Tissue specimens and patient-derived cell lines

After institutional ethics board approval (CHEO Research Ethics Board, protocol CHEOREB# 19/49X), archived frozen pediatric muscle samples were obtained, as secondary use of clinical samples, from the Department of Pathology and Laboratory Medicine, Children’s Hospital of Eastern Ontario (CHEO), Ottawa, Ontario, Canada. Experiments were carried out in accordance with the CHEO Ethics Board guidelines and regulations.

Immortalized human skeletal muscle myoblasts (HSMM) generated from biopsies obtained from 8 DMD patients and 3 controls were acquired from Dr. Bénédicte Chazaud and previously described in Massenet et al. [31]. HSMM were cultured in Ham’s F14 media supplemented with 30% FBS, 10 µg/ml of insulin (16634, Sigma-Aldrich, St. Louis MO, USA), 25 ng/ml FGF-2 (SRP4037, Sigma-Aldrich), 10 ng/ml hEGF (E9644, Sigma-Aldrich), 2 µg/ml amphotericin B (A2942, Sigma-Aldrich), and 1% penicillin/streptomycin at 37 °C, 5% CO2.

Transfection

Human skeletal muscle myoblasts (HSMM) on collagen-coated 35 mm dishes were transfected with 5 nM of Silencer® Select siRNA targeting PANX1 (5’ CGAUCAGUUUCAGUGCAAAtt 3’) or 5 nM of the Silencer® Select Negative Control No. 1 (ambion 4390843, Thermo Fisher Scientific, Carlsbad CA, USA) using Lipofectamine 2000 (Thermo Fisher Scientific) as per the manufacturer’s protocol. Dye uptake assays were performed 48 h post-transfection. Once completed, cell lysates were collected to confirm successful knockdown via western blotting.

Dye uptake assays

The low [K+] solution (145 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 10 mM HEPES diluted in ddH2O) and high [K+] solution (60 mM NaCl, 50 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 10 mM HEPES diluted in ddH2O) were prepared as described by [41]. Cells were washed twice with either the low [K+] or high [K+] solution, then incubated in fresh low [K+] or high [K+] solution for 5 min at 37 °C, in 5% CO2. Next, cells were incubated with 4 mg/ml of sulforhodamine B dye (S1307, Invitrogen, Waltham MA, USA) dissolved in low [K+] or high [K+] solution for 15 min at 37 °C, in 5% CO2, washed, and imaged immediately with the EVOS FL Auto fluorescent microscope (Thermo Fisher Scientific) under 10X magnification. Ten random fields of view were imaged per slide. Images were counted for the incidence of dye uptake defined as the percentage of cells that took up the dye.

Experimental animals

All experiments were conducted in accordance with the University of Ottawa Animal Care Guidelines and the Canadian Council of Animal Care Guidelines. Mice were housed under a 12 h light/dark cycle with food and water ad libitum. Mdx (C57BL/10ScSn-Dmdmdx/J) mice were purchased from The Jackson Laboratory (strain #001801). Panx1−/− mice were generated by Dr. Valery Shestopalov [16] and back-crossed onto C57BL/6J for another six times in the Dr. Leigh Anne Swayne laboratory [39]. To generate mdx mice lacking Panx1, male Panx1−/− mice were crossed with female mdx (DMDmdx/mdx) mice to generate Panx1+/-/DMDmdx/Y male mice. Male Panx1+/-/DMDmdx/Y mice were then crossed with female mdx mice. Resulting Panx1+/-/DMDmdx/Y male mice and Panx1+/-/DMDmdx/mdx female mice were crossed to generate the experimental male Panx1−/−/mdx (Panx1−/−/ DMDmdx/Y) mice and their control Panx1+/+/mdx (Panx1+/+/ DMDmdx/Y) mice. Genotypes were determined by PCR screening of ear notch DNA for the Panx1 deletion as described by Sanchez-Arias et al. [39] and for the nonsense mutation in exon 23 of the dystrophin gene as described by Shin et al. [40]. All measurements and assessments were done in a blinded fashion. Body weights were recorded as well as survival.

