Inho Choi

INVERSE RELATIONSHIP BETWEEN FUNCTIONAL MATURITY AND EXPONENTIAL GROWTH RATE OF AVIAN SKELETAL MUSCLE: A CONSTRAINT ON EVOLUTIONARY RESPONSE

In this study, we investigate whether a tissue‐level constraint can explain the general inverse relationship between growth rate and precocity of development in birds. On the whole, altricial (dependent) chicks grow three to four times faster than the less dependent, more able chicks of precocial species of similar adult mass. We suggest that an antagonism between growth and acquisition of mature function in skeletal muscle constrains postnatal growth and development in most species of birds. Altricial species, represented by European starlings in this study, hatch with skeletal muscle having low capacity for generating force but grow rapidly. Conversely, precocial species (northern bobwhite quail and Japanese quail), hatch with relatively mature skeletal muscle, especially in their legs, but grow more slowly. As development proceeds in all species, exponential growth rates decrease as muscles acquire adult levels of function. Among four variables associated with muscle function, exponential growth rate (EGR) was negatively correlated with pyruvate kinase activity (glycolysis), potassium concentration (electrical potential), and dry weight fraction (contractile proteins) in both pectoral and leg muscles but not with citrate synthase activity (aerobic metabolism) in either set of muscles. For pectoral muscle, these variables accounted for 87% of the total variation in EGR in all three species combined despite a twofold difference in growth rates between the starling and quail. EGRs of leg muscle (51% of variation accounted for) were less than predicted by the pectoral‐muscle equation in quail during the early part of the postnatal period and in starlings during the late postnatal period. This result would not contradict a growth rate/maturity constraint hypothesis if EGRs were down‐regulated for allometric or other considerations.

Skeletal Muscle Growth, Enzyme Activities, and the Development of Thermogenesis: A Comparison between Altricial and Precocial Birds

We examined activities of enzymes indicative of aerobic capacity (citrate synthase [CS]) and glycolytic capacity (pyruvate kinase [PK]) in pectoral and leg muscles of chicks of the European starling, northern bobwhite, and Japanese quail. The starling exhibits altricial development; the nestlings are highly dependent on their parents for food and maintenance of a high body temperature. In contrast, the quail species exhibit precocial development; at hatching, the chicks can feed themselves and thermoregulate under mild cold stress. In all three species, the proportion of muscle in the body increased with age; in addition, the proportion of pectoral muscle increased relative to that of leg muscle. Pyruvate kinase activity increased in all muscles, but especially in the pectoral muscles of the quail (from approximately 50 to 800 μmol/min · g wet mass [IU/g] through day 21). In contrast, CS increased rapidly in the pectoral muscle of the starling (from 10 to 150 IU/g between hatching and 16 d) but not in quail. Citrate synthase activity initially was high in the leg muscle of quail (40-70 IU/g) but decreased with age; in starling leg muscle, CS activity increased through day 8 and then remained constant at about 45-50 IU/g. In the bobwhite, lactate dehydrogenase activity, which is associated with glycolytic metabolism, closely paralleled that of PK Muscle-mass-specific metabolic scope, estimated from peak oxygen consumption under cold stress, was higher in neonates of both quail species than in starling neonates, but within 2 wk it increased to a higher level in the starling (30-38 mL/h · g muscle). In general, total metabolic scope per individual varied in proportion to the total CS activity, not PK activity, of the pectoral and leg muscles, regardless of species and age. However, increases in the activities of both CS and PK activities in the starling during the first week were not accompanied by development of thermogenic capacity.

Activation of stress signaling molecules in bat brain during arousal from hibernation

Induction of glucose‐regulated proteins (GRPs) is a ubiquitous intracellular response to stresses such as hypoxia, glucose starvation and acidosis. The induction of GRPs offers some protection against these stresses in vitro, but the specific role of GRPs in vivo remains unclear. Hibernating bats present a good in vivo model to address this question. The bats must overcome local high oxygen demand in tissue by severe metabolic stress during arousal thermogenesis. We used brain tissue of a temperate bat Rhinolopus ferrumequinum to investigate GRP induction by high metabolic oxygen demand and to identify associated signaling molecules. We found that during 30 min of arousal, oxygen consumption increased from nearly zero to 11.9/kg/h, which was about 8.7‐fold higher than its active resting metabolic rate. During this time, body temperature rose from 7°C to 35°C, and levels of TNF‐α and lactate in brain tissue increased 2–2.5‐fold, indicating a high risk of oxygen shortage. Concomitantly, levels of GRP75, GRP78 and GRP94 increased 1.5–1.7‐fold. At the same time, c‐Jun N‐terminal protein kinase (JNK) activity increased 6.4‐fold, and extracellular signal‐regulated protein kinase (ERK) activity decreased to a similar degree (6.1‐fold). p38 MAPK activity was very low and remained unchanged during arousal. In addition, survival signaling molecules protein kinase B (Akt) and protein kinase C (PKC) were activated 3‐ and 5‐fold, respectively, during arousal. Taken together, our results showed that bat brain undergoes high oxygen demand during arousal from hibernation. Up‐regulation of GRP proteins and activation of JNK, PKCγ and Akt may be critical for neuroprotection and the survival of bats during the repeated process.

