Polar bears experience skeletal muscle atrophy in response to food deprivation and reduced activity in winter and summer.

When reducing activity and using stored energy during seasonal food shortages, animals risk degradation of skeletal muscles, although some species avoid or minimize the resulting atrophy while experiencing these conditions during hibernation. Polar bears may be food deprived and relatively inactive during winter (when pregnant females hibernate and hunting success declines for other demographic groups) as well as summer (when sea ice retreats from key foraging habitats). We investigated muscle atrophy in samples of biceps femoris collected from free-ranging polar bears in the Southern Beaufort Sea (SBS) throughout their annual cycle. Atrophy was most pronounced in April-May as a result of food deprivation during the previous winter, with muscles exhibiting reduced protein concentration, increased water content, and lower creatine kinase mRNA. These animals increased feeding and activity in spring (when seal prey becomes more available), initiating a period of muscle recovery. During the following ice melt of late summer, ~30% of SBS bears abandon retreating sea ice for land; in August, these 'shore' bears exhibited no muscle atrophy, indicating that they had fully recovered from winter food deprivation. These individuals subsequently scavenged whale carcasses deposited by humans and by October, had retained good muscle condition. In contrast, ~70% of SBS bears follow the ice north in late summer, into deep water with less prey. These 'ice' bears fast; by October, they exhibited muscle protein loss and rapid changes in myosin heavy-chain isoforms in response to reduced activity. These findings indicate that, unlike other bears during winter hibernation, polar bears without food in summer cannot mitigate atrophy. Consequently, prolonged summer fasting resulting from climate change-induced ice loss creates a risk of greater muscle atrophy and reduced abilities to travel and hunt.

Increase in cardiac myosin heavy-chain (MyHC) alpha protein isoform in hibernating ground squirrels, with echocardiographic visualization of ventricular wall hypertrophy and prolonged contraction.

Deep hibernators such as golden-mantled ground squirrels (Callospermophilus lateralis) have multiple challenges to cardiac function during low temperature torpor and subsequent arousals. As heart rates fall from over 300 beats min(-1) to less than 10, chamber dilation and reduced cardiac output could lead to congestive myopathy. We performed echocardiography on a cohort of individuals prior to and after several months of hibernation. The left ventricular chamber exhibited eccentric and concentric hypertrophy during hibernation and thus calculated ventricular mass was ~30% greater. Ventricular ejection fraction was mildly reduced during hibernation but stroke volumes were greater due to the eccentric hypertrophy and dramatically increased diastolic filling volumes. Globally, the systolic phase in hibernation was ~9.5 times longer, and the diastolic phase was 28× longer. Left atrial ejection generally was not observed during hibernation. Atrial ejection returned weakly during early arousal. Strain echocardiography assessed the velocity and total movement distance of contraction and relaxation for regional ventricular segments in active and early arousal states. Myocardial systolic strain during early arousal was significantly greater than the active state, indicating greater total contractile movement. This mirrored the increased ventricular ejection fraction noted with early arousal. However, strain rates were slower during early arousal than during the active period, particularly systolic strain, which was 33% of active, compared with the rate of diastolic strain, which was 67% of active. As heart rate rose during the arousal period, myocardial velocities and strain rates also increased; this was matched closely by cardiac output. Curiously, though heart rates were only 26% of active heart rates during early arousal, the cardiac output was nearly 40% of the active state, suggesting an efficient pumping system. We further analyzed proportions of cardiac myosin heavy-chain (MyHC) isoforms in a separate cohort of squirrels over 5 months, including time points before hibernation, during hibernation and just prior to emergence. Hibernating individuals were maintained in both a 4°C cold room and a 20°C warm room. Measured by SDS-PAGE, relative percentages of cardiac MyHC alpha were increased during hibernation, at both hibernacula temperatures. A potential increase in contractile speed, and power, from more abundant MyHC alpha may aid force generation at low temperature and at low heart rates. Unlike many models of cardiomyopathies where the alpha isoform is replaced by the beta isoform in order to reduce oxygen consumption, ground squirrels demonstrate a potential cardioprotective mechanism to maintain cardiac output during torpor.

Interspecific and intragenic differences in codon usage bias among vertebrate myosin heavy-chain genes.

