Muscular dystrophy with myositis (mdm) mice carry a deletion in the N2A region of the gene for the muscle protein titin (TTN), shiver at low frequency, fail to maintain body temperatures (Tb) at ambient temperatures (Ta) <34°C, and have reduced body mass and active muscle stiffness in vivo compared with wild-type (WT) siblings. Impaired shivering thermogenesis (ST) could be due to the mutated titin protein causing more compliant muscles. We hypothesized that non-shivering thermogenesis (NST) is impaired. To characterize the response to cold exposure, we measured Tb and metabolic rate (MR) of WT and mdm mice at four nominal temperatures: 20, 24, 29 and 34°C. Subsequently, we stimulated NST with noradrenaline. Manipulation of Ta revealed an interaction between genotype and MR: mdm mice had higher MRs at 29°C and lower MRs at 24°C compared with WT mice. NST capacity was lower in mdm mice than in WT mice. Using MR data from a previous study, we compared MR of mdm mice with MR of Perognathus longimembris, a mouse species of similar body mass. Our results indicated low MR and reduced NST of mdm mice. These were more pronounced than differences between mdm and WT mice owing to body mass effects on MR and capacity for NST. Correcting MR using Q10 showed that mdm mice had lower MRs than size-matched P. longimembris, indicating that mutated N2A titin causes severe thermoregulatory defects at all levels. Direct effects of the titin mutation lead to lower shivering frequency. Indirect effects likely lead to a lower capacity for NST and increased thermal conductance through decreased body size.

Defense of a constant core body temperature (Tb) by generating or dissipating heat is the defining feature of homeothermic endotherms. Thermoregulatory heat production and heat loss allows small animals to maintain thermal constancy even when challenged with temperatures outside of their thermal neutral zone. Large deviations in Tb can have severe consequences, such as reduced enzyme efficiency, altered diffusion capacity and changes in membrane fluidity. These critical cellular functions can result in loss of consciousness, inability to coordinate and execute motor functions, and even death (Morrison and Nakamura, 2011). Tb is maintained during cold exposure by vasoconstriction of peripheral vessels, piloerection, postural changes, shivering thermogenesis (ST) and regulatory non-shivering thermogenesis (NST; Hemingway, 1963). A previous study found that mice with a mutation in the gene for the muscle protein titin (TTN) exhibit decreased tremor frequency during ST and could not maintain euthermic Tb when exposed to ambient temperatures (Ta) <34°C (Taylor-Burt et al., 2015). In this study, we investigated the effect of the titin mutation on the other heat-generating mechanism, NST.

NST is a heat-generating mechanism that occurs through the uncoupling of oxidative phosphorylation and ATP production by uncoupling protein-1 (UCP1) in brown adipose tissue (BAT). UCP1 is expressed among many animals, including rodents (Cannon and Nedergaard, 2004; Depocas, 1960; Golozoubova, 2006; Lowell et al., 1993; Meyer et al., 2010; Nedergaard et al., 2001; Nicholls and Locke, 1984). Like basal metabolic rate (BMR), the contribution of NST to summit metabolism (summit oxygen consumption rate, O2sum) as well as O2sum itself, scales with body mass (Wunder and Gettinger, 1996). Thermogenic capacity, which can be approximated by O2sum, provides an estimate of an animal's ability to thermoregulate and refers to the total capacity for heat production during cold exposure. Thermogenic capacity can be measured when both ST and NST are maximally activated during acute cold exposure. The house mouse (Mus musculus), has a small body mass, which leads to relatively high thermal conductance and a greater contribution of NST to VO2sum in order to offset heat loss in comparison with larger animals (Wunder and Gettinger, 1996).

The muscular dystrophy with myositis (mdm) mouse has a 779-bp deletion in the N2A region of the titin gene TTN (Garvey et al., 2002), and a previous study demonstrated severe thermoregulatory deficiencies (Taylor-Burt et al., 2015). Mice homozygous for the mdm mutation exhibit severe and progressive degeneration of skeletal muscles and lower body mass, stiffer gait and reduced lifespan compared with wild-type (WT) littermates (Garvey et al., 2002; Huebsch et al., 2005; Lopez et al., 2008). Muscle in mdm mice has higher passive stiffness than WT muscle (Hessel et al., 2017; Lopez et al., 2008; Monroy et al., 2017). Taylor-Burt et al. (2015) demonstrated in vitro that mdm mice have lower active muscle stiffness as well and a lower tremor frequency during ST. It is expected that if titin did not contribute to active muscle stiffness, then the titin mutation in mdm mice would not affect tremor frequency. However, tremor frequencies are dramatically reduced in mdm mice (Taylor-Burt et al., 2015). The deficiency in shivering frequency likely leads to the observed hypothermic state of mdm mice at temperatures below 34°C. The combination of increased thermal conductance and lower heat generation via ST results in an offset between heat production and heat loss, resulting in a failure to adequately thermoregulate even at Ta values modestly below the Tb set-point. Although no studies have previously investigated NST in mdm mice, the hypothermic state of mdm mice motivated us to determine whether an impairment in NST is also contributing to the decreased heat production.

