All procedures were approved by the University of Louisville Institutional Animal Care and Use Committee. C57BL/6J and FVB/NJ mice were ordered from Jackson Laboratory (Bay Harbor, ME) at 12 weeks of age and allowed to acclimate at the University of Louisville animal facility for 3 weeks. At 15 weeks of age, male mice were assigned randomly by strain to sedentary (SED) or exercise (EXE-Day or EXE-Night) groups. Food and water were provided ad libitum, and the mice were maintained on a 12:12-h light-dark schedule. Because the majority of published studies regarding exercise in mice have utilized male mice, we chose this gender for our study, which enables comparison with the literature. At the conclusion of the study and 24 h after the last exercise session, the mice were fully anesthetized with sodium pentobarbital (40 mg/kg, i.p.), followed by euthanasia via excision of the heart. These procedures are consistent with the AVMA Guidelines on Euthanasia.

To assess for differences in basal metabolism and diurnal/nocturnal behavior, metabolic cage analyses were performed in naïve 15-week-old C57BL/6J and FVB/NJ mice, essentially as described (Sansbury et al., 2012; Cummins et al., 2014). Body weight was recorded prior to the initial and final exercise capacity tests to assess for changes in total body mass. Oxygen consumption rates, carbon dioxide production rates, respiratory exchange ratios, food consumption, water consumption and activity (sum of ambulatory and fine movements) were measured using a physiological/metabolic cage system (TSE PhenoMaster System, Bad Homberg, Germany) as described previously (Sansbury et al., 2012; Cummins et al., 2014).

We performed exercise familiarization and capacity testing in a manner similar to that outlined previously (Massett and Berk, 2005), with minor modifications (Figures 1A,B). Mice were familiarized to the motorized rodent treadmill (Columbus Instruments, Columbus OH) on the Wednesday and Thursday before the first week of training. Familiarization consisted of an initial 10 min period where the treadmill speed and incline were set to zero with shock grid settings of 25 V, 0.34 mA, and 2 Hz. The treadmill speed was then increased steadily to 10 m/min (Wed) and 12 m/min (Thurs) for an additional 10 min.

On the Friday immediately following familiarization to the treadmill, we subjected mice to an exercise capacity test (Figure 1B). For this, the mice were acclimated to the treadmill for 10 min, with the speed and incline set initially to zero. The treadmill speed was then increased to 8.5 m/min with an angle of inclination set to 0° for 9 min. Next, the treadmill speed and incline were increased to 10 m/min and 5°, respectively, for 3 min. The speed was then increased by 2.5 m/min every 3 min to a maximum speed of 40 m/min, while inclination increased by 5° every 9 min until a maximum incline of 15° was achieved.

We developed strict a priori criteria for exercise-induced exhaustion. These criteria were: (1) 10 consecutive seconds on the electric grid; (2) spending more than 50% of time on the grid; and/or (3) lack of motivation to manual prodding. Each mouse was removed immediately from their respective lane once one or more of these criteria was reached. Following the protocol, the mice were housed separately for 30 min to avoid noticeable aggressive behavior following exercise.

Following 4 weeks of training, we repeated this testing protocol to assess changes in exercise capacity. Exercise capacity was measured using the parameters of distance run (meters achieved prior to exhaustion) and work accomplished [calculated as the product of body weight (kg) and vertical distance (m); vertical distance = (distance run) (sinθ), where θ = the angle of inclination of the treadmill from 0° to 15°] as outlined previously (Massett and Berk, 2005).

