*2.4. Tissue Collection*

Mice were anesthetized with isoflurane and subsequently euthanized by cervical dislocation 48 h after sham or CLP procedures. The gastrocnemius (GAS) muscles were carefully removed from both legs and cut in half; one half was mounted for histology and small strips were prepared for transmission electron microscope (TEM) analyses, as previously described [34]. The rest of the GAS was quickly frozen in liquid nitrogen and stored −80 ◦C until use for immunoblotting and qPCR experiments.

#### *2.5. Fiber Size Determination*

Muscles samples were mounted on plastic blocks in tragacanth gum and frozen in liquid isopentane cooled in liquid nitrogen. The samples were then stored until use at −80 ◦C. The samples were cut into 10 μm cross-sections using a cryostat (Leica Biosystem Inc., Concord, ON, Canada) at −20 ◦C and then mounted on lysine coated slides (Superfrost) to assess muscle fiber size, as described in [32,34]. To this end, the muscle cross-sections were first allowed to reach room temperature and were rehydrated with phosphate buffered saline (PBS, pH 7.2) and then blocked with goat serum (10% in PBS). The sections were then incubated with primary rabbit IgG polyclonal anti-laminin antibody (MilliporeSigma, Oakville, ON, Canada, L9393, 1:750) for 1 h at room temperature. The sections were then washed three times in PBS before being incubated for 1 h at room temperature with an Alexa Fluor 594 goat anti-rabbit IgG antibody (Invitrogen, Burlington, ON, Canada A-11037, 1:500). The sections were then washed three times in PBS and the slides were cover-slipped using Prolong Gold (Invitrogen, P36930) as mounting medium. The slides were imaged with a Zeiss Axio Imager 2 fluorescence microscope (Zeiss, Dorval, QC, Canada). The median minimum Feret's diameter of the muscle fibers, a reliable marker of myofiber size [35], was determined for each muscle sample using at least 200 fibers per muscle sample (average number ± SD of fiber analyzed for each group: sham AAV-GFP, 317 ± 62; sham AAV-Parkin, 300 ± 15; CLP AAV-GFP, 345 ± 30; CLP AAV-Parkin, 304 ± 46). Analyses were performed using ImageJ (NIH, Bethesda, MD, USA, https://imagej.nih.gov/ij/).

#### *2.6. Transmission Electron Microscopy (TEM)*

The samples for TEM were prepared as described in [34,36,37]. Briefly, small strips prepared from GAS were incubated in 2% glutaraldehyde buffer solution in 0.1 M cacodylate (pH 7.4) and were subsequently post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer. Tissues were then dehydrated via increasing the concentrations of methanol to propylene oxide and infiltrated and embedded in EPONTM resins at the Facility for Electron Microscopy Research (FEMR) of McGill University. Ultrathin sections (60 nm) were cut longitudinally using an ultramicrotome (Ultracut III, Reichert-Jung, Leica Biosystem Inc., Concord, ON, Canada) and mounted on nickel carbon-formvar-coated grids for electron microscopy. Uranyl acetate and lead citrate stained sections were then imaged using a FEI Tecnai 12 transmission electron microscope at 120 kV, and images were digitally captured using a XR80C CCD camera system (AMT, Woburn, MA, USA) at a magnification of 1400×. Individual intermyofibrillar (IMF) mitochondria from all groups were manually traced in longitudinal orientations using ImageJ 2.0.0 software (NIH, Bethesda, MD, USA, https://imagej.nih.gov/ij/) to measure the following morphological characteristics: area (in μm2), perimeter (μm), circularity (4π·(surface area/perimeter2)), Feret's diameter (longest distance (μm) between any two points within a given mitochondrion), aspect ratio (major axis/minor axis)—a measure of the "length to width ratio" and form factor (perimeter/4π·surface area)—a measure sensitive to the complexity and branching aspect of mitochondria [34,36,37]. An index of mitochondrial morphological complexity was finally calculated as follows: Mitochondrial complexity index = Aspect ratio × Form Factor. Details on the number of IMF mitochondria that were traced are available in the figure legends.

### *2.7. Immunoblotting*

Frozen skeletal muscle tissues (15–30 mg) were homogenized in an ice-cold lysis buffer (50 mM Hepes, 150 mM NaCl, 100mM NaF, 5 mM EDTA, 0.5% Triton X-100, 0.1 mM DTT, 2 μg/mL leupeptin, 100 μg/mL PMSF, 2 μg/mL aprotinin, and 1 mg/100 mL pepstatin A, pH 7.2) using Mini-beadbeater (BioSpec Products) with a ceramic bead at 60 Hz. The muscle homogenates were kept on ice for 30 min with periodic agitation and were then centrifuged at 5000 *g* for 15 min at 4 ◦C, the supernatants were collected, and the pellets were discarded. The protein contents in each sample were determined using the Bradford method. The aliquots of crude muscle homogenates were mixed with Laemmli buffer (6×, reducing buffer, # BP111R, Boston BioProducts, Ashland, MA, USA) and subsequently denatured for 5 min at 95 ◦C. Equal amounts of protein extracts (30 μg per lanes) were separated by SDS-PAGE, and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Saint-Laurent, QC, Canada) using a wet transfer technique. The total proteins on the membranes were detected with Ponceau-S solution (MilliporeSigma #P3504). The membranes were blocked in PBS + 1% Tween® 20 + 5% bovine serum albumin (BSA) for 1 h at room temperature and then incubated with the specific primary antibodies overnight at 4 ◦C. The complete list of antibodies used for immunoblots analysis can be found in Supplementary Table S1. Membranes were washed in PBST (3 × 5 min) and incubated with HRP-conjugated secondary anti-rabbit or anti-mouse secondary antibodies (Abcam, Toronto, ON, Canada, cat# Ab6728, Ab6721) for 1 h at room temperature, before further washing in PBST (3 × 5 min). Immunoreactivity was detected using an enhanced chemiluminescence substrate (Pierce™, Thermo Fisher Scientific, Saint-Laurent, QC, Canada) with the ChemiDoc™ XRS+ Imaging System. The optical densities (OD) of the protein bands were quantified using ImageLab software (Bio-Rad Laboratories) and normalized to loading control (Ponceau-stained PVDF membranes). Immunoblotting data are expressed as relative to Sham AAV-GFP.