List of symbols and abbreviations
     
  • BAT

    brown adipose tissue

  •  
  • BMR

    basal metabolic rate

  •  
  • mdm

    muscular dystrophy with myositis

  •  
  • MR

    metabolic rate

  •  
  • NST

    non-shivering thermogenesis

  •  
  • RMR

    resting metabolic rate

  •  
  • ST

    shivering thermogenesis

  •  
  • Ta

    ambient temperature

  •  
  • Tb

    body temperature

  •  
  • UCP1

    uncoupling protein-1

  •  
  • O2

    oxygen consumption rate

  •  
  • O2sum

    summit oxygen consumption rate

  •  
  • WT

    wild type

We set out to determine whether mdm mice have impaired NST and whether this contributes to their observed hypothermia in addition to impaired ST and higher thermal conductance as compared with the WT mouse. We tested two hypotheses: (1) MR of mdm mice is greater at higher Ta than WT mice owing to a shifted thermoneutral zone (TNZ) of mdm mice; and (2) the inability to defend Tb cannot be explained solely by lower shivering frequencies; therefore, an impairment in the other heat-production mechanism, NST, is present.

Animal experiments were approved by the Institutional Animal Care and Use Committee of Northern Arizona University, under protocol 05-014-R3, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International.

Mice

Breeding pairs of B6C3Fe a-/a-mdm mice (Mus musculus Linnaeus 1758) from the Jackson Laboratory (Bar Harbor, ME, USA) were housed on a 14 h:10 h light:dark cycle at 23–24°C at Northern Arizona University. In this study, all animals were housed in an environmental chamber set to 34°C upon weaning, and experiments were conducted during the dark phase of the light cycle, when mice are active. WT mice were fed LabDiet 5001 Laboratory Rodent Diet and mdm mice were fed LabDiet 5LJ5 PicoLab High Energy Mouse Diet (LabDiet, St Louis, MO, USA). mdm mice were provided the high-fat-diet chow owing to high mortality rates when fed standard laboratory chow. WT mice were housed singly or with siblings and mdm mice were housed with the mother and other mdm siblings owing to mortality if separated. Each cage was equipped with bedding and enrichment.

Sample sizes for WT and mdm mice at each experimental temperature were as follows: 20°C (n=7 WT, n=5 mdm), 24°C (n=6 WT, n=7 mdm), 29°C (n=6 WT, n=8 mdm) and 34°C (n=7 WT, n=8 mdm), unless otherwise noted. Body mass differed significantly between genotypes (Welch's test, P<0.001), with mdm mice (7.2±0.21 g; n=9) at approximately one-third of the mass of the WT mice (26.0±1.82 g; n=7).

Surgery

To measure core Tb, recording devices were surgically placed in each mouse's peritoneal cavity at the age of 25–30 days. Different temperature recording devices were used because of the smaller size of the mdm mice compared with WT siblings. mdm mice were implanted with a Biothermo13 passive integrated transponder (PIT) tag (0.109±0.030 g; Biomark, Boise, ID, USA). The PIT tag measured and recorded Tb at a resolution of ±0.5°C. WT mice were implanted with a PhysioTel TA-F10 telemetry device (1.6 g; Data Sciences International, St Paul, MN, USA), which measured Tb at a resolution of ±0.05°C. The surgical protocol was modified from Richter et al. (2017). Briefly, mice were anesthetized with isoflurane USP (MWI Veterinary Supply, Boise, ID, USA) using a Forane vaporizer (Ohio Medical Corporation, Gurnee, IL, USA). Anesthesia was induced by placing the mouse in a small chamber supplied with a 1–2% isoflurane–oxygen mixture. Upon unconsciousness, the mouse was transferred to a heating pad, where anesthesia was administered using a nosecone that delivered a 1–2% oxygen–isoflurane mixture. Once pedal reflex was absent, the abdominal fur was shaved and the exposed skin was scrubbed with povidone iodine and 70% ethanol. The skin and muscle were incised and bluntly dissected to expose the peritoneal cavity. The device was then placed freely inside the peritoneal cavity and the muscle and skin were closed with 3-0 Maxon absorbable monofilament suture. Buprenorphine, an analgesic agent, was administered subcutaneously (0.10 mg kg−1) for 72 h following surgery. Post-surgery recovery lasted for 7 days at 34°C before further experiments were conducted.