Synonymous codon usage bias is a broadly observed phenomenon in bacteria, plants, and invertebrates and may result from selection. However, the role of selective pressures in shaping codon bias is still controversial in vertebrates, particularly for mammals. The myosin heavy-chain (MyHC) gene family comprises multiple isoforms of the major force-producing contractile protein in cardiac and skeletal muscles. Slow and fast genes are tandemly arrayed on separate chromosomes, and have distinct patterns of functionality and expression in muscle. We analyze both full-length MyHC genes (~5400 bp) and a larger collection of partial sequences at the 3' end (~500 bp). The MyHC isoforms are an interesting system in which to study codon usage bias because of their length, expression, and critical importance to organismal mobility. Codon bias and GC content differs among MyHC genes with regards to functional type, isoform, and position within the gene. Codon bias even varies by isoform within a species. We find evidence in favor of both chromosomal influences on nucleotide composition and selection against nonsense errors (SANE) acting on codon usage in MyHC genes. Intragenic variation in codon bias and elongation rate is significant, with a strong trend for increasing codon bias and elongation rate towards the 3' end of the gene, although the trend is dependent upon the degeneracy class of the codons. Therefore, patterns of codon usage in MyHC genes are consistent with models supporting SANE as a major force shaping codon usage.

Muscle plasticity in hibernating ground squirrels (Spermophilus lateralis) is induced by seasonal, but not low-temperature, mechanisms.

During hibernation, ground squirrels (Spermophilus lateralis) show unusually altered expression of skeletal muscle myosin heavy-chains. Some muscle groups show transitions from fast to slower myosin isoforms despite atrophy, which are not predicted from other mammalian studies of inactivity. We measure myosin protein and mRNA expression, and the mRNA expression of genes important in atrophy and metabolism in a time-course of muscle plasticity prior to, and during extended hibernation. We also investigate the role of strictly low-temperature processes by comparing torpid individuals at 20 and 4°C. Shifts in myosin isoform expression happen at both temperatures, before the onset of torpor, or within the first month of torpor, in all muscles demonstrating isoform remodeling. Skeletal muscle atrophy is greatly attenuated in this hibernating species, and even may be absent in some muscles. When present, atrophy develops early in hibernation, and does not progress in the final 3 months of torpor. Myostatin mRNA is down-regulated 50-75% in the soleus and diaphragm, two important muscles that are spared of atrophy. The transcription factor FOXO1, which spurs proteolytic degradation of contractile proteins through regulation of the ubiquitin ligase MAFbx, is also generally down-regulated, and may contribute to reduced atrophy. Hypoxia-inducible factor (HIF-1α) mRNA expression was reduced 50% in some muscles, while elevated more than 300% in others. Our collective findings most strongly support early, seasonal, phenotype changes in skeletal muscles which are not uniquely confined to, or prompted by, torpor at 4°C. Such seasonal control of myosin would be a novel mechanism in mammalian skeletal muscle, which otherwise is most susceptible to mechanical loading and limb-activity patterns.

Adaptive thermogenesis and thermal conductance in wild-type and UCP1-KO mice.

We compared maximal cold-induced heat production (HPmax) and cold limits between warm (WA; 27°C), moderate cold (MCA; 18°C), or cold acclimated (CA; 5°C) wild-type and uncoupling-protein 1 knockout (UCP1-KO) mice. In wild-type mice, HPmax was successively increased after MCA and CA, and the cold limit was lowered to -8.3°C and -18.0°C, respectively. UCP1-KO mice also increased HPmax in response to MCA and CA, although to a lesser extent. Direct comparison revealed a maximal cold-induced recruitment of heat production by +473 mW and +227 mW in wild-type and UCP1-KO mice, respectively. The increase in cold tolerance of UCP1-KO mice from -0.9°C in MCA to -10.1°C in CA could not be directly related to changes in HPmax, indicating that UCP1-KO mice used the dissipated heat more efficiently than wild-type mice. As judged from respiratory quotients, acutely cold-challenged UCP1-KO mice showed a delayed transition toward lipid oxidation, and 5-h cold exposure revealed diminished physical activity and less variability in the control of metabolic rate. We conclude that BAT is required for maximal adaptive thermogenesis but also allows metabolic flexibility and a rapid switch toward sustained lipid-fuelled thermogenesis as an acute response to cold. In both CA groups, expression of contractile proteins (myosin heavy-chain isoforms) showed minor training effects in skeletal muscles, while cardiac muscle of UCP1-KO mice had novel expression of beta cardiac isoform. Neither respiration nor basal proton conductance of skeletal muscle mitochondria were different between genotypes. In subcutaneous white adipose tissue of UCP1-KO mice, cold exposure increased cytochrome-c oxidase activity and expression of the cell death-inducing DFFA-like effector A by 3.6-fold and 15-fold, respectively, indicating the recruitment of mitochondria-rich brown adipocyte-like cells. Absence of functional BAT leads to remodeling of white adipose tissue, which may significantly contribute to adaptive thermogenesis during cold acclimation.

