Animal models

All animal procedures were conducted in accordance with guidelines for the care and use of laboratory animals as approved by the Institutional Animal Care and Use Committee of the University of Kentucky. Mice were housed in a temperature and humidity-controlled room and maintained on a 14:10 h light-dark cycle with food and water ad libitum. We utilized a transgenic Pax7 CreER/CreER × Rosa26 DTA/DTA (Pax7/DTA) mouse model that allows for the conditional depletion of >90 % of satellite cells following tamoxifen treatment as previously described [6, 10, 15, 18]. To control for any potential adverse effects of tamoxifen, the parental strain Pax7CreER/CreER (Pax7CreER) was employed as a treatment control. Furthermore, to assess the potential for tamoxifen to induce recombination in the brain, a Pax7 reporter mouse was generated by crossing the Rosa26ZsGreen/ZsGreen × Pax7CreER/CreER creating a Pax7/ZsGreen mouse in which Pax7+ nuclei express Zoanthus sp. Green Fluorescent Protein (ZsGreen) upon tamoxifen-induced recombination [19].

Experimental design

Adult (4-month old) female Pax7/DTA mice (n = 8–12 per group) received either an intraperitoneal injection of tamoxifen at a dose of 2.5 mg/day for five consecutive days or were injected with a vehicle control (15 % ethanol in sunflower seed oil) as described previously [6, 10, 15], followed by an 8 week washout period. At 6 months of age vehicle- or tamoxifen-treated mice were singly housed and randomly divided into two groups which either remained ambulatory or were provided with running wheels. The Pax7CreER mice were used to assess the potential independent effects of tamoxifen on running performance. Furthermore, to assess the potential for tamoxifen to cause recombination in the brain, the Pax7/ZsGreen reporter mouse was utilized [19]. Pax7CreER and Pax7/ZsGreen mice received identical tamoxifen treatment as the Pax7/DTA mice.

Voluntary wheel running protocol

Female Pax7/DTA or Pax7CreER mice at approximately 6 months of age were housed individually in plastic cages measuring 30.5 × 15.2 × 12.7 cm. The mice had open access to running wheels that were mounted within each cage. Ambulatory controls were singly housed in cages of equal dimensions, but without running wheels. A mechanical counter was used to record wheel rotations and was connected to a desktop computer via ClockLab software (Actimetrics, Wilmette, IL). The software analyzed running speed (km/h), total distance run (km/day), total time run (h/day), peak running rate (counts/min), and running bout length (min). The animals had access to food and water ad libitum and were checked daily for health and wellness. Following the 8-week (Pax7/DTA), or 6-week (Pax7CreER), running period, the animals were sacrificed and their plantaris muscles were dissected, processed, and stored at −80 °C for further analyses as described below. Plantaris muscles were chosen for analysis due to their known activation in rodent wheel running protocols [20] and to directly compare the current study’s results with our previous investigations regarding synergist ablation and 8 weeks of plantaris overload in the Pax7/DTA mouse [15].

Histochemistry/Immunohistochemistry (IHC)

Plantaris muscles were dissected from surrounding connective tissue, weighed, pinned to a cork block at resting length, covered with a thin layer of Tissue Tek OCT compound (Sakura Finetek, Torrance, CA) and quickly frozen in liquid nitrogen-cooled isopentane and stored at −80 °C prior to sectioning. Muscles were sectioned on a cryostat (MicromHM 525) at 7 μm and either used immediately (succinate dehydrogenase (SDH) and cluster of differentiation 31 (CD31)), or frozen at −20 °C for subsequent analysis. Muscles were immunohistochemically analyzed for Pax7 (satellite cells), dystrophin (sarcolemma), myosin heavy chain (MyHC) isoforms, Oil Red O (lipid content) and DAPI (10nM) (4', 6-diamidino-2-phenylindole, Invitrogen, Carlsbad, CA) to visualize nuclei. Additionally, tibialis anterior (TA) muscles and brain tissue were collected from Pax7/ZsGreen mice, cryosectioned and immunohistochemically analyzed for Pax7 and dystrophin. All images were captured using an Axioimager MI upright fluorescent microscope (Zeiss, Göttingen, Germany), and analyses were performed using AxioVision Rel software (v4.8).