Temperature manipulations

Respirometry was conducted as in Richter et al. (2015) and modified for mice. Briefly, mice were placed in 9 liter metabolic cages during the experiments. Oxygen consumption rate (O2) and carbon dioxide (CO2) production were measured using an open-flow respirometry system (Promethion, Sable Systems International, Las Vegas, NV, USA). Each cage had its own gas analysis chain for respiratory gases comprising a flow controller, capacitive water vapor partial pressure analyzer, spectrophotometric CO2 analyzer and fuel cell O2 analyzer. Water vapor was continuously measured and its dilution effect on O2 and CO2 was compensated mathematically. The flow rate was maintained at 2 l min−1 and subsampled at 250 ml min−1, resulting in ∼12 complete air exchanges per hour. The gas analyzers were calibrated using 100% N2 and a span gas with a known concentration of CO2 mixed with N2 prior to each set of experiments for an animal. The cages were housed inside an environmental chamber (Percival Scientific, Perry, IA, USA) so that Ta could be controlled. Bedding was removed from each cage prior to metabolic trials. Cages sat on top of either an antennae or telemetry receiver (Biomark; Data Sciences International) to collect Tb data throughout the experiment. Tb was recorded once per minute using either PhysioTel RPC-1 Receivers for the PhysioTel TA-F10 telemetry devices or HPR Plus PIT Tag Reader and Antennae for the BioTherm13 PIT tags (Data Sciences International; Biomark). Ta was recorded once per minute using a datalogger (Onset HOBO Data Logger, Bourne, MA, USA) with a resolution of ±0.14°C.

O2 and CO2 were recorded once per second using SableScreen v3.3.11 acquisition software, with 60 samples averaged per minute for final analysis (Sable Systems International). O2 (ml min−1) is expressed as ml O2 g−1 body mass h−1 for each animal and is referred to hereafter as MR. Raw data were processed using ExpeData v1.8.4 (Sable Systems International). A subset of 10 min of each metabolic trial was selected for analysis based upon the following criteria: (1) recordings occurred after the fasting period; (2) Ta did not exceed the specified temperature range for that experiment (20, 24, 29 and 34°C); and (3) the 10 data points for oxygen consumption (ml O2 g−1 h−1) occurred when animal movement was minimal. Movement was assessed by BXYZ-R beam arrays (Sable Systems International).

All mice underwent four 4-h (mdm) or 5-h (WT) experiments where Ta was manipulated once per day for four consecutive days. To minimize the effect of diet-induced thermogenesis, WT mice were fasted for 2 h at the onset of the experiment and mdm mice were fasted for only 1 h due to their fragility. All mice were weighed before and after each experiment. When calculating mass-specific MR, mass was assumed to be lost linearly across the experimental period. The experimental temperatures (20, 24, 29 and 34°C) were chosen based on a previous study which found that 34°C was the lower critical limit of the TNZ in mdm mice (Taylor-Burt et al., 2015). The Ta=34°C was used to establish resting MR for this study.

Q10 effects on MR

Because mdm mice fail to maintain euthermic Tb below 34°C (Taylor-Burt et al., 2015), we corrected MR of mdm mice assuming a Q10 of 2.4 (Hudson and Scott, 1979) using the following equation:
formula
(1)
where MRexpected is MR expected if Tb is 37°C, MRobserved is observed MR and Tobserved is observed Tb. Once MRexpected was calculated, it was subtracted from MRobserved to estimate Q10 effects and is referred to hereafter as E–O MR.

Noradrenaline-stimulated thermogenesis

To compare the capacity for NST between genotypes, we first fasted mice either for 1 or 2 h (mdm and WT, respectively; Speakman, 2013) at 34°C, measured MR (as above) and then briefly removed them from the metabolic cages to subcutaneously administer 1.2 mg kg−1 of noradrenaline (Wunder and Gettinger, 1996). Mice were then placed back into the metabolic cages and measured for MR and Tb over the next 2–3 h. The duration of the metabolic curve, referred to as total effect time, as well as time to peak MR and peak MR were measured to test for differences in noradrenaline response between genotypes. In addition, peak Tb and average Tb were compared between genotypes after noradrenaline injection to test whether Tb followed the same trend as MR. Peak Tb was defined as the highest 60 s average, and average Tb was the average Tb during the test.

