Model sd9

Transfection of plasmids in FDB muscles of adult mice was performed as described (DiFranco et al., 2009 (link)). In brief, mice were anesthetized using 5% isoflurane, footpads were injected with 2 mg/ml hyaluronidase type IV (Sigma-Aldrich), and 1 h later, ~20 μg total cDNA was injected (10 μl) followed by electroporation using acupuncture needles as electrodes and delivering 20 pulses of 60 V and 30-ms duration at 1 Hz using a square pulse stimulator (model SD9; Grass Technologies) visualized with an oscilloscope (model TDS 220; Tektronix). Six–10 d after electroporation, mice were euthanized and FDB muscles were harvested. Muscles were digested using 4 mg/ml collagenase type 2 (16101015, ThermoFisher) for 1 h at 36°C under agitation, followed by mechanical dissociation, adapted from Casas et al. (2010) (link). Isolated fibers were plated onto Matrigel (BD Biosciences)-coated coverslips in DMEM medium supplemented with Pen/Strep and 10% horse serum (HS), and used within 24 h after plating.

Transfection of plasmids in FDB muscles of adult rats was performed as described previously (DiFranco et al., 2009 (link)). In brief, the animals were anesthetized using 5% isoflurane to inject their footpads with 2 mg/ml hyaluronidase type IV (Sigma-Aldrich), and 1 h later, 40–60 µg total cDNA was injected (20–40 µl) followed by electroporation using acupuncture needles as electrodes and delivering 20 pulses of 100 V and 30-ms duration at 1 Hz using a square pulse stimulator (model SD9; Grass Technologies) combined with an oscilloscope (model TDS 220; Tektronix). Mfn1 or Mfn2 silencing was performed by injection of the fluorescent proteins expressing cDNAs, plus 400 pmol of siRNA. In every experiment, NT-siRNA was injected to the left footpad and Mnf1 or Mfn2 siRNA to the right footpad. 7–10 d after electroporation, the animals were euthanized and the FDB muscles were harvested. FDB muscles were digested using 3 mg/ml collagenase type 2 (Worthington Biochemical Corporation) for 2–3 h at 37°C under agitation, followed by mechanical dissociation, adapted from Casas et al. (2010) (link). Isolated fibers were plated onto CELL-TAK–coated (BD) coverslips in DMEM/F12 medium (Lonza) supplemented with penicillin/streptomycin and 10% horse serum (HS), and used within 24 h after plating.