Behavior and Muscle Performance in Heterothermic Bats

Body temperatures of winter‐resident Korean bats typically range from 10° to 40°C between August and September and from 3° to 15°C between January and April. To learn how behavior and the motor systems of heterothermic bats respond to this body‐temperature variation, we examined whole‐organism performance and the temperature‐dependence of contractile properties of flight muscle in Murina leucogaster ognevi. In winter and midspring, the lowest limits of body temperature were 8°C for biting and crawling, 16°C for visually observable shivering, 22°C for wing flapping (without powered flight), and 28°C for aerial flight. In summer, the lowest temperature limits changed little for biting and wing flapping, but the temperature limits increased about 3°C for crawling, shivering, and flight. Maximum isometric tetanic tension of the isolated biceps brachii muscle was almost insensitive to tissue temperatures between 10° and 40°C, with an average temperature coefficient of 1.02 in summer and of 0.96 in winter. Rate of tetanic tension production between 10° and 40°C and shortening velocity and power between 15° and 25°Cwere temperature sensitive, with average temperature coefficients of 1.3–2.3. Seasonal differences in contractile properties within each temperature were not significant, except for maximum tetanic tension at 30°–40°C. Thus, the motor system of the bats had functional capacity over the range of body temperature experienced in winter to summer. The temperature‐dependence of behavior was consistent with muscle physiology. The defensive behaviors, like biting and crawling, observed at 8°–12°C body temperature could be exerted by using temperature‐independent tetanic tension, whereas activities, such as flight, that require power generation would be restricted to higher body temperatures by temperature‐sensitive rate properties. Some rate processes appeared to be more temperature sensitive in summer than in winter.

Molecular mechanism underlying muscle mass retention in hibernating bats: Role of periodic arousal

Hibernators like bats show only marginal muscle atrophy during prolonged hibernation. The current study was designed to test the hypothesis that hibernators use periodic arousal to increase protein anabolism that compensates for the continuous muscle proteolysis during disuse. To test this hypothesis, we investigated the effects of 3‐month hibernation (HB) and 7‐day post‐arousal torpor (TP) followed by re‐arousal (RA) on signaling activities in the pectoral muscles of summer‐active (SA) and dormant Murina leucogaster bats. The bats did not lose muscle mass relative to body mass during the HB or TP‐to‐RA period. For the first 30‐min following arousal, the peak amplitude and frequency of electromyographic spikes increased 3.1‐ and 1.4‐fold, respectively, indicating massive myofiber recruitment and elevated motor signaling during shivering. Immunoblot analyses of whole‐tissue lysates revealed several principal outcomes: (1) for the 3‐month HB, the phosphorylation levels of Akt1 (p‐Akt1) and p‐mTOR decreased significantly compared to SA bats, but p‐FoxO1 levels remained unaltered; (2) for the TP‐to‐RA period, p‐Akt1 and p‐FoxO1 varied little, while p‐mTOR showed biphasic oscillation; (3) proteolytic signals (i.e., atrogin‐1, MuRF1, Skp2 and calpain‐1) remained constant during the HB and TP‐to‐RA period. These results suggest that the resistive properties of torpid bat muscle against atrophy might be attained primarily by relatively constant proteolysis in combination with oscillatory anabolic activity (e.g., p‐mTOR) corresponding to the frequency of arousals occurring throughout hibernation. J. Cell. Physiol. 222: 313–319, 2010.