Thermogenic capacity

Thermogenic capacity can be approximated by O2sum (Wunder and Gettinger, 1996) as:
formula
(2)
where O2sum is summit metabolism, BMR is basal MR, ST is shivering thermogenesis and NST is non-shivering thermogenesis. O2sum is referred to hereafter as thermogenic capacity. Relative contributions of ST, NST and BMR to thermogenic capacity were calculated by rearranging the above equation. MR at 34°C was used for calculations of BMR. To calculate NST, BMR was subtracted from peak MR during noradrenaline-stimulated thermogenesis (n=6 WT, n=6 mdm) to parse out MR due solely to NST. Because the mice may not have been post-absorptive during either experiment, NST was likely underestimated and metabolic measurements may have been overestimated. Therefore, we will refer to BMR hereafter as resting metabolic rate (RMR). Taylor-Burt et al. (2015) previously reported thermogenic capacity; therefore, ST was calculated by subtracting peak MR during noradrenaline-stimulated thermogenesis (RMR+NST) from thermogenic capacity.

Statistical analysis

Statistical analysis was performed using JMP v12 (SAS Institute, Inc., Cary, NC, USA). Values are reported as means±s.e.m. and an alpha level of 0.05 was used for all tests. For statistical analysis, body mass was normally distributed but variance of residuals was unequal between groups, so a Welch's test, where the means are weighted by the reciprocal of the group mean variances, was used to identify significant differences. Throughout the study, the mdm group at 20°C had a large within-group variance with an outlier, leading to unequal variances when compared with WT mice. Therefore, we used two statistical methods to analyze O2 with and without the outlier. For our primary and more conservative method, data were ranked using temperature as a blocking variable (20, 24, 29 and 34°C). A mixed-model ANOVA with subject nested within genotype and fixed effects of temperature and genotype was conducted for Tb, MR and Q10 effects data. Steel–Dwass multiple comparisons were used to identify significant differences between genotypes at all temperatures. In our secondary method with the outlier removed, residuals of the O2 data were normally distributed with equal variance; therefore, data were analyzed using two-way ANOVA with subject nested within genotype and fixed effects of temperature and genotype. Tukey’s HSD test was conducted post hoc. Removing the outlier for the E–O MR data did not change statistical significance, so this was not reported. For the noradrenaline-stimulated thermogenesis data, Welch's test for unequal variances was used to identify significant differences in peak Tb, average Tb and time to peak. t-tests were used for peak MR, total effect time and NST capacity. To identify significant differences in absolute contributions to thermogenic capacity, t-tests were used to compare ST and NST between genotypes.

For calculations of noradrenaline-stimulated thermogenesis, subsequent rise in MR after noradrenaline injection yielded a metabolic curve from which thermogenic capacity due to NST was calculated by taking the integral of the increase in MR after noradrenaline injection, computed numerically using the trapezoidal rule. The average of 10 consecutive minutes of MR measures that met the criteria (as above) before the animal was removed from the cage was considered their baseline MR. This was subtracted from the calculation to account for animals that had higher or lower O2 before injection. The area calculation began with the onset of injection and ended when O2 reached previously calculated baseline values.

Body temperature

mdm mice exhibited hypothermic characteristics, as shown by their lower Tb values in comparison with WT mice at lower Ta. There was a significant effect of genotype and Ta on Tb (P<0.001) but no interaction between Ta and genotype. We found a significant effect of subject (P<0.05). Post hoc tests revealed significant differences between genotypes at all temperatures (P<0.05; Fig. 1). At 20, 24, 29 and 34°C, we observed the following differences in Tb between WT and mdm mice, respectively: 8.8, 4.2, 1.9 and 1.4°C.

Metabolic rate

At 24°C, thermoregulatory mechanisms in mdm mice are insufficient to maintain constant and euthermic Tb, resulting in significantly lower MR (Fig. 2) and Tb (Fig. 1) as compared with WT mice. In addition, mdm mice activate ST and NST more than WT mice at 29°C, as demonstrated by their higher MR. Despite their higher MR, their Tb remained lower than that of WT mice.

There was a significant interaction between genotype and temperature on MR (P<0.05). The MRs of WT and mdm mice did not significantly differ at 34°C (P>0.05). At 29°C, the MR of mdm mice (2.4±0.12 ml O2 g−1 h−1) was significantly higher than that of WT mice (1.9±0.09 ml O2 g−1 h−1; P<0.05). At 24°C, the MR of WT mice (3.9±0.19 ml O2 g−1 h−1) was significantly higher than that of mdm mice (3.1±0.34 ml O2 g−1 h−1). WT mice (4.8±0.29 ml O2 g−1 h−1) did not have significantly different MRs from mdm mice (3.5±1.51 ml O2 g−1 h−1) at 20°C (P=0.1939); this was likely due to the large variability in mdm MR at 20°C compared with WT mice (Fig. 2). When the mdm outlier at 20°C was removed, MRs of WT mice at 20°C were significantly higher than those of mdm mice. Additionally, the mdm mean MR dropped to 2.9±0.89 ml O2 g−1 h−1 as opposed to 3.5±1.51 ml O2 g−1 h−1.

Q10 effects on MR

In general, mdm mice had MRobserved values far lower than MRexpected values, with the largest differences in E–O MR at the lower temperatures (Fig. 3). WT mice tended to remain close to MRexpected, except at lower temperatures, where MRobserved was higher than predicted. The higher E–O MR values for WT mice at lower temperatures are likely a result of certain subjects having higher activity, and thus higher MR at lower temperatures (observational).

A significant effect of genotype was found on E–O MR: mdm mice had significantly higher E–O MR compared with WT mice at all temperatures (P<0.001). The largest differences in E–O MR were seen at the two lowest temperatures. At 24°C, mdm mice (1.0±0.08 ml O2 g−1 h−1) had significantly higher E–O MR than WT mice (−0.4±0.10 ml O2 g−1 h−1; P<0.05), which had higher observed values than predicted, making their E–O MR negative. At the lowest temperature of 20°C, mdm mice (3.0±0.46 ml O2 g−1 h−1) failed to attain their expected MR. WT mice, in contrast, had higher observed than expected values (0.4±0.32 ml O2 g−1 h−1; P<0.05).

Noradrenaline-stimulated thermogenesis

Both WT and mdm mice exhibited similar metabolic profiles following noradrenaline injection, but WT mice had a significantly higher capacity for NST, found using area under the curve (143.9±20.01 ml O2 g−1 h−1), than mdm mice (62.3±22.78 ml O2 g−1 h−1; P<0.05; Fig. 4). There were no significant differences between genotypes in either latency to reach peak MR (P=0.60) or the total effect time (P=0.08; Fig. 4). WT mice took 17.3±2.20 min to reach peak MR and mdm mice took 22.8±9.65 min. Total effect time for WT and mdm mice was 66.3±4.92 min and 48.3±10.44 min, respectively. Interestingly, genotypes did not differ in peak MR reached after injection (P=0.21), with WT mice reaching 7.2 ml O2 g−1 h−1 and mdm mice reaching 6.4 ml O2 g−1 h−1. However, WT mice (39.7±0.08°C) reached a higher peak Tb (P<0.05) than mdm mice (38.5±0.35°C) during the trial. In addition, WT mice had a significantly higher average Tb (38.7±0.24°C) than mdm mice (37.3±0.33°C; P<0.05).

In this study, we examined the capacity for NST in mdm mice to investigate whether this component of heat production is impaired. Our results demonstrate that a deletion in the titin gene TTN not only results in a deficiency in ST, but it impacts NST as well, likely contributing to lower Tb, MR and thermogenic capacity. Previously, it was shown that the capacity for ST of mdm mice is compromised (Taylor-Burt et al., 2015). We confirmed the inability of mdm mice to maintain homeothermy by measuring their Tb and MR across a range of Ta values. Compared with WT mice, mdm mice had lower Tb values at all Ta values and lower MRs at 24°C. Because mdm mice could not defend core Tb, we observed significant Q10 effects on MR. E–O MR was significantly higher for mdm mice. Further, we found that the capacity for NST was significantly lower in mdm mice compared with WT mice, indicating that this component of heat generation is also impaired. The relative contributions of basal MR, ST or NST to O2sum did not differ between WT and mdm mice.

Body temperature

mdm mice had significantly lower Tb values than WT mice, even at 34°C (Fig. 1). These findings suggest that in order for mdm mice to have a euthermic Tb set-point similar to that of WT mice, Ta needs to be even higher than 34–35°C, as previously reported by Taylor-Burt et al. (2015) for mdm mice to maintain euthermy.

We do not think that mdm mice are entering torpor at the temperatures they were tested. A reduction in Tb set-point due to torpor is unlikely owing to the pattern of MR and Tb observed. Torpor is characterized by a ‘decided’ reduction in Tb set-point that leads to a precipitous decline in MR and Tb (Geiser et al., 2014). During hypothermia, Tb set-point is at euthermic levels, but the animal cannot support the metabolic heat production needed to maintain euthermia; therefore, both Tb and MR decrease slowly as thermoregulatory mechanisms fail to meet demand and then decline rapidly once the body cools further. mdm mice exhibited increases in MR upon cold exposure followed by a gradual reduction, which was likely hypothermia. This indicates that thermoregulatory mechanisms of mdm mice are insufficient to maintain Tb. Hudson and Scott (1979) measured Tb and MR in M. musculus and found that torpid mice with Tb of 32°C had a metabolism 50% of what was observed at euthermic Tb levels. The mice in our study had similar Tb values of ∼29.3°C at 20°C but had higher MRs of ∼3.5 and ∼2.0 ml O2 g−1 h−1 at 20 and 34°C, respectively, suggesting that mdm mice lack sufficient thermoregulatory capacity at Ta values <34°C and do not adopt the behavioral strategy of hypometabolism through torpor.

Studies have shown thermoregulatory deficiencies specific to NST in UCP1-null mice, mice with induced obesity and Type II diabetes due to leptin alterations (ob/ob and db/db), and mice with inherited defects of fatty acid oxidation. UCP1-null mice were able to acclimate and tolerate 18°C with a well-defended Tb that was not significantly different from that of WT mice (Golozoubova, 2006). Similarly, administration of leptin to ground squirrels failed to alter their thermogenic capacity (Boyer et al., 1997). However, adaptive NST was significantly altered in UCP1-null mice (details below). Mice with induced rapid early-onset obesity (ob/ob) and diabetes (db/db) have markedly reduced capacity for NST (Yen, et al., 1974). Tb of ob/ob and db/db mice was 30±1.4°C and 26.8±8°C, respectively, after 90 min of exposure to 4°C. This is comparable to mdm mice at 20°C, with Tb values of 29.3±1.2°C. In addition, mice homozygous for the inactivated allele BALB/cByJ, which encodes the short-chain acyl CoA dehydrogenase, have abnormal NST (Guerra et al., 1998). In BALB/cByJ mice, Tb decreased by 10°C in less than 4 h at 4°C, which is similar to mdm mice at 20°C.

The impact of ST on maintenance of Tb can be assessed by blocking muscular activity via curare-like drugs, which competitively block the binding of acetylcholine to the motor endplates of striated muscles (Bowman, 2006; Kashimura et al., 1992). In a study that reduced shivering by 50% via curare, WT mice were still able to maintain a Tb of 35.4±0.4°C at 4°C (Bal et al., 2012), likely because of their ability to compensate via NST. It is difficult to compare thermoregulatory deficits of mdm mice with those of genetically altered mice in other studies owing to 4°C being the standard cold temperature versus 20°C, which was used in our study as this is the low temperature limit of mdm mice. Nonetheless, the mdm mice in our study expressed more severe thermoregulatory defects than mice with deficits in either ST or NST discussed above. This is likely due to the combined defects of both ST and NST in mdm mice.

Metabolic rate

mdm and WT mice maintained similar RMRs at a Ta of 34°C (Fig. 2). These values are slightly higher but comparable to resting MRs reported for M. musculus that were fasted between 5 and 30 h (Hudson and Scott, 1979). The mice in our study were fasted for only 2 h (WT) or 1 h (mdm), which could account for the slightly higher MRs we found.

mdm mice reach thermoregulatory failure at Ta values below 29°C and are more cold-stressed than WT mice at this Ta. This is demonstrated by significantly higher MRs of mdm mice compared with WT mice (Fig. 2). WT mice had comparable MRs at 34°C and 29°C likely because these Ta values are near or within their TNZ (31–35°C; Hudson and Scott, 1979). Interestingly, although mdm mice have higher MRs than WT mice at 29°C, they maintained a significantly lower Tb. These results indicate that at this Ta, mdm mice work harder than WT mice to thermoregulate, yet they still cannot maintain or attain euthermic Tb and are thus hypothermic. The failure to maintain a higher Tb despite higher MRs is likely due to the combination of increased thermal conductance of the much smaller mdm mice and their compromised thermoregulatory capacity.

At 24°C, there was a large difference in MR between mdm and WT mice, with WT mice having significantly higher MRs (Fig. 2). There were no significant differences in MRs between WT and mdm mice at 20°C, which was interesting considering Tb differences were even more different between genotypes. This is likely due to the effect of a single outlier in the mdm group that greatly increased the standard error of this group as compared with the WT group. When we analyzed the data with the outlier removed, WT mice had significantly higher MRs than mdm mice.

As previously stated, MR scales with body size and it is necessary to account for the much smaller size (7 g) of mdm mice and its impact on MR. A similarly sized mouse, Perognathus longimembris (little pocket mouse), is highly abundant and one of the smallest rodents in North America (Bartholomew and Cade, 1957). Previous groups have studied this species because of its ability to maintain homeothermy despite its small size. A previous study investigating MR of the little pocket mouse reported regressions across Ta values (Chew et al., 1967) to investigate allometric scaling of MR with Ta (Kleiber, 1932). This allowed us to compare MR of the little pocket mouse with MR of mdm mice (using Q10-corrected MR) at our desired Ta. For each of the temperature ranges (20, 24, 29 and 34°C), differences in MRexpected between the little pocket mouse and mdm mice were as follows: 0.54, 1.69, 1.65 and 0.85 ml O2 g−1 h−1. Surprisingly, MRexpected of mdm mice was less than predicted MRs for the size-matched little pocket mouse at all of the test temperatures.

Our data indicate that even if mdm mice could obtain euthermic Tb, they would still have a deficit in MR. E–O MR of WT and mdm mice differed significantly at all Ta values. In general, WT mice had MRexpected close to MRobserved, leading us to conclude that their Q10 approximated 2.4, which is typical of euthermic M. musculus (Hudson and Scott, 1979). The Tb data also confirm that WT mice were euthermic for all trials (Fig. 1). mdm mice, in contrast, had much higher MRexpected than MRobserved, leading to high E–O MR.

Non-shivering thermogenesis

Maximal O2 following noradrenaline injection correlates well with body mass and is affected by acclimation temperature, with cold-acclimated animals having higher O2 responses. Allometric relationships for maximal metabolic response to noradrenaline in small mammals have been described by Wunder and Gettinger (1996) and include a BMR component that can be used for animals acclimated to 23°C. Using this equation, the O2 response of WT and mdm mice to noradrenaline was predicted to be 6 and 11 ml O2 g−1 h−1, respectively. Observed maximal O2 responses to noradrenaline values for WT and mdm mice were 7.2 and 6.4 ml O2 g−1 h−1, which did not significantly differ. WT mice did not differ from the Wunder model output and mdm mice failed to increase MR after noradrenaline injection accordingly. These conclusions are also reflected in the NST contributions to thermogenic capacity (Fig. 5). These comparisons provide further evidence that mdm mice have a lower O2 response to noradrenaline than WT mice.

Although mdm mice have a lower capacity for NST, the latency from noradrenaline injection to peak MR, total effect time of noradrenaline, and peak MR were similar to WT mice (Fig. 4). Peak Tb and average Tb of WT mice were significantly higher after noradrenaline injection than in mdm mice (Fig. 4). These results are consistent with the finding that mdm mice have a reduced capacity for NST. Similarly, mdm mice were unable to maintain euthermic Tb. This could have been due to their increased thermal conductance or to a depletion of metabolic fuels needed for NST, primarily free fatty acids and glucose (Townsend and Tseng, 2014).

Based on the severe phenotype of the mdm mice compared with UCP1-null mice, the null hypothesis is that the lower capacity of NST in mdm mice is due to the absence of UCP1. In WT and UCP1-null mice that were warm-acclimated to 30°C, noradrenaline evoked large increases in MR for WT mice and small increases for UCP1-null mice (Golozoubova, 2006). The small increase seen in UCP1-null mice was attributed to either UCP1-independent adrenergic thermogenesis or a general activation of adrenergic receptors. The mice in our study did not have a significantly different peak O2 response to noradrenaline compared with WT mice (Fig. 4); therefore, a solely adrenergic effect causing MR increase in mdm is not likely. In this study, we did not quantify expression of UCP1 or excise putative BAT from mice in the study; however, an interesting line of thought is the possibility for decreased UCP1 expression or decreased sensitivity in BAT to noradrenaline in mdm mice.

Although only MR and Tb were measured, mdm mice appear to be compromised in heat production at the BAT level. It is unlikely they are affected at the sympathetic outflow level based on the results from noradrenaline injection. The NST response was inferred from indirect calorimetry by mimicking a maximal sympathetic response through administration of noradrenaline, which is known to stimulate UCP1 activity in BAT. Because noradrenaline injection resulted in a lower than expected metabolic response and animals could not maintain euthermic Tb, we hypothesize that mdm mice are impacted at the level of BAT.

Titin has been implicated as a regulator in mitochondrial respiration as well as bioenergetics. The sallimus (sls) gene in Drosophila, whose product is homologous to the NH2-terminal half of titin in vertebrates, has been identified as a transcription regulator of mitochondrial respiration (Jumbo-Lucioni et al., 2012). Jumbo-Lucioni et al. (2012) observed the natural variation between state 3 and state 4 mitochondrial respiration and found a direct effect of sls on mitochondrial function. Homozygous slsd00134 flies, which have a missing allele from the sls gene, had 17% lower mitochondrial state 3 and 18% higher state 4 rates than controls. These results reveal that sls is a novel gene hub for regulation of mitochondrial respiration and that specific alleles of this gene can control naturally occurring variation in mitochondrial function. In addition, truncating titin variants (TTNtv) that cause genetic dilated cardiomyopathy (DCM) have been shown to alter mitochondrial bioenergetics (Verdonschot et al., 2018). In patients with TTNtv, increased expression of genes across all electron transport chain complexes as well as ATP synthase was found. These findings suggest a compensatory response of increased oxidative phosphorylation components in order to counteract limited contractile ability of cardiac tissue as an indirect effect of a titin mutation.