Functional overload in ground squirrel plantaris muscle fails to induce myosin isoform shifts.

We performed 2 wk of mechanical overload by synergist ablation on plantaris muscles from a small rodent hibernator, Spermophilus lateralis. While this muscle displays prominent myosin heavy-chain (MyHC) isoform shifts during hibernation, sensitivity to mechanical loading as a stimulus for muscle mass and isoform plasticity has not been demonstrated. Squirrel muscles, whether during hibernation or not, potentially are less sensitive to mechanical unloading, but we hypothesized that increased loading would produce the typical mammalian response of greater plantaris mass and MyHC shifts. Mechanical overload produced a 50% increase in muscle mass but, surprisingly, no changes in MyHC isoform protein or mRNA expression, despite previously observed fast-to-slow MyHC isoform switching during hibernation. Citrate synthase enzyme activity, as well as mRNA expression of creatine kinase and the muscle growth factor myostatin, were all unchanged. The mRNA expression of critical muscle atrophy genes decreased by 50% during hypertrophy, including ubiquitin ligases MuRF1 and MAFbx, and the related transcription factor FOXO-1a. Insulin-like growth factor (IGF-1) and hypoxia-inducible factor (HIF-1alpha) mRNA expression was elevated by 400% and 150%. Fast-to-slow MyHC isoform shifts appear unnecessary to support the increased recruitment of the plantaris muscle, shifts which are seen in other rodent models. Our results are consistent with muscular activity during interbout arousals as a potential mechanism to preserve muscle mass, but illustrate the primary importance of other seasonal factors besides patterns of muscle activation which must act in concert to alter MyHC isoforms and muscle fiber type during hibernation.

Exhaustive exercise training enhances aerobic capacity in American alligator (Alligator mississippiensis).

The oxygen transport system in mammals is extensively remodelled in response to repeated bouts of activity, but many reptiles appear to be 'metabolically inflexible' in response to exercise training. A recent report showed that estuarine crocodiles (Crocodylus porosus) increase their maximum metabolic rate in response to exhaustive treadmill training, and in the present study, we confirm this response in another crocodilian, American alligator (Alligator mississippiensis). We further specify the nature of the crocodilian training response by analysing effects of training on aerobic [citrate synthase (CS)] and anaerobic [lactate dehydrogenase (LDH)] enzyme activities in selected skeletal muscles, ventricular and skeletal muscle masses and haematocrit. Compared to sedentary control animals, alligators regularly trained for 15 months on a treadmill (run group) or in a flume (swim group) exhibited peak oxygen consumption rates higher by 27 and 16%, respectively. Run and swim exercise training significantly increased ventricular mass (~11%) and haematocrit (~11%), but not the mass of skeletal muscles. However, exercise training did not alter CS or LDH activities of skeletal muscles. Similar to mammals, alligators respond to exercise training by increasing convective oxygen transport mechanisms, specifically heart size (potentially greater stroke volume) and haematocrit (increased oxygen carrying-capacity of the blood). Unlike mammals, but similar to squamate reptiles, alligators do not also increase citrate synthase activity of the skeletal muscles in response to exercise.

Maintenance of slow type I myosin protein and mRNA expression in overwintering prairie dogs (Cynomys leucurus and ludovicianus) and black bears (Ursus americanus).

Hibernating mammals have the remarkable ability to withstand long periods of fasting and reduced activity with dramatic maintenance of skeletal muscle function and protein composition. We investigated several hindlimb muscles of white-tailed prairie dogs (Cynomys leucurus) and black bears (Ursus americanus), two very different hibernators who are dormant and fasting during winter. The black-tailed prairie dog (C. ludovicianus) remains active during winter, but suffers minor skeletal muscle atrophy; nevertheless, they also demonstrate apparent skeletal muscle adaptations. Using SDS-PAGE, we measured myosin protein isoform profiles before and after the hibernation season. All species maintained or increased levels of slow myosin, despite the collective physiological challenges of hypophagia and reduced activity. This contrasts markedly with standard mammalian models of skeletal muscle inactivity and atrophy predicting significant loss of slow myosin. A mechanism for changes in myosin isoforms was investigated using reverse-transcription PCR, following partial sequencing of the adult MHC isoforms in C. leucurus and U. americanus. However, mRNA expression was not well correlated with changes in MHC protein isoforms, and other synthesis and degradation pathways may be involved besides transcriptional control. The muscles of hibernating mammals demonstrate surprising and varied physiological responses to inactivity and atrophy with respect to slow MHC expression.

Postprandial increase of oleoylethanolamide mobilization in small intestine of the Burmese python (Python molurus).

Oleoylethanolamide (OEA) is an endogenous lipid mediator that inhibits feeding in rats and mice by activating the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPAR-alpha). In rodents, intestinal OEA levels increase about threefold upon refeeding, a response that may contribute to the induction of between-meal satiety. Here, we examined whether feeding-induced OEA mobilization also occurs in Burmese pythons (Python molurus), a species of ambush-hunting snakes that consume huge meals after months of fasting and undergo massive feeding-dependent changes in gastrointestinal hormonal release and gut morphology. Using liquid chromatography/mass spectrometry (LC/MS), we measured OEA levels in the gastrointestinal tract of fasted (28 days) and fed (48 h after feeding) pythons. We observed a nearly 300-fold increase in OEA levels in the small intestine of fed compared with fasted animals (322 +/- 121 vs. 1 +/- 1 pmol/mg protein, n = 3-4). In situ OEA biosynthesis was suggested by the concomitant increase of N-acyl phosphatidylethanolamine species that serve as potential biosynthetic precursors for OEA. Furthermore, we observed a concomitant increase in saturated, mono- and diunsaturated, but not polyunsaturated fatty-acid ethanolamides (FAE) in the small intestine of fed pythons. The identification of OEA and other FAEs in the gastrointestinal tract of Python molurus suggests that this class of lipid messengers may be widespread among vertebrate groups and may represent an evolutionarily ancient means of regulating energy intake.

Physiology: postprandial cardiac hypertrophy in pythons.

Oxygen consumption by carnivorous reptiles increases enormously after they have eaten a large meal in order to meet metabolic demands, and this places an extra load on the cardiovascular system. Here we show that there is an extraordinarily rapid 40% increase in ventricular muscle mass in Burmese pythons (Python molurus) a mere 48 hours after feeding, which results from increased gene expression of muscle-contractile proteins. As this fully reversible hypertrophy occurs naturally, it could provide a useful model for investigating the mechanisms that lead to cardiac growth in other animals.

Myosin isoform expression and MAFbx mRNA levels in hibernating golden-mantled ground squirrels (Spermophilus lateralis).

Hibernating mammals present many unexplored opportunities for the study of muscle biology. The hindlimb muscles of a small rodent hibernator (Spermophilus lateralis) atrophy slightly during months of torpor, representing a reduction in the disuse atrophy commonly seen in other mammalian models. How torpor affects contractile protein expression is unclear; therefore, we examined the myosin heavy-chain (MHC) isoform profile of ground squirrel skeletal muscle before and after hibernation. Immunoblotting was performed first to identify the MHC isoforms expressed in this species. Relative percentages of MHC isoforms in individual muscles were then measured using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The soleus and diaphragm did not display differences in isoforms following hibernation, but we found minor fast-to-slow isoform shifts in MHC protein in the gastrocnemius and plantaris. These subtle changes are contrary to those predicted by other models of inactivity but may reflect the requirement for shivering thermogenesis during arousals from torpor. We also measured mRNA expression of the Muscle Atrophy F-box (MAFbx), a ubiquitin ligase important in proteasome-mediated proteolysis. Expression was elevated in the hibernating gastrocnemius and the plantaris but was not associated with atrophy. Skeletal muscle from hibernators displays unusual plasticity, which may be a combined result of the intense activity during arousals and the reduction of metabolism during torpor.

Cloning and sequencing of myosin heavy chain isoform cDNAs in golden-mantled ground squirrels: effects of hibernation on mRNA expression.

The golden-mantled ground squirrel is a small rodent hibernator that demonstrates unusual myosin heavy chain (MHC) isoform plasticity during several months of torpor, punctuated by bouts of rewarming and shivering thermogenesis. We measured MHC mRNA levels to determine whether pretranslational control mechanisms were responsible for differences in MHC2x protein expression, as we previously observed between active and hibernating ground squirrels. We first cloned cDNA using the 3' rapid amplification of cDNA ends (3' RACE) technique and identified three sequences corresponding to MHC1, MHC2x, and MHC2b. A DNA control fragment was developed to be used in conjunction with a coupled RT-PCR reaction to simultaneously measure MHC mRNA levels for each isoform in the skeletal muscle of ground squirrels. MHC mRNA and protein expression were strongly correlated, and type IIx and IIb mRNA levels were significantly different between active and hibernating ground squirrels. Pretranslational control of MHC protein is apparently an important process during hibernation, although the exact stimulus is not known. The techniques presented can be used to obtain MHC cDNA sequences and to measure mRNA expression in many vertebrate groups.