Pax7

Pax7 was immunodetected on air-dried frozen sections fixed in 4 % paraformaldehyde (PFA) as described previously [6, 10, 15]. Briefly, following fixation sections underwent an epitope retrieval protocol at 92 °C using sodium citrate buffer (10 mM, pH 6.5). Endogenous peroxidase activity was blocked with 3 % hydrogen peroxide in PBS, followed by incubation with the Mouse-on-Mouse Blocking Reagent (Vector Laboratories, Burlingame, CA). Sections were then incubated in Pax7 primary antibody (Developmental Studies Hybridoma Study Bank, Iowa City, IA) at a 1:100 dilution followed by incubation with a goat anti-mouse biotin-conjugated secondary antibody (1:1000) (Jackson ImmunoResearch, West Grove, PA) and subsequently streptavidin-HRP (1:100) included as part of the Tyramide Signal Amplification kit (TSA) (Invitrogen). TSA-Alexa Fluor 488 or 594 (Invitrogen) was used to visualize antibody-binding. Sections were counterstained with DAPI (10 nm) (Invitrogen) for nuclear detection and mounted with Vectashield fluorescent mounting medium (Vector Laboratories). Pax7+/DAPI+ nuclei were counted and normalized per number of fibers. Brain tissue was collected from mice (n = 3 per group per mouse strain analyzed) following intracardiac perfusion with 4 % PFA and then subsequently put through sequential sucrose incubations before being frozen, sectioned (10 μm), and allowed to air dry at room temperature. Pax7 IHC was performed on brain tissue using the same protocol as plantaris muscle sections.

Dystrophin/DAPI

Air-dried frozen sections were blocked in Mouse-on-Mouse IgG blocking solution (Vector Laboratories). Immediately following the block dystrophin primary antibody ((1:50) Vector Laboratories)) was added to the sections overnight at 4 °C. Sections were then incubated with Texas Red-conjugated goat anti-mouse secondary antibody (1:200) (Rockland Immunochemicals Inc., Gilbertsville, PA). Lastly, sections were post-fixed in 4 % PFA and stained with DAPI. Myonuclear number was assessed by counting DAPI+ nuclei, within the dystrophin boundary. The data are presented as the myonuclei per fiber. Additionally, the dystrophin boundary around each fiber was traced to assess fiber cross-sectional area (μm2) and is reported as the mean fiber cross-sectional area per plantaris muscle.

MyHC composition

To evaluate fiber-type distribution, frozen sections were air-dried, fixed in methanol, and then incubated with isoform-specific MyHC antibodies: type I (1:100) (BA.D5), type IIa (SC.71), and type IIb (BF.F3) from Developmental Studies Hybridoma Study Bank (Iowa City, IA) overnight at 4 °C. Following washes in PBS, secondary antibodies were applied to the sections at RT as follows: Gt anti-Ms IgG2b, Alexa Fluor 647 conjugated 2 Ab (1:250) (Invitrogen) Gt anti-Ms IgG1, Alexa Fluor 488 conjugated 2°Ab (1:500) (Invitrogen) Gt anti-Ms IgM, and biotin conjugated 2°Ab (1:150) (Invitrogen). Lastly, the sections were incubated in streptavidin-Texas red (1:150) (Vector) and mounted with Vectashield mounting medium (Vector). MyHC isoform expression was manually assessed from ×20 images and expressed as relative fiber-type frequency to account for any fluctuations in fiber number within muscle cross sections.

