Animals

All procedures were approved by the Animal Use Subcommittee at the University of Western Ontario (London, ON, Canada) where the experiments were performed. Adult thirteen-lined ground squirrels, Ictidomys tridecemlineatus (Mitchill 1821), were live-trapped (Tomahawk, Hazelhurst, WI, USA) from late May to early June in the wild (Carman, MB, Canada; 49°30′N, 98°01′W) and were transported by automobile to London, ON, Canada. Individuals were housed separately in shoebox-style cages (26.7×48.3×20.3 cm high) with dried corncob bedding, paper towels for nest building and a transparent red plastic tube (Bio-Serv, Frenchtown, NJ, USA) for enrichment. Animals arrived at the lab on 6 June 2011, where they were housed at 23±3°C with a photoperiod adjusted weekly to match that of Carman, MB, until October. Water and food (LabDiet 5P00, St Louis, MO, USA) were supplied ad libitum. Food was supplemented with 10–12 sunflower seeds three times weekly. Animals were weighed weekly at the time of cage changing and all animals displayed mass gain, from June through to August, characteristic of hibernators. In early July, 5–6 weeks following capture, all animals underwent minor surgery as described previously (Muleme et al., 2006) during which temperature-sensitive radio-telemetric transmitters were implanted intraperitoneally. These transmitters were used to measure body temperature to monitor torpor bouts.

Six animals (3 males, 3 females) were randomly selected and were killed in late July (summer animals) by barbituate overdose (Euthanyl, 270 mg ml⁻¹, 0.2 ml 100 g⁻¹). Euthanyl has no effect on mitochondrial metabolism (Takaki et al., 1997). Tissue samples were subsequently removed from these summer animals.

By early October, the remaining six animals (3 males, 3 females) had ceased to gain further mass, and many appeared to be torpid despite being held at 23°C. At this time, these animals were transferred to a controlled environment chamber and the temperature was reduced by 3°C each day until 5°C was reached. At this time, the photoperiod was reduced to 2 h light:22 h dark (Brown et al., 2012) (lights on at 09:00 h). These conditions induced torpor in all individuals within a week, at which time food was removed but water was supplied ad libitum. The six torpid animals were killed (see below), and skeletal tissue samples removed, ~3 months after their first torpor bouts were observed. In accordance with animal care protocols, torpid animals were euthanised by cervical dislocation to prevent arousal from torpor.

Dissection

Ground squirrel body mass was determined to the nearest 0.1 g using an electronic balance (model PJ300, Mettler, Zurich, Switzerland). Both hindlimbs were removed and were used for dissection. Gastrocnemius muscle samples were rapidly isolated from the right hindlimb, while kept at room temperature (19–22°C), then flash frozen in liquid nitrogen prior to storage in a −80°C freezer in preparation for subsequent analyses of glycogen content and antioxidant capacity. As much skeletal muscle as possible was removed from the hindlimb to produce a mixed hindlimb muscle sample for subsequent isolation of mitochondria. Soleus muscle was dissected from the left hindlimb in oxygenated (95% O₂: 5% CO₂) ice-cold Krebs–Henseleit solution (composition, in mmol l⁻¹: 118 NaCl, 4.75 KCl, 1.18 MgSO₄, 24.8 NaHCO₃, 1.18 KH₂PO₄, 10 glucose, 2.54 CaCl₂; pH 7.4).

Isometric studies

For each soleus muscle preparation, one tendon was clamped to a strain gauge (UF1, Pioden Controls, Canterbury, Kent, UK) whilst the other tendon was clamped to a motor arm (V201, Ling Dynamics Systems, Royston, Herts, UK) attached to an LVDT (linear variable displacement transformer, DFG 5.0, Solartron Metrology, Bognor Regis, West Sussex, UK). The muscle was then maintained at 36.5±0.5°C in circulating oxygenated (95% O₂: 5% CO₂) Krebs solution. The preparation was stimulated via parallel platinum electrodes while held at constant length to generate a series of twitches. Stimulus amplitude, pulse width (pulse duration) and muscle length were adjusted to determine the stimulation parameters and muscle length corresponding to maximal isometric twitch force. The muscle length that yielded maximal twitch force was measured to the nearest 0.1 mm using vernier calipers. An isometric tetanic force response was then elicited by subjecting the soleus muscle to a 350 ms train of stimulation. Time to half-peak tetanic force and time from last stimulus to half-tetanic force relaxation were measured. A rest period of 5 min was allowed between each tetanic response. Stimulation frequency was then altered to determine maximal tetanic force.