Variations in take-off velocity of anuran amphibians: Relation to morphology, muscle contractile function and enzyme activity☆

The relationship between variability of take-off velocity and variation in skeletomuscular features was examined in three anuran species, Rana nigromaculata, R. rugosa and Bombina orientalis. Video analyses on “maximal” take-off trials of individuals indicated that average take-off velocity (m · s−1) of R. nigromaculata (2.35 ± 0.17 SD, n = 14) and R. rugosa (2.33 ± 0.11 SD, n = 8) was significantly greater than that of Bombina (1.74 ± 0.12, n = 8). Body mass (9.2 ± 3.3 SD for R. nigromaculata; 11.5 ± 5.6 SD for R. rugosa and 6.5 ± 0.8 SD for B. orientalis) did not affect take-off velocity within each species. Compared to Bombina, the two ranid species showed longer hindlimbs relative to body length, greater mass of thigh muscles relative to body mass and a narrower interilial width (at the sacral vertebra) relative to body length. The two ranid species also exhibited significantly shorter tetanic rise time (TRT) and greater rate of force production (dFdt) examined on the gastrocnemius muscle. Isometric tetanic force, ranging between 189 and 272 mN · mm−2, was essentially the same among the three species. Activity of lactate dehydrogenase, an enzyme involved in anaerobic metabolism, of the gastrocnemius muscle, was statistically indistinguishable among the three species. The activity of citrate synthase (CS), indicative of aerobic catalytic capacity, was significantly lower in R. nigromaculata than in either R. rubosa or B. orientalis, while the CS activity was not different between the latter two species. These results indicate that the faster jumpers have a more effective skeletomuscular system (relatively longer hindlimbs and more musculature, higher contraction rate) that can generate a faster out-velocity and a greater out-force against gravity. The enzyme assay study was not sufficient to support the observed relationships in these species.

Nuclear factor kappa B-mediated kainate neurotoxicity in the rat and hamster hippocampus

Administration of the excitotoxin kainate produces seizure activity and selective neuronal death in various brain areas. We examined the degeneration pattern of hippocampal neurons following systemic injections of kainate in the hamster and the rat. As reported, treatment with kainate resulted in severe neuronal loss in the hilus and CA3 in the rat. While the hilar neurons were also highly vulnerable to kainate in the hamster, neurons in the CA1 area, but not CA3, were highly sensitive to kainate. In both animals, immunoreactivity to anti-p50 nuclear factor kappa B antibody was increased in nuclei of the hilar neurons within 4 h following administration of kainate. Kainate treatment also increased the nuclear factor kappa B immunoreactivity in hamster CA1 neurons and rat CA3 neurons 24 h later. Neurons showing intense nuclear factor kappa B signal were stained with acid fuchsin. Kainate also increased DNA binding activity of p50 and p65 nuclear factor kappa B in the nuclear extract of the hippocampal formation as analysed by electrophoretic mobility shift assay in the hamster, suggesting that activation of nuclear factor kappa B may contribute to kainate-induced hippocampal degeneration. Administration of 100 nmol dizocilpine maleate 3 h prior to kainate attenuated kainate-induced activation of nuclear factor kappa B and neuronal death in CA1 in the hamster. The present study provides evidence that the differential vulnerability of neurons in the rat and the hamster hippocampus to kainate is partly mediated by mechanisms involving N-methyl-D-aspartate-dependent activation of nuclear factor kappa B.

Stress and signaling responses of rat skeletal muscle to brief endurance exercise during hindlimb unloading: a catch-up process for atrophied muscle.

At times, exercise accompanied by its anabolic effects is not a tractable countermeasure to muscle atrophy. Instead, training is often attempted after the affected muscle has atrophied greatly as a result of unloading. This study was designed to elucidate stress and signaling mechanisms underlying a process of muscle catch-up growth as a result of transitory exercise during unloading. Rats were exercised daily with a routine of 20- or 40-minute treadmill running (at 60% of maximum oxygen uptake) during the second week of a two-week hindlimb suspension. We examined the expression and activation of heat shock proteins and anabolic and proteolytic markers in the rat soleus muscle. Muscle mass relative to body mass decreased 2.4-fold in the unloaded group (HU) with respect to controls but decreased only 1.7-fold in the 40-min trained group (HT40) (P < 0.05) - equivalent to a 1.4-fold increase in the relative muscle mass over HU. Immunoblotting analyses on whole-tissue lysates demonstrated the following: (1) HSP72 and αB-crystallin were upregulated 7- and 2.5-fold, respectively, in HT40 versus HU; (2) phosphorylation of Akt1 and p70/S6K decreased only slightly in HU; (3) when compared to HU, HT40 phosphorylation of Akt1, S6K, and FoxO1 increased 1.4- to 3.0-fold while phosphorylation of FoxO3 was unchanged; and (4) activities of the ubiquitin E3 ligases, calpain 1 and caspase-3 increased 2- to 4-fold in the unloaded groups regardless of exercise duration. These results suggest that the significant upregulation of chaperