Animals

This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Rochester called the University Committee on Animal Resources (UCAR). C57BL/6J juvenile/adult (1–3-month-old, Jackson Labs) male mice were used for all experiments and were singly housed at 8 weeks of age in accordance with the UCAR protocol. Mice were maintained on a 12:12 light/dark cycle and provided ad libitum access to pelleted feed and standard drinking water (Hydropac)

SARRP radiation

All ionizing X-ray irradiation was delivered using the Small Animal Radiation Research Platform (SARRP, XStrahl) with a 10 × 25 mm variable collimator as previously described [3, 4]. Mice were anesthetized with vaporized isoflurane. Fractionated irradiation was administered to 4-week-old mice using 3 fractions of 8.2-Gy radiation delivered on Monday, Wednesday, and Friday. Radiation was delivered locally to the lower right hindlimb from the footpad to the tibial plateau.

Voluntary wheel running exercise

Low-profile wireless rodent wheels (ENV-047 wheels, Med Associates, Fairfax, VT, USA) were used in singly housed mouse cages to track chronic endurance exercise running activity over a 4-week period, with sedentary control animals placed in cages having wheels that were locked and unable to rotate. Wheels were connected to a wireless central hub that recorded running activity every 30 s in the Wheel Manager software, with the Wheel Analysis software (Med Associates) used to report data as the total distance run each day (km/day). The percentage of daytime running activity was reported as the total running activity that was conducted during the 12 h daily that the lights were on. Once mice were removed from the wheels, terminal experiments were performed immediately thereafter.

Ex vivo muscle force generation assessment

Muscle force generation capacity was analyzed for EDL and soleus muscles using the Aurora Scientific (ASI) muscle contraction system [21, 24, 25]. Briefly, mice were anesthetized with vaporized isoflurane, the tibialis anterior (TA) muscle removed, and then the proximal and distal tendons of the EDL or soleus muscles were sutured and removed. The muscles were adjusted to their optimal length (Lo), electrically stimulated at increasing frequencies, subjected to three 150-Hz warm-up stimulations, and then finally assessed for force generation capacity. Muscle force data were recorded and analyzed with the Dynamic Muscle Control (DMC) and Analysis (DMA) software. Physiologic cross-sectional area (P-CSA) was calculated as (muscle weight [mg])/(1.056 × (0.44 or 0.71) × length [mm]), where 1.056 = muscle density [g/cm3], 0.44 = EDL angular factor, and 0.71 = soleus angular factor [26].

RNA extraction and RT-qPCR

RNA isolation and RT-qPCR were performed as previously described [21]. Briefly, the gastrocnemius muscles were removed, flash-frozen in TRIzol Reagent (Life Technologies), homogenized, and RNA isolated using RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocols. Then, cDNA was synthesized using the qScript cDNA SuperMix (QuantaBio). RT-qPCR was performed on a Step One Plus Real-Time PCR machine (Applied Biosystems) using SYBR Green FastMix (QuantaBio). Transcript levels from each experiment were standardized to their internal Gapdh gene expression and then normalized to the control condition.

For qPCR, we used the following primers:

Dnm1l forward primer 5′-TTACGGTTCCCTAAACTTCACG-3′

Dnm1l reverse primer 5′-GTCACGGGCAACCTTTTACGA-3′

Fis1 forward primer 5′-TGTCCAAGAGCACGCAATTTG-3′

Fis1 reverse primer 5′-CCTCGCACATACTTTAGAGCCTT-3′

Mfn1 forward primer 5′-CCTACTGCTCCTTCTAACCCA-3′

Mfn1 reverse primer 5′-AGGGACGCCAATCCTGTGA-3′

Pgc1α forward primer 5′-TATGGAGTGACATAGAGTG-3′

Pgc1α reverse primer 5′-CCACTTCAATCCACCCAGAAAG-3′

Tissue sectioning and immunostaining

The muscles were removed, placed in 30% sucrose overnight at 4 °C, embedded in OCT (Tissue Tek), flash-frozen using dry ice-cooled isopentane, stored at − 80 °C, and sectioned at 10 μm thickness. Prior to immunostaining, tissue sections were fixed in 4% PFA (except muscle fiber type sections), permeabilized with PBS-T (0.2% Triton-x-100 in PBS) for 10 min, and blocked in 10% normal goat serum (NGS, Jackson ImmunoResearch) for 30 min at room temperature (RT), then primary antibodies were applied. If using mouse primary antibodies, sections were blocked in 3% AffiniPure Fab fragment goat anti-mouse (Jackson ImmunoResearch) with 2% NGS at RT for 1 h. Primary antibody incubation in 2% NGS/PBS was performed for 2 h at RT or overnight at 4 °C followed by secondary antibody incubation for 1 h at RT. DAPI staining was performed to identify the nuclei. All slides were mounted with Fluoromount-G (SouthernBiotech). The sections were imaged at × 4, × 10, and × 20 magnifications on the Echo Revolve microscope and analyzed using ImageJ (NIH). Sample analyses were performed by investigators blinded to the experimental group.

Single FDB myofiber isolation

All resting cytosolic, SR store Ca2+, and MitoSOX experiments were conducted using single, acutely dissociated flexor digitorum brevis (FDB) myofibers, as previously described [19]. FDB muscles were dissected from the hind limb footpads and placed in Ringer’s solution (145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4) supplemented with 1 mg/mL collagenase A (Roche Diagnostics, Indianapolis, IN, USA) while rocking gently at 37 °C for 1 h. FDB myofibers were then liberated on glass-bottom dishes by gentle trituration in Ringer’s solution using three sequentially increasing gauge glass pipettes and then allowed to settle for 20 min. Only healthy fibers with clear striations and no observable damage were used for experiments.

Resting Ca2+ measurements

Resting free cytosolic Ca2+ concentration was determined as previously described [19]. Briefly, isolated FDB myofibers were loaded with 4 μM fura-2 AM (Thermo Fisher, Carlsbad, CA, USA) in Ringer’s solution at RT for 30 min followed by a 30-min washout in dye-free Ringer’s solution. Loaded fibers were placed on the stage of an inverted epifluorescence microscope (Nikon Instruments) and alternatively excited at 340 and 380 nm (20 ms exposure per wavelength, 2 × 2 binning) using a monochromator-based illumination system with fluorescence emission at 510 nm captured using a high-speed QE CCD camera (TILL Photonics, Graefelfing, Germany). 340/380 ratios from cytosolic areas of interest were calculated using TILL vision software (TILL Photonics Graefelfing, Germany), analyzed using ImageJ and converted to resting free Ca2+ concentrations using a fura-2 calibration curve approach described previously [27].

Total releasable SR Ca2+ store content measurements

Total Ca2+ store content was determined as previously described [19]. Briefly, FDB myofibers were loaded with 5 μM fura-FF AM (AAT Bioquest, Sunnyvale, CA, USA), a low-affinity ratiometric Ca2+ dye, at RT for 30 min, followed by a 30-min washout in dye-free Ringer’s. Total releasable SR Ca2+ store content was calculated from the peak change in the fura-FF ratio (ΔRatio340/380) upon application of ICE Ca2+ release cocktail (10 μM ionomycin, 30 μM cyclopiazonic acid, and 100 μM EGTA) in Ca2+-free Ringer’s solution. Peak change in the fura-FF ratio was calculated using Clampfit 10.0 (Molecular Devices, Sunnyvale, CA, USA).

Mitochondrial ROS production

Mitochondrial ROS production was assessed using a procedure modified from Lee et al. [28]. Briefly, single FDB myofibers were incubated with 5 μM MitoSOX Red (Thermo Fisher) in Tyrode’s solution (121 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 500 μM MgCl2, 400 μM NaH2PO4, 5.5 mM glucose, 24 mM NaHCO3, 100 μM EDTA) for 10 min at RT, followed by incubation in dye-free Tyrode’s solution for 10 min at RT. Loaded myofibers were excited at 488 nm, and emission was captured at 605 nm. Images were taken after the 10-min dye-free incubation and 10 min after taking the initial image. Images were taken using a Nikon Digital Eclipse C1 confocal microscope using a × 40 objective, and image analysis was done using the EZ-C1 software and ImageJ. Change in fluorescence after 10 min was normalized to the original measurement and reported as Δf/f0.

Western blot analyses

TA and soleus muscles were flash-frozen in liquid nitrogen and stored at − 80 °C until ready for use. The muscles were mechanically homogenized in RIPA lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 1 mM Na3VO4, 10 mM NaF) and supplemented with Halt protease inhibitor, as recommended by the manufacturer. Samples were centrifuged at 13,000g for 30 min; supernatants were retained and then protein concentration was determined using the Bio-Rad DC assay (500-0116). Ten micrograms of total protein was separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membranes were briefly stained with a 0.1% Ponceau S solution (Sigma-Aldrich, P3504) to ensure equal protein loading. The membranes were probed with primary antibodies for 2 h at RT or overnight at 4 °C while shaking, diluted in TBS-T (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH 7.6, 3% bovine serum albumin). Secondary antibodies were applied, diluted in TBS-T supplemented with 5% non-fat dry milk for 1 h at RT while shaking. Blots were imaged on a LI-COR Odyssey gel imaging system, and band intensity was quantified using Image Studio Lite (LI-COR Biosciences, Lincoln, NE, USA) using GAPDH as a loading control for each sample. Analysis for 3-NT and oxyblot data was conducted by assessing the overall intensity of the entire lane.

Oxyblot for oxidized proteins

Oxidized proteins were quantified by the Oxyblot Protein Oxidation Kit (Millipore) using 10 μg of muscle lysate for each sample following the manufacturer’s protocol.

Antibodies

The following antibodies were used: rat anti-laminin-α2 (1:1500, Sigma-Aldrich, L0663), DAPI (1:3000), mouse anti-BA-D5 (MyHC-I, IgG2b, 1:40, Developmental Studies Hybridoma Bank (DSHB)), mouse anti-SC-71 (MyHC-IIA, IgG1, 1:40, DSHB), mouse anti-BF-F3 (MyHC-IIB, IgM, 1:40, DSHB), rabbit anti-PGC1α (1:1000, Novus Biologicals), rabbit ant-MCU (1:1000, Cell Signaling, D2Z3B), mouse anti-PMCA (1:2000, Thermo Fisher, 5F10), mouse anti-NCX (1:500, Swant, R3F1), mouse anti-MFN1 (1:500, Neuromab, 75-162), mouse anti-GAPDH (1:50,000, Thermo Fisher, AM4300), mouse anti-CASQ1 (1:5000, Affinity BioReagents, MA3-913), rabbit anti-pan SERCA (1:10,000, Santa Cruz, sc-30110), rabbit anti-VDAC (1:5000, Sigma Aldrich), rabbit anti-DNP (1:150, Sigma Aldrich), mouse anti-3-NT (1:1000, Sigma Aldrich, N5538), Alexa Fluor 405-conjugated goat anti-mouse IgG2b (1:1500, Thermo Fisher, A-21141), Alexa Fluor 488-conjugated goat anti-mouse IgM (1:1500, Thermo Fisher, A-21042), Alexa Fluor 594-conjugated goat anti-mouse IgG1 (1:1500, Thermo Fisher, A-21125), AlexaFluor 488-conjugated goat anti-mouse IgG (1:1500, Thermo Fisher, A-11001), AlexaFluor 488-conjugated goat anti-rabbit IgG (1:1500, Thermo Fisher, A-11034), AlexaFluor 647-conjugated goat anti-rat IgG (1:1500, Thermo Fisher, A-21247), goat anti-rabbit IRDye800 (1:10000, LiCor), goat anti-mouse IRDye800 (1:10,000, LiCor), and goat anti-mouse IRDye700 (1:10,000, LiCor).

Statistical analyses

Statistical calculations were performed using the GraphPad Prism 9 software. Statistical significance was determined through Student’s t-tests with Welch’s corrections (unpaired, two-tailed, 95% confidence interval) or ANOVA (one-way or two-way, followed by Tukey multiple comparisons test), where p < 0.05 was considered statistically significant (*/#p < 0.05, **/##p < 0.01, ***/###p < 0.001, ****/####p < 0.0001). Error bars are represented as ± standard error of the mean (SEM).