Defects in oxidative phosphorylation can affect BAT thermogenic activity (Kajimura and Saito, 2014). During BAT-mediated thermogenesis, UCP1 uncouples heat production from ATP synthase; therefore, it is likely that defects in oxidative phosphorylation could affect this output. As mentioned above, titin can modulate mitochondrial bioenergetics and transcription of oxidative phosphorylation components, and therefore could be a potential explanation for the reduced NST capacity of mdm mice. Taylor-Burt et al. (2015) demonstrated a direct effect of titin stiffness on the rate of ST, and our results indicate that an NST deficiency could reflect a regulatory effect of titin on metabolic processes. Whether these effects are due to titin signaling or other pathways is a question that could be explored in future work.

Thermogenic capacity and its components

Thermogenic capacity is significantly reduced in mdm mice compared with expected values for their body size (Fig. 5) (Taylor-Burt et al., 2015). Many small mammals, including deer mice, increase their thermogenic capacity solely by increasing NST after cold acclimation (Van Sant and Hammond, 2008). Although NST is regarded as the most plastic component of O2sum, animals can also increase their capacity for ST after cold acclimation (Nespolo, et al., 1999). This leads to the hypothesis that animals with deficient ST would increase NST capacity to compensate. It is interesting that mdm mice have a reduced thermogenic capacity for both ST and NST.

Our results show significant differences in MR as well as the capacity for NST even after accounting for body mass differences (WT mice are approximately four times larger than mdm mice). Because of the allometric relationship between body mass, MR and capacity for NST, our comparisons allowed us to conclude that the severe defects in thermoregulation are not just due to body size. Comparisons between mdm mice and the little pocket mouse demonstrate that mdm mice do not exhibit similar MRs to size-matched animals, as shown by their smaller MRs, even at thermoneutrality (Chew et al., 1967). Additionally, as animals become smaller, the contribution of NST to thermogenic capacity increases to offset the balance in increased thermal conductance due to increased surface area (Wunder and Gettinger, 1996). O2 responses to NE in mdm mice were less than half the predicted value (11 ml O2 g−1 h−1; Wunder and Gettinger, 1996). Our findings demonstrate that mdm mice do not activate more heat-generating mechanisms like other small animals to offset the increased heat loss owing to small body size, and that their thermoregulatory defects are either due to direct or indirect effects of the N2A deletion in titin.

Conclusions

A previous study showed an unexpected link between a small titin mutation, ST and thermoregulation (Taylor-Burt et al., 2015). The present study expands on the previous study through continuous monitoring of Tb and investigation of NST and MR. A link between titin mutations and metabolic deficits might have implications for understanding inherited diseases due to titin mutations, which are increasingly found in humans. We conclude that a deletion in the N2A titin gene TTN reduces not only the rate of ST in mdm mice (Taylor-Burt et al., 2015), but also NST. Collectively, mdm mice exhibit a significantly compromised thermogenic capacity. When comparisons are made to account for the small body size of mdm mice, these deficiencies are exacerbated. It is not clear how titin modulates both NST and body size, but it could be through an indirect role in regulating oxidative phosphorylation or other pathways. Future studies should investigate these possible links.

We thank Anthony Hessel, Johhn Lighton, Uzma Tahir, Tinna Traustadóttir and our reviewers for their helpful comments on this manuscript. We would also like to give a special thanks to Danielle Dillon, Destiny Simpson and the team at Sable Systems International for their technical help with this project.

Author contributions

Conceptualization: C.A.M., C.L.B., K.C.N.; Methodology: C.A.M., C.L.B., K.C.N.; Validation: C.A.M.; Formal analysis: C.A.M., K.C.N.; Investigation: C.A.M.; Resources: C.A.M., C.L.B., K.C.N.; Data curation: C.A.M., S.F.; Writing - original draft: C.A.M.; Writing - review & editing: C.A.M., S.F., C.L.B., K.C.N.; Visualization: C.A.M., C.L.B., K.C.N.; Supervision: C.L.B., K.C.N.; Project administration: C.L.B., K.C.N.; Funding acquisition: C.L.B., K.C.N.

Funding

Our funding sources include the National Science Foundation (IOS-0742483, IOS-1025806 and IOS-1456868), the W. M. Keck Foundation, and the Technology Research Initiative Fund of Northern Arizona University.

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Competing interests

The authors declare no competing or financial interests.