualized by counterstaining with DAPI. Primary antibodies used were against: Laminin (Sigma; 1:100), embryonic MyHC (Developmental Studies Hybridoma Bank, DSHB; 1:20), MF-20 (DSHB, 1:20), MyoD (Santa Cruz, 1:20), GFP (Invitrogen, 1:500). To stain lipids, 10% formalin fixed cells and tissues were rinsed with water and then with 60% isopropanol, stained with Oil red O in 60% isopropanol and rinsed with water. Images were acquired with a Leica confocal microscope and edited using the Photoshop software. Fields reported in the figures are representative of all examined fields. For Hematoxylin and Eosin staining, cryosections were fixed in 4% PFA, then washed in PBS-1X and then they were stained in haematoxylin for 4 min and eosin for 6 min. Then the cryosections were dried in ethanol and at the end they were fixed in xylene and mounted with EUKITT mounting (O. Kindler GmbH & CO). To stain fibrotic tissue Masson's trichrome analyses was used. Muscle cryosections were stained in Working Weigert's Iron Hematoxilin Solution for 5 min, washed in running tap water for 5 min and stained in Biebrich Scarlet-Acid Fucsin for 5 min. Rinsed in de-ionized water, placed in Working Phosphotungstic\Phosphomolybdic acid solution for 5 min, stained in Aniline Blue solution for 5 min and in acid acetic 1% for 2 min. The slides were mounted with EUKITT mounting. For immunofluorescence after cell transplantation, tibialis anterior muscles were excised and frozen prior to the collection of transverse muscle sections using a Leica CM 3050S cryostat. To identify donor-derived GFP+ myofibres, sections were incubated in the following primary antibodies: rabbit anti-GFP (Invitrogen), rat anti-laminin (Millipore) and Hoechst 33258 (Invitrogen) for the identification of nuclei.
Images were acquired using an inverted fluorescent microscope (Nikon TE300), 10× objective lens, CCD SPOT RT camera and SPOT imaging software (Diagnostic Instruments, Inc.). Images were composed, edited and modifications applied to the whole image using Photoshop CS4 (Adobe).
The cross-sectional area (CSA) was calculated using the ImageJ software downloaded from http://rsb.info.nih.gov/ij
. Fibrotic areas were measured by selecting four representative and non-adjacent sections and photographing up to three microscopic fields. The total fibrosis was calculated from sections evaluating image analysis algorithms for colour deconvolution. ImageJ was used for image processing, the original image was segmented with three clusters and the plugin assumes images generated by colour subtraction (white representing background, blue collagen, and magenta non collagen regions; (Krajewska et al, 2009
; Ruifrok & Johnston, 2001
). Oil Red O areas were quantified by selecting four representative and non-adjacent sections and photographing up to three microscopic fields. Oil red O areas were measured by using ImageJ, calculating the area of red pixels (pixel2
) per field. To assess the efficiency of transplantation, 8-µm thick transverse sections were obtained at 100 µm intervals throughout the muscle and then stained. The total number of GFP+
myofibres per transplanted muscle was recorded. Statistical significance was determined by the student t
Non-invasive bioluminescence imaging
Recipient mice were anaesthetized via isoflurane inhalation and imaged using a Xenogen IVIS 200 device. Briefly, the system contains a light-tight imaging chamber, a charge-coupled device camera and the appropriate computer system equipped with Living Image 3.2 software. Following intraperitoneal injection of 0.1 mmol/g body weight luciferin, images were acquired every 60 s for 15 min. In subsequent analysis, images containing peak luminescence values over this time period were used.
Total RNA was extracted with Trizol, and 0.5–1 µg were retrotranscribed using the Taqman reverse transcription kit (Applied Biosystems). Real time quantitative PCR was performed to analyse relative gene expression levels using SYBR Green Master mix (Applied Biosystems) following manufacturer indications. Relative expression values were normalized to the housekeeping gene GAPDH.
Primers sequences are as follow:
- Fw: 5′-CACCATCTTCCAGGAGCGAG-3′
- Rev: 5′-CCTTCTCCATGGTGGTGAAGAC-3′
- Fw: 5′-CAATAAACTGCGGGCAAAGAC-3′
- Rev: 5′-CTTGCTCACTCCTCGCTTTCA-3′
- Fw: 5′-GAACTTGAAGGAGAGGTCGA-3′
- Rev: 5′-GAGCACATTCTTGCGGTCTT-3′
- Fwd: 5′-AGTCCTACACGGTCTTCAAGG-3′
- Rev: 5′-AGGAAGTGGTCATCAATGAGC-3′
- Fwd: 5′-GCCTGCCACCTGAGAAAGG-3′
- Rev : 5′-CGCCACACTGGATATCTTCACA-3′
Data are presented as mean ± SEM. Comparisons between two groups were made using the Student's t-test assuming a two-tailed distribution, with significance being defined as *p < 0.05; **p < 0.01; ***p < 0.001. Comparisons between three or more groups were made using one-way Anova test, with Tukey's multiple comparisons post-test and with significance being defined as *p < 0.05; **p < 0.01; ***p < 0.001.