Mice assigned to exercise training groups were subjected to a 4-week protocol of forced treadmill running. The training protocol commenced the Monday after the initial exercise capacity testing with mice exercising 5 days/week (Mon-Fri) at 70 or 75% of the maximal speed achieved during the initial exercise capacity test and an inclination appropriate to the speed (Figure 1C). Prior to each training bout, we provided mice with a “warm-up” period of 10 min at 0 m/min and 10 min at 12 m/min to promote exercise protocol compliance and to minimize risk of injury. For comparison between the groups, training intensity was set at 20.8 m/min at a 10° incline for FVB/NJ mice and 16.4 m/min at a 5° incline for C57BL/6J mice, which corresponded to 70% of the maximal speed and the appropriate incline at the calculated speed for each strain during the initial exercise capacity test. In subsequent studies of FVB/NJ mice, mice were exercised only during the day, and in these groups, we implemented a more intensive training protocol to further examine systemic and cardiac adaptations to treadmill running. For this, training intensity was set at 75% of the maximal initial exercise capacity, which corresponded to 22.3 m/min at a 10° incline. In all groups, we progressively increased the workload of the mice, such that they trained for 40 min during week 1, 50 min during week 2, and 60 min during weeks 3 and 4. We chose this progressive intensity protocol to prevent training plateau and to stimulate exercise-induced adaptations (Dudley et al., 1982; Hildebrandt et al., 2003; De Angelis et al., 2004; Massett and Berk, 2005).

To prevent injury and record protocol compliance, we monitored the mice carefully during each exercise session. Upon meeting pre-established indicators of exhaustion, mice were removed from the treadmill, and the time run was recorded (See Supplemental Table 1). We used the percentage of total sessions and total minutes completed throughout the 4-week training program as a measure of compliance. Before and after the initial exercise capacity test, we measured blood lactate levels, which provided a biochemical indicator of exercise-induced exhaustion at or near maximal oxygen consumption (VO_2max) (McConnell, 1988; Pederson et al., 2005; Ferreira et al., 2007; Hakimi et al., 2007). We recorded lactate concentration in 0.7 μl of blood from a small tail clip (Lactate Plus meter; Nova Biomedical) prior to the protocol and upon meeting the exhaustion criteria defined above. High lactate levels increase confidence in a successful exercise capacity test by ensuring that failure to continue is due to exhaustion at or near VO₂max and not a failure to comply with the protocol (Von Wittke et al., 1994; Gladden, 2004; Billat et al., 2005).

Following euthanasia, tissue was excised and rapidly fixed for immunohistochemical analysis or immediately snap frozen in liquid nitrogen and stored at −80°C. Tissue was fixed in 10% formalin, paraffin embedded, and sectioned at 4 μm. Heart cross-sections were stained with 4′6-diamidino-2-phenylindole (DAPI; Invitrogen) and wheat germ agglutinin (WGA; ThermoFisher) for quantification of cardiomyocyte cross-sectional area. Quantitative measurements were determined using Nikon Elements software.

Mitochondrial abundance in heart tissue was estimated by measuring mitochondrial DNA (mtDNA) abundance relative to nuclear DNA (nDNA), similar to our previous studies (Cummins et al., 2014; Salabei et al., 2016). Briefly, total DNA was isolated using a QIAamp DNA Mini Kit (Qiagen). A 25-mg aliquot of the tissue was homogenized, followed by overnight digestion in proteinase K at 55°C. Following isolation, relative amounts of mtDNA and nDNA were compared using quantitative real-time PCR, using 2 ng of the isolated DNA. Primers for cytochrome b (mtDNA) and β-actin (nDNA) were used. The sequences are: cytochrome b, word5′-TTGGGTTGTTTGATCCTGTTTCG-3′ and 5′-CTTCGCTTTCCACTTCATCTTACC-3′; and β-actin, 5′-CAGGATGCCTCTCTTGCTCT-3′ and 5′-CGTCTTCCCCTCCATCGT-3′.

Unpaired or paired Student's t-test was used for direct comparisons; multiple groups were compared by one-way and two-way ANOVA followed by Bonferroni or Sidak Multiple Comparison test, as appropriate. For ratio-based statistical comparisons, the data were log-transformed, and unpaired Student's t-test was applied for assessing statistical significance between groups; we used a one-sample t-test for intragroup differences from a ratio of 1 (used to determine significant chronobiological differences in metabolic cage endpoints, i.e., food and water intake, physical movement, VO₂, VCO₂, RER). A p ≤ 0.05 was considered statistically significant.