SDH

SDH content within muscle fibers was visualized following 1-h incubation at 37 °C in the following solution: nitro blue tetrazolium (Sigma) and succinate acid disodium (Sigma) and dissolved in 0.2 M of phosphate-buffered saline (PBS). The reaction was performed in a light protected coplin jar, and sections were then sequentially rinsed with 30 and 60 % acetone before being mounted with aqueous mounting media. Images were captured at ×20 magnification, were manually assessed for SDH staining intensity, and were categorized as positive, weakly positive, or negative fibers (++, +, −). The assessor was blinded to the treatment of the animals.

CD31

To quantify capillary density, an antibody recognizing the endothelial cell marker CD31 (BD-Pharmingen, 550274) was applied to sections following fixation in ice-cold (4 °C) acetone. Sections were incubated with a rat anti-mouse CD-31 antibody, followed by α-Rat HRP 2° antibody (Impress Kit, Vector Labs MP-7404) and Alexa Fluor 555 conjugated 2°antibody (1:200) in amplification buffer. Images were captured, and CD31+ events were analyzed using the automated thresholding feature of AxioVision Rel software (v4.8) and are reported normalized per muscle fiber.

Oil Red O

Lipid content within muscle fibers was assessed by staining with Oil Red O. Oil Red O was dissolved in 60 % triethyl phosphate. Freshly cut sections were air dried, fixed briefly in 37 % formaldehyde at RT, and rinsed in ddH 2 0 prior to staining. Sections were allowed to incubate in Oil Red O solution for 2 h at RT and were subsequently rinsed and mounted in Vectashield. Lipid droplets were visualized fluorescently, and images were captured and analyzed using the automated thresholding feature of AxioVision Rel software (v4.8). Lipid content is reported as total Oil Red O area as a percent of muscle area measured.

WGA

Detection of N-acetyl-d-glucosamine was evaluated on frozen muscle sections using Texas Red-conjugated wheat germ agglutinin (WGA) (eBiosciences, San Diego, CA). Sections were fixed in 4 % PFA and then incubated with WGA conjugate for 2 h at RT. Images were captured at ×10 magnification, and the staining was quantified using the thresholding feature of the AxioVision Rel software. The area occupied by WGA was expressed either relative to muscle fiber number, or in the case of spindle fiber analysis, it was normalized to spindle fiber circumference to account for total spindle size and reported as the extracellular matrix (ECM) index. Furthermore, WGA images were used to trace the cross-sectional area of intrafusal fibers located within spindle fibers. The average cross-sectional area of all intrafusal fibers within a given spindle fiber was averaged and reported as the mean intrafusal fiber area (μm2) per spindle fiber.

Voltage dependent anion channel (VDAC) Western blot

Plantaris muscles were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentrations were quantified using a BCA (Thermo) assay. Thirty micrograms of protein were separated on a 4–15 % SDS polyacrylamide gel (Bio-Rad, Hercules, CA) and subsequently transferred onto nitrocellulose membranes. Membranes were blocked in Odyssey blocking buffer (LI-COR, Lincoln, NE) and immunoblotted with a VDAC (1:1000) primary antibody (Cell Signaling) overnight at 4 °C. Following incubation with goat anti-rabbit IgG (Alexa 680) (LI-COR) secondary antibody, immunoreactive bands were visualized using the Odyssey Infrared Imaging System. Band intensity was quantified using Odyssey Infrared Imaging System Application Software Version 3.0.21, and results are expressed as arbitrary densitometric values of VDAC normalized to actin to assure equal protein loading.

Functional outcomes

A battery of functional measurements were performed on 6-month-old female Pax7/DTA mice 8 weeks following either vehicle or tamoxifen treatment (n = 10 vehicle, 10 tamoxifen-treated).

Grip strength

Forelimb grip strength was measured by allowing mice to grab a bar attached to a force transducer as it was pulled horizontally by the tail away from the bar (Model 1027CSM; Columbus Instrument Co., Columbus, Ohio) [21]. The test was performed five times, and the average peak force (N) for each mouse was normalized to body mass (g) to determine grip strength for each mouse.

Gait analysis

The gait of each mouse was evaluated during part of a standard rodent functional observational battery [22]. Gait assessment was given a score of 0 if the mouse exhibited a fluid gait with pelvic elevation and a score of 1 if the mouse presented with an irregular gait and/or an abnormal pelvic tilt. The examiner was blinded to the treatment of the mice.

Rota-Rod test

Sensorimotor coordination was assessed using a Rotor-Rod apparatus consisting of a rotating rod suspended 18 in. above a padded floor (San Diego Instruments, San Diego, CA). This system uses a mouse’s natural fear of falling as a motivational tool to test gross motor function. Mice were placed on the rotating rod at a speed of 4 rpm for 60 s for their training sessions (two training sessions separated by at least 10 min). After successful completion of the training sessions and adequate rest, the speed of the rod is gradually increased to a maximum of 40 rpms for each of the three testing sessions. The trial is complete when the animal falls, or the time period ends (300 s max). Latency to fall (s) and distance traveled (cm) were recorded, and the average of the three testing trials was reported for each animal.

Balance beam

A beam walking protocol was used to evaluate mice on their ability to traverse beams of decreasing widths. The beam walking protocol evaluates motor balance and coordination by assessing both the time it takes the mice to traverse the beam and the number of foot slips that the mouse experiences during the crossing. This test is more precise in detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rota-Rod [23]. Following standard acclimation and training, three beam widths were used to assess balance (28, 17, and 11 mm) and the mice were given 1 min per trial to complete the crossing of the beam to a bedding filled safe-room on the far end of the beam. The mean of all values per beam obtained in the five trials was used for analysis.

Muscle spindle activity assays

Extensor digitorum longus (EDL) muscles were chosen for this assay because they have two tendons and nerve endings that can be easily dissected and have similar fiber-type composition as the plantaris muscle. Electrophysiological methods were performed as described and modified as needed [24]. Briefly, EDL muscles with the associated peroneal nerve were carefully dissected from euthanized animals and bathed in Lily’s solution (see [24]). The proximal and distal tendons of the EDL were then double tied with silk thread; one tendon was attached to a stationary position with stainless steel staples and the other to a stainless steel hook attached to a speaker (Model SF-9324, Pasco Scientific, Denmark) driven by a DC source from a stimulator (Grass S88, Grass Products, Natus Neurology). Movement excursions were calibrated by observing the attachment of the thread to the tendon with a millimeter grid placed under the recording dish and used to accurately and reproducibly stretch the muscle 1 mm (10 % of muscle length). A suction electrode made from glass pipettes fitted with plastic tips was used to record extracellular signals from the cut nerve and the extracellular signals were amplified via a P-15 (Grass Products, Natus Neurology). A PowerLab 4SP (AD Instruments) in conjunction with a computer was used for data acquisition at 20 kHz on line. LabChart software (AD Instruments) was used for offline analysis of firing frequency (evoked activity) in response to the stretch. Analysis was analogous to the procedure previously described [24]. The 1-mm stretches were given 10 s apart while the muscle was pulled taut to a set length where background muscle spindle firing was minimal to allow for reproducibility with each stretch. Five stretches of 4-s duration were provided in one series. The last three consistent stretches were used for analysis for consistency. Within each stretch period approximately the last second was used to count the number of spikes recorded above a set threshold over the noise, to ensure that the dynamic response of the muscle spindle had a consistent firing during the static phase. An average firing frequency of the three stretches in the series was calculated per muscle and the average reading of two legs for the same animal is reported (n = 2 per treatment group).

Statistics

Data were analyzed with SigmaPlot software (Systat Software, San Jose, CA) via a two factor ANOVA, or a two factor repeated measures ANOVA (running data). If a significant interaction was detected, an applicable post hoc analysis was employed to determine the source of the significance. Additionally, non-paired Student’s t tests were used where appropriate. Statistical significance was accepted at P ≤ 0.05. Data are reported as mean ± standard error of the mean.