Tissue processing

Male (12‐week‐old) mice were euthanized and their tibialis anterior (TA) and soleus (Sol) muscles were harvested. Muscles were either embedded in OCT compound (Thermo Fisher Scientific) by freezing in isopentane pre-cooled in liquid nitrogen or fixed in 4% paraformaldehyde for 24 h at room temperature then stored in 70% ethanol until paraffin embedding. Cross-sectional area (CSA), fiber number, and central nuclei analysis were completed in frozen muscle sections stained with Hematoxylin and Eosin, while Masson trichrome staining was done on paraffin embedded sections. Paraffin embedding, tissue processing, and staining of paraffin-embedded tissue were performed at the Louise Pelletier Histology Core Facility at the University of Ottawa.

Hematoxylin & eosin, masson trichrome, and oil red o staining

Hematoxylin and Eosin (H&E) (Sigma‐Aldrich) staining was performed on muscle sections as described in [19]. The Masson trichrome and Oil Red O staining was done according to manufacturer’s protocols at the Louise Pelletier Histology Core Facility. Entire tissue sections were scanned using the EVOS FL Auto (Thermo Fisher Scientific) under 10X magnification. CSA was measured from randomly selected fibers (100–250 fibers for the Sol and 200–500 for the TA). Fiber number counts were performed on the entire section and represented as fibers per mm2. Central nuclei counts were performed on entire muscle scans and data represents the average of three serial sections. For fat infiltration, data represents the percentage of area stained in red by Oil Red O. For the Masson trichrome staining, data represents the percentage of muscle section area stained in blue (collagen) [45]. All measurements were calculated using the ImageJ (FIJI) software.

Maximum force and force frequency curve

Control solution contained (in mM): 118.5 NaCl, 4.7 KCl, 2.4 CaCl2, 3.1 MgCl2, 25 NaHCO3, 2 NaH2PO4, and 5.5 d-glucose. Solutions were continuously bubbled with 95% O2 – 5% CO2 to maintain a pH of 7.4. Experiments were performed at room temperature. Total flow of solutions in the muscle chamber was 15 ml/min being split just above and below the muscle to prevent any buildup of reactive oxygen species.

Mice (12-week-old) were euthanized, and their soleus were then dissected out. Muscle length was adjusted to give maximal tetanic force. Muscles were positioned horizontally in a Plexiglas chamber. One end of the muscle was fixed to a stationary hook, whereas the other end was attached to a force transducer (model400A; Aurora Scientific Canada). Similarly to methods described in Ammar et al. [2] the transducer was connected to a data acquisition system (KCP13104,Keithley), and data were recorded at 5 kHz [2]. Electrical stimulations were applied across two platinum wires (4 mm apart) located on opposite sides of the fibers. They were connected to a Grass S88 stimulator and a Grass SIU5 isolation unit (Grass Technologies, Cheshire UK). Tetanic contractions were elicited with 200-ms trains of 0.3-ms, 10-V (supramaximal voltage) pulses. Stimulation frequencies were set to give maximum tetanic force (140 Hz during maximization and equilibrium stages.) The force–frequency relationship was measured after a 30 min equilibrium period over a range from 1- 200 Hz. Twitch and tetanic force, defined as the force developed following a single stimulation pulse or a train of pulses, respectively, was calculated as the difference between the maximum force during a contraction and the force measured 5 ms before the contraction was elicited. Forces are presented in N/cm2.

EchoMRI, forelimb grip strength, and pole descending test

EchoMRI was performed by a technician at the Animal Behavior Core Facility at the University of Ottawa on the EchoMRI-700 body composition analyzer and associated software (Houston, TX, USA).

Forelimb grip strength measurements of 12‐week‐old mice were completed using the Chatillon DFE II digital meter (Columbus Instruments, Columbus, USA), as we have previously described [19]. Following acclimatization to the workspace, each mouse was allowed to grip the grid firmly and then gently pulled horizontally relative to the grid until release. The maximum peak force value was recorded. This was repeated for a total of five times for each mouse with 10–15 s intervals between trials.

For the pole descending test, 12-week-old mice were placed near the top of a textured metal pole (diameter: ~ 8 mm; height: ~ 55 cm) with their nose facing upwards. The time taken to turn completely around (time to turn) and to climb down the pole face down were recorded (time to descend). If the animal fell from the pole, the maximum value of 120 s was given. Mice were tested in 5 consecutive trials. [21]. Grip strength measurements and the pole test were also performed at the Animal Behavior Core Facility at the University of Ottawa.

Differentiation assays

For the differentiation assays, HSMM (70 000 cells/well) were seeded in 24-well dishes containing one collagen coated glass cover slip. Cells were transduced with a lentivector containing either GFP or PANX1 [46] one day prior to induction of differentiation. Based on GFP, a transduction efficiency of approximately 50% was achieved. Differentiation was initiated by switching cells into differentiation medium which consisted of DMEM supplemented with 5% horse serum and 1% penicillin/streptomycin. Cells were fixed in 3.7% PFA on day 5 of differentiation. Cells were stained for myosin heavy chain (MHC) (1:500, MF20, MAB4470, R&D Biosystems, Minneapolis, MN, USA), and mounted with Fluoromount-DAPI (Southern Biotech, Birmingham, AL, USA). Cells were imaged with the EVOS FL Auto fluorescent microscope (Thermo Fisher Scientific) under 20X magnification. The differentiation index (number of MHC positive cells/ total number of nuclei (%)) and fusion index (number of nuclei in myotubes/total number of nuclei (%)) were analyzed from 3–5 randomly selected fields of view [34].

Immunofluorescence

Tissue and myoblasts were fixed in 3.7% PFA diluted in PBS for 20 min and permeabilized in 0.1 M glycine and 0.1% Triton X-100 in PBS for 10–15 min. Non-specific labeling was blocked for 1 h in 5% horse serum, 2% BSA and 0.1% Triton X-100 in PBS. Samples were incubated with antibodies against PANX1 (1:200, HPA016930 Sigma, St. Louis, MO, USA), Pax7 (1:2, hybridoma cell supernatant, DSHB, Iowa city, IA), MHC (1:500, MF20, cat# MAB4470, R&D Biosystems, Minneapolis, MN, USA), laminin (1:250, ab11575 Abcam, Cambridge, UK) at either room temperature for 1 h. Incubation with anti-rabbit Alexa Fluor 488, anti-mouse Alexa Fluor 488 or anti-rabbit Alexa Fluor 594, anti-mouse Alexa Fluor 594 (1:500–1:1000, A11008, A11017, A11005 and A11012, Life technologies, Eugene, OR, USA) antibodies were done at room temperature for 1 h. Myoblasts nuclei were stained with Hoescht 33,342 (Invitrogen). Tissue specimens were mounted with Fluoromount-DAPI (Southern Biotech, Birmingham, AL, USA). Samples were visualized with either the EVOS FL Auto (ThermoScientific) under 20X magnification or with the Olympus Fluoview FV1000 confocal microscope under either 20X or 60X magnification.

Western blotting

Cells were lysed in 150 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM EGTA, 1% Triton, 0.5% NP-40 and protease/phosphatase inhibitors (5872S, Cell Signaling, Danvers, MA, USA) for 1 h on ice then centrifuged at 12 000 rpm to remove cell debris. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and non-specific binding was blocked with 5% bovine serum albumin in PBS + 0.01% Tween 20. Membranes were incubated with anti-PANX1 (1:1000, HPA016930, Sigma),anti-β-actin (1:5000, 8H10D10, Cell Signaling), or anti-GAPDH (1:5000; 14C10; Cell Signaling) followed by Alexa 680- (1:5000, A21009, Thermo Fisher Scientific, Carlsbad CA, USA) or infrared fluorescent-labeled secondary antibodies IRDye800 (1:5000, 925–32210, Li-COR Biosciences, Lincoln, NE, USA). Immunoblots were scanned with the Odyssey infrared-imaging system (LI-COR biosciences) and quantified using the ImageJ software.

Statistics

The data were analyzed using unpaired or paired two-tailed student’s t-tests, one-way or two-way ANOVA followed by Tukey’s or Sidak’s post hoc tests as specified in the figure legends. All data are represented as mean ± s.d. unless otherwise stated. The number of animals used for each experiment is indicated in the figure legends. The individual data (each animal) points are also displayed on the graphs.