Laboratory mice, in general, are a nocturnal species (Kohsaka et al., 2007; Laposky et al., 2008; Arble et al., 2009), and they typically choose to participate in voluntary exercise at night (Verwey et al., 2013). Thus, the time at which treadmill protocols are executed could be critical for ensuring exercise regimen compliance and adaptation. Therefore, we first examined the circadian characteristics of the mouse strains by measuring their food and water intake, locomotion, VO₂, VCO₂ and RER by placing untrained (naïve) mice in metabolic chambers. To assess differences between day and night behavior, a ratio (night values:day values) was calculated for each parameter. Although, overall food consumption was not different between the two strains, the C57BL/6J mice consumed, on average, 3-fold more food at night. In contrast, the FVB/NJ mice showed no difference in food consumption in the night compared with the day (Figure 2A). Cumulative water intake was not different between the strains; however, C57BL/6J mice showed significantly higher water intake at night (Figure 2B). Similarly, C57BL/6J mice were more active at night compared with FVB/NJ mice (Figure 2C). In general, FVB/NJ mice had fewer total beam breaks per hour (C57BL/6J = 2583 ± 711; FVB/NJ = 1736 ± 582; p < 0.01), indicating that cage behavioral activity is lower in this strain.

Metabolic analysis yielded analogous results. In C57BL/6J mice, oxygen consumption (VO₂; Figure 2D), carbon dioxide production (VCO₂; Figure 2E), and respiratory exchange ratios (RER; Figure 2F) were higher at night compared with the day. Conversely, the FVB/NJ mice did not show metabolic differences in the day vs. the night. Thus, the behavioral and metabolic qualities of C57BL/6J mice are supportive of their known nocturnal nature and are in stark contrast to FVB/NJ mice which demonstrate a relative lack of features characteristic of the nocturnal phenotype. Additionally, FVB/NJ mice had higher rates of oxygen consumption (C57BL/6J = 3713 ± 327 ml/h/kg; FVB/NJ = 4058 ± 346 ml/h/kg; p < 0.05) which contributed to lower respiratory exchange ratios (C57BL/6J = 0.93 ± 0.02; FVB/NJ = 0.86 ± 0.02; p < 0.0001).

To determine the effects of mouse genetic background on initial exercise capacity, we subjected 15-week-old C57BL/6J and FVB/NJ mice to exercise capacity testing. The mice were exercised either during the day (i.e., between 9 a.m. and 12 p.m.) under normal laboratory lighting or at night (i.e., between 7:00 p.m. and 10:00 p.m.) under dark room conditions (safelight red lamp). We chose the latter time based on metabolic cage activity data, which showed increased voluntary locomotor activity starting at 6 p.m. Compared with C57BL/6J mice, the FVB/NJ strain ran ~1.5-fold farther (Figure 3A) and demonstrated ~2-fold greater initial capacity for treadmill work (Figure 3B), regardless of when the mice were tested. Despite an apparent trend toward lower initial exercise capacity at night, we found no statistically significant differences between C57BL/6J mice exercised during the day or the night (Figures 3A,B); however, FVB/NJ mice showed significantly lower levels of work performed at night (Figure 3B).

In preliminary assessments, we observed that C57BL/6J mice received more shocks than FVB/NJ mice during treadmill exercise. Therefore, to confirm that reliable exercise capacity values were obtained and to rule out non-compliance to the testing protocol, we recorded blood lactate levels prior to and immediately following the initial exercise capacity test. Resting blood lactate values were similar in C57BL/6J and FVB/NJ mice (Figure 3C). Although, it should be noted that the night C57BL/6J group had a slightly higher resting lactate level, in general, blood lactate increased by 2–4-fold in both strains upon meeting criteria for exhaustion (Figure 3C); both resting and maximal lactate abundances are within the ranges previously published for resting mice and mice exercising at or near their VO_2max, respectively (Pederson et al., 2005; Ferreira et al., 2007; Hakimi et al., 2007). Collectively, these results suggest that FVB/NJ mice demonstrate a superior ability to perform treadmill work compared with C57BL/6J mice, and they have higher initial exercise capacities when tested during the day.