Work loop studies

The work loop technique was used to determine the power output of muscles during cyclical length changes (Josephson, 1985; James et al., 1995). Each muscle preparation was subjected to a set of four sinusoidal length changes symmetrical about the length that was optimal for maximal twitch force production. The muscle stimulation parameters found to yield maximal isometric force were used (stimulation frequency, amplitude and pulse width). Electrical stimulation and length changes were controlled via a data acquisition board (KUSB3116, Keithley Instruments, Cleveland, OH, USA) and a custom-designed program developed with TestPoint software (CEC Testpoint version 7, Measurement Computing, Norton, MA, USA). For each work loop cycle, muscle force was plotted against muscle length to generate a work loop, the area of which equated to the net work produced by the muscle during the cycle of length change (Josephson, 1985). Instantaneous power output was calculated for every data point in each work loop (1000 data points per work loop), then these instantaneous power output values were averaged to generate a net work value for each work loop. The net work produced was multiplied by the frequency of length change cycles to calculate net power output (average power per cycle). A total strain of length change cycles of 0.10 was initially used in each experiment (i.e. ±5% of resting muscle length). During each work loop the muscle was subjected to phasic stimulation (active work loop cycle). Every 5 min the muscle was subjected to a further set of four work loop cycles with strain, stimulation duration and stimulation phase parameters being altered until maximum net work was achieved at each cycle frequency. The cycle frequency of length change was altered up and down within the range 1–5 Hz to generate power output–cycle frequency curves. Before the fatigue run, a set of control sinusoidal length change and stimulation parameters was imposed on the muscle every three to five sets of work loops to monitor variation in the muscle's ability to produce power/force. Any variation in power was found to be due to a matching change in the ability to produce force. Therefore, the power produced by each preparation, prior to the fatigue run, was corrected to the control run that yielded the highest power output, assuming that alterations in power generating ability were linear over time. On completion of the power output–cycle frequency curve, each muscle was subjected to a fatigue run consisting of 60 work loop cycles at a cycle frequency of 2 Hz, using the strain and stimulation parameters found to generate maximal power output. Within 5 min of completion of the fatigue run muscles on average had recovered to 61% of their pre-fatigue power output.

At the end of the muscle mechanics experiments, bones, tendons and connective tissue were removed and each soleus muscle was blotted on absorbent paper to remove excess Krebs solution. Wet muscle mass was determined to the nearest 0.1 mg using an electronic balance (model TR-204, Denver Instrument Company, Bohemia, NY, USA). Mean muscle cross-sectional area was calculated from muscle length and mass assuming a density of 1060 kg m⁻³ (Méndez and Keys, 1960). Maximum isometric muscle stress was then calculated as maximum tetanic force divided by mean cross-sectional area (kN m⁻²). Normalised muscle power output was calculated as power output divided by wet muscle mass (W kg⁻¹).

Antioxidant capacity assay

An antioxidant assay kit was used (Cayman Chemical Company, Ann Arbor, MI, USA), which relies on the ability of antioxidants in the sample to inhibit oxidation of ABTS [2,2′-azino-di-(3-ethylbenzthiazoline sulphonate)]. A vial containing lyophilised powder of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was reconstituted in 1 ml of HPLC-grade water (final concentration, 1.5 mmol l⁻¹) and vortexed. The preparations for the standard curve combined increasing amounts of the reconstituted Trolox diluted in antioxidant assay buffer (5 mmol l⁻¹ potassium phosphate, pH 7.4, containing 0.9% sodium chloride and 0.1% glucose). The following concentrations of Trolox were used to create the standard curve; 0, 0.045, 0.090, 0.135, 0.18, 0.225 and 0.330 mmol l⁻¹. The standard wells were prepared by adding 10 μl of a Trolox preparation, 10 μl of metmyoglobin (lyophilised powder reconstituted in antioxidant assay buffer) and 150 μl of chromogen (containing ABTS reconstituted in HPLC-grade water) per well. Samples of frozen gastrocnemius muscle were crushed into small pieces under liquid nitrogen, weighed, and then homogenised 1:4 w:v in ice-cold antioxidant assay buffer (containing protease inhibitor) as per the manufacturer's instructions. The samples were centrifuged at 10,000 r.p.m. (10 min, 4°C), and the supernatant collected and stored at −80°C until use. Sample wells were prepared by adding 10 μl of a 10-fold dilution of each muscle sample (diluted in antioxidant assay buffer), 10 μl of metmyoglobin, and 150 μl of chromogen. All samples and standards were analysed in duplicate. The reactions were initiated by addition of 40 μl of 441 μmol l⁻¹ hydrogen peroxide followed by incubation on a shaker for 5 min at room temperature. The absorbance was read at 750 nm, using a plate reader, and quantified as Trolox equivalents (mmol l⁻¹ mg⁻¹ wet mass). Antioxidants measured by the assay include glutathione, ascorbate (vitamin C), vitamin E, bilirubin, BSA and uric acid.

Glycogen assay

A glycogen assay kit was used (Cayman Chemical Company). A vial containing lyophilised powder of glycogen was reconstituted in glycogen assay buffer (phosphate-buffered saline, pH 7.0) to a final concentration of 200 μg ml⁻¹ and vortexed. The preparations for the standard curve combined increasing amounts of the reconstituted glycogen diluted in glycogen assay buffer. The following concentrations of glycogen were used to create the standard curve: 0, 2.5, 5, 10, 15, 20, 30 and 40 μg ml⁻¹. Samples of frozen gastrocnemius muscle were crushed into small pieces under liquid nitrogen, weighed and homogenised 1:4 w:v in ice-cold glycogen assay buffer (containing protease inhibitor) as per the manufacturer's instructions. The samples were centrifuged at 3000 r.p.m. (10 min, 4°C), and the supernatant collected and stored at −80°C until use. The standard and sample wells were prepared by adding 10 μl of standard or 10 μl of muscle sample per well, and 50 μl of reconstituted hydrolysis enzyme solution (lyophilised powder of amyloglucosidase reconstituted in a 50 mmol l⁻¹ acetate glycogen hydrolysis buffer, pH 4.5). The sample background wells were prepared by adding 10 μl of the sample and 50 μl of the glycogen hydrolysis buffer (without amyloglucosidase). The plate was covered and incubated for 30 min at 37°C to allow for full glycogen hydrolysis. During the incubation, the developer was prepared by combining the following components: 0.5 ml fluorometric detector (lyophilised powder of 10-acetyl-3,7-dihydroxyphenoxazine, reconstituted in 100 μl DMSO and 400 μl assay buffer), 2.5 ml enzyme mixture (lyophilised enzyme mixture reconstituted in 2.5 ml assay buffer) and 5 ml assay buffer. Following a 30 min incubation, 150 μl of developer was added to all wells including the standard, sample and sample background wells. The plate was covered and incubated for 15 min at 37°C for maximum fluorescence development. The plate was read in a FluoStar plate reader (BMG Labtech, Allmendgruen, Ortenberg, Germany) using an excitation wavelength of 530–540 nm and an emission wavelength of 585–595 nm.

Mitochondrial respiration rates

Crude skeletal muscle mitochondria were isolated from mixed hindlimb skeletal muscle samples following a modified protocol (Bhattacharya et al., 1991) and purified using a previous method (Yoshida et al., 2007). Mixed muscle tissue was washed in ice-cold muscle homogenisation buffer (MHB, in mmol l⁻¹; 100 sucrose, 10 EDTA, 100 Tris-HCl, 46 KCl; pH 7.4 at 4°C). Fat, connective tissue, nerves and hair were removed. The remaining muscle tissue was decanted and suspended in nine volumes of MHB with protease (from Bacillus licheniformis, 5 mg g⁻¹ wet muscle mass; Sigma, St Louis, MO, USA) and minced with fine scissors. After 5 min, muscle tissue was homogenised with three passes of a loose-fitting Teflon pestle in a glass mortar. The homogenate was then incubated on ice for 5 min, followed by further homogenisation with three passes of a tight-fitting Teflon pestle in a glass mortar. The resulting homogenate was filtered through one layer of cheesecloth and centrifuged at 2000 g for 10 min at 4°C. The supernatant was filtered through four layers of cheesecloth and centrifuged at 10,000 g for 10 min at 4°C. The pellet was suspended in 5 ml MHB with 0.5% BSA and centrifuged again at 10,000 g for 10 min at 4°C. This pellet was then resuspended in 5 ml MHB, and this raw mitochondrial suspension was layered on top of 5 ml of 60% Percoll solution (made in MHB) and centrifuged at 21,000 g for 1 h at 4°C. Purified muscle mitochondria accumulated at the boundary between the MHB and 60% Percoll solution, and were removed and suspended in MHB. To remove residual Percoll, this suspension was centrifuged at 21,000 g for 10 min at 4°C. This washing step was repeated three times. The final pellet was used for all measurements of respiration rate.

Mitochondrial respiration rates were determined using a high-resolution respirometer (Oxygraph O2K, Oroboros, Innsbruck, Austria) calibrated with air-saturated buffer and oxygen-depleted buffer (obtained by addition of yeast suspension) using published oxygen solubilities (Forstner and Gnaiger, 1983), corrected for local atmospheric pressure. Unless otherwise noted, all compounds were dissolved in MiR05 assay buffer (in mmol l⁻¹: 110 sucrose, 0.5 EGTA, 3 MgCl₂, 60 potassium lactobionate, 20 taurine, 10 KH₂PO₄, 20 Hepes; pH 7.1 at 30°C, 1% BSA) (Gnaiger and Kuznetsov, 2002). Respiration of skeletal muscle mitochondria was measured by adding ~0.03 mg protein to 2 ml of MiR05 assay buffer equilibrated to 37°C. Succinate (10 mmol l⁻¹; in the presence of rotenone, 2 μg ml⁻¹, dissolved in ethanol) was added to stimulate state 2 respiration, and state 3 respiration was achieved by adding ADP (0.2 mmol l⁻¹).

Conditions approximating state 4 respiration were subsequently achieved by adding oligomycin (2 μg ml⁻¹, dissolved in ethanol).

Statistical analysis

Any dispersion measurements are given as s.e.m. In most cases, summer (active) and winter (torpid) results were compared using independent samples t-tests. Levene's test demonstrated that variances were equal in all cases. Comparison of power output–cycle frequency curve data between summer and torpid samples was performed using two-factor ANOVA with experimental treatment and cycle frequency as the factors. This approach allowed the interaction term to indicate whether torpor had affected the shape of the power output–cycle frequency curve. Hypothetically, if during torpor the contractile properties of a muscle became faster, due to a shift towards a faster muscle fibre type, there might be a rightward and upward shift in the peak of that muscle's power output–cycle frequency curve.

Two-factor ANOVA was also used to analyse the effects of torpor on fatigue resistance, with hibernation and loop number (essentially, time elapsed during the fatigue run) used as the two factors. The interaction term in this analysis should indicate whether the shape in the curve is different, hence indicating a difference in the pattern of fatigue between torpid and summer animals. Independent samples t-tests were used to determine whether power output was significantly different between torpid and summer animals at each time point during the fatigue run.

The truncated product method (Zaykin et al., 2002) was used to combine all the P-values in this study to determine whether there was a bias from multiple hypothesis testing. The truncated product method P-value was <0.001, indicating that the results were not biased by multiple comparisons.