Biopsies of skeletal muscles from 16 healthy dogs (labelled with identification number (id) 1–16) of various breeds and ages not affected by muscular disease as determined by a detailed clinical investigation (e.g., no clinical signs of a muscle disease, normal blood cell count, normal serum creatinine kinase and lactate levels) were taken during routine surgical procedures such as laminectomy or fracture fixation with the owners’ agreement. All procedures fulfilled the requirements of the German Animal Welfare Act and were approved by the Federal State Office for Consumer Protection and Food Safety of Lower Saxony, Germany (reference number: Az 42502_1). From each dog one biopsy, 1 cm3 in size, was taken from different striated skeletal muscles (see Table 1) and transported to the laboratory in 15 mL tubes filled with 10 mL of a sterile transport medium. This transport medium consisted of: 3.6 g N- [2-hydroxyethyle] piperazine-N-2- ethansulphonic acid (HEPES), 3.8 g sodium chloride (NaCl), 0.112 g potassium chloride, 0.99 g glucose and 0.000567 g phenol red dissolved in 500 mL distilled water (aqua dest.). All components were obtained from Sigma-Aldrich. After adding the substances to 400 mL aqua dest. the pH of 7.6 was obtained by using 1 M NaOH. The volume was increased to 500 mL with aqua dest. and the solution was filtered through a 0.22 μm syringe membrane filter (Vivasience AG).
Further processing steps were performed under sterile conditions in a laminar flow bench (CA/R Clean Air). Tissue samples were washed once with fresh transport medium and the adhering connective tissue was removed with forceps and scissors. All biopsies were mechanically minced with scissors to a size of 2 mm3. The minced muscle biopsies were digested with a 0.25% trypsin solution. The trypsin-stock-solution (2.5%, Invitrogen) was diluted using the transport medium to a ratio of 1:10. The mechanically dissociated tissue pieces were resuspended in 7 mL of the 0.25% trypsin solution, transferred into a trypsinization flask (Wheaton) and incubated for 7 min. (Shoubridge et al., 1996). During the incubation time the tissue mash was mixed on a magnetic stirrer using a feather-edged magnetic stirring bar. After 7 min. the stirring was stopped to allow the larger tissue pieces to drop to the bottom of the trypsinization flask and the supernatant was poured into a 50 mL tube filled with 10 mL washing medium. The latter was composed of Dulbecco’s modified Eagle Medium (DMEM) (Invitrogen) and 10% Foetal Bovine Serum (FBS) (Invitrogen). 7 mL of 0.25% trypsin solution was added to the remaining tissue mash in the trypsinization flask and stirring and incubation was repeated for 7 min. Thereafter, the supernatant was effused again into a new 50 mL tube filled with washing medium and the whole procedure was repeated. The supernatants of three 50 mL tubes were combined in one tube followed by separation centrifugation as described elsewhere (Greene and Raub, 1992). The remaining pellet was resuspended in proliferation medium and transferred to a 25 cm2 tissue culture flask coated with 0.1% PSG. The proliferation medium consisted of Skeletal Muscle Cell Basal Medium (Cat. No. C-23260, PromoCell), Skeletal Muscle Cell Growth Medium Supplement Pack (Cat. No. C-39360, PromoCell), 10% FBS and 1.5% L-alanyl-L-glutamine (Invitrogen).
All cultures were incubated at 37°C in a 5% CO2 enriched humid atmosphere and the proliferation medium was changed every other day.
Freezing and recultivation of myotubes
For freezing and long-term storage the proliferating cells were harvested from the tissue culture flask and resuspended in 1 mL of freezing medium (DMEM, 10% Dimethyl sulfoxide (DMSO), 20% Fetal Calf Serum) at 4°C. Prior to storage in liquid nitrogen the tubes were kept for 24 hours in a −80°C freezer. After several weeks (up to 8 weeks) in liquid nitrogen, the tubes were briefly pre-thawed in a water bath (37.0°C) and with the centre of the pellet still frozen decanted in warm proliferation medium. After 12 hours the proliferation medium was discarded, the cells were washed with PBS and fresh proliferation medium was added.
Differentiation of myotubes
The mixed cell cultures, consisting of fibroblasts and myoblasts, were kept under proliferation conditions until the cells had reached confluence of up to 80% (approx. 8 to 14 days). In order to induce differentiation of myoblasts into myotubes, cultures with a confluence of up to 80% were not split and proliferation medium was replaced by differentiation medium composed of Skeletal Muscle Cell Basal Medium, 5% horse serum (Invitrogen) and 5 mg Insulin (PromoCell). The different morphological stages of the differentiating cells were documented with an ocular camera (Microscope Systems). The spontaneously occurring contractions of the matured myotubes were filmed as .mpg files using the same camera.
Cells were fixed with cold (−20°C) methanol and washed with PBS (Biochrom). In order to prevent non-specific binding of the primary antibody 2% of horse serum in PBS was added to the cells overnight at 4°C. Thereafter, cells were washed with PBS before the primary antibody, 1:1 diluted in a PBS /- 0.3% Triton®/- 2% horse serum-solution (anti-Human Desmin Clone D33, Cat. No. M 0760, DakoCytometion) was added. After incubation at room temperature (RT) for 60 min., followed by several washing steps with PBS, the secondary antibody (Cy3- conjugated goat anti mouse IgG, Cat. No. 115165020, Dianova) diluted 1:100 in PBS was added for 60 min. (RT). Cells were washed again three times with PBS prior to nuclear staining with 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI, Molecular Probes) at a dilution of 1:20 in PBS and incubated for 4 min. at room temperature (RT). After a final washing step with aqua dest. slides were mounted with 1, 4-Diazabicyclo [2.2.2] octane (DABCO, Sigma). Staining was visualised and photographed with a Leica DMLB microscope, Leica DC 300 camera and Leica software-IM1000 (Leica microsystem AG). Canine fibroblast cultures were used as negative and human myoblast cultures as positive controls for the primary antibodies. Human myoblast cultures were obtained from the Muscle Tissue Culture Collection at the Friedrich-Baur-Institute (Department of Neurology, Ludwig-Maximilians-University, Munich, Germany; part of the German network on muscular dystrophies, MD-NET, service structure S1, 01GM0601; partner of Eurobiobank .
Cells were removed from the tissue culture flask with a cell scraper (Greiner cell scraper, Sigma) and transferred to an Eppendorf tube and centrifuged by 700 × g for 1 min. Supernatant was discharged and the remaining pellet was fixed with 10% paraformaldehyde (PFA, Sigma) for 24 hours at 4°C. After fixation the cell pellet was embedded in a paraffin wax and 5 μm thick sections were obtained. Sections were mounted on Glass Plus slides (Menzel Gläser), rinsed twice with TRIS-buffered saline (TBS) for 10 min. and blocking of the endogenous peroxidase was performed with 0.03% H2O2 diluted in TBS for 30 min. Prior to application of the primary antibody, sections were incubated with undiluted goat serum (normal goat serum, Cat. No. S-1000, Vector Laboratories) for 10 min. to block non-specific binding sites. As primary antibody an anti-human desmin monoclonal antibody (clone D33, Cat. No. M 0760, DakoCytometion) was used. The slides were incubated overnight at 4°C with the primary antibody diluted 1:50 in TBS containing 20% of goat serum.
After a washing step with TBS the secondary antibody (biotinylated goat anti-mouse IgG, Cat. No. BA-9200, Vector Laboratories) and the Avidin-Biotin-Complex (Vector Laboratories) was applied for 30 min. (RT). The positive antigen-antibody reaction was visualised by incubating the slides with 3,3′-diaminobenzidine-tetrahydrochloride (DAB)-H2O2 in 0.1 M imidazole, pH 7.1, for 10 min. (RT) (Sigma). Ascites of non-immunised BALB/cJ mice, diluted 1:1000, were used as negative control for the staining. As positive control normal canine skeletal muscle tissue was used.
For the electrophysiological experiments myotube cultures were transferred into an extra-cellular solution containing 140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM HEPES, and 5 mM Dextrose. Patch pipettes were pulled from borosilicate glass tubes (GC150TF, Cat. No.: 30–0066, Harvard Apparatus LTD) with a DMZ-Universal Puller (Zeitz Instruments). They had a series resistance between 1.7 and 2 MΩ when filled with an intracellular solution containing 130 mM KCl, 10 mM HEPES, 2 mM MgCl2, 5 mM EGTA. The pH of the extra- and intra-cellular solution was adjusted with KOH at 7.4 and the osmolarity to 290 mosmL-1 with mannitol.
Only cells morphologically identified as canine myotubes (40 – 60 μm, more than 3 nuclei) were used for patch-clamp experiments. All measurements were performed with an EPC-9 patch-clamp amplifier (List Electronics) and the data were recorded online with HEKA software.
For recording voltage-gated channels in a giga-sealed whole-cell configuration a holding potential of – 100 mV was applied to the myotubes. After hyperpolarisation of the cell to −120 mV (for 15 ms) the first test pulse (15 ms duration) depolarised the membrane to −50 mV. The consecutive series of 19 test pulses of the same duration depolarised the membrane in + 5 mV steps up to + 45 mV.