To determine if mouse strain or the time of training (i.e., day vs. night) influences exercise protocol compliance and adaptive responses to exercise, C57BL/6J and FVB/NJ mice were subjected to a 4-week training program, with one group from each strain training at night, and one group training during the day. Each day, we recorded compliance to the training program for individual mice (Supplemental Table 1). The C57BL/6J mice completed only 50–60% of their training sessions (Figures 4A,C), resulting in less time exercising in general (Figure 4D). Interestingly, compliance in C57BL/6J mice diminished progressively with duration of the training protocol (Figure 4A, Supplemental Table 1). While FVB/NJ mice were generally compliant when exercised during the day or the night, 100% of the day FVB/NJ group complied with the protocol, whereas FVB/NJ mice exercising at night appeared to show modestly compromised compliance (Figures 4B,D); however, this did not achieve statistical significance.

Pre-training and post-training exercise capacity tests showed that, while both FVB/NJ exercise groups significantly increased exercise capacity after 4 weeks of training, the C57BL/6J mice showed either no improvement or a decrease in the distance run or the work accomplished following training (Figures 5A,B). This lack of response to exercise in the C57BL/6J mice is likely due to poor compliance to the protocol.

To determine if compliance of C57BL/6J mice to the protocol correlated with improvements in exercise capacity, we plotted the percent improvement in distance and work against the percent time completed during the exercise regimen. As shown in Figures 5C,D, there is a significant, albeit weak, correlation between the percent time completed and exercise capacity in this strain. Collectively, these results show that, unlike FVB/NJ mice, C57BL/6J mice are not compliant with this extended treadmill exercise protocol, and that the time at which FVB/NJ mice exercise does not markedly influence their exercise capacity.

Physiological cardiac growth is a common endpoint used to verify cardiometabolic adaptation to exercise (Maillet et al., 2013). Exercise training increased heart weight to tibia length (HW/TL) in both FVB/NJ exercise groups, whereas the C57BL/6J mice showed no changes in heart size (Table 1). We did not find a significant correlation between exercise time completed and HW/TL in the C57BL/6J strain (data not shown). These results indicate that exercise causes cardiac growth in FVB/NJ mice and that C57BL/6J mice, likely due to lack of compliance to this treadmill protocol, fail to demonstrate physiological cardiac growth.

It is known that, compared with the C57BL/6J strain, FVB/NJ mice can achieve higher critical running speeds on the treadmill (Billat et al., 2005), which indicates that their training regimen could be intensified further to evoke more robust adaptations to exercise. To test this, we trained an independent group of FVB/NJ mice at a slightly higher intensity (75% of their initial maximum exercise capacity) for 4 weeks. Compared with pre-training exercise capacity values, this more intensive protocol yielded a ~1.7-fold improvement in distance run and a ~2.4-fold increase in work in the mice (Figures 6A,B). As expected, cardiac size was significantly higher in exercised mice (Figures 6C,D). In exercise-adapted hearts, myocyte cross sectional area was found to be 22% higher than that found in hearts of sedentary mice, and the myocyte area distribution curves were shifted to the right (Figures 6E–G), indicating that the exercise-induced cardiac growth in this strain is due primarily to an increase in cardiomyocyte size. Furthermore, mitochondrial biogenesis, estimated using the ratio of relative mtDNA to nDNA content, was 53% higher in the exercise-adapted mice (Figure 6H). Collectively, these results indicate that treadmill exercise in FVB/NJ mice elicits robust systemic and cardiac muscle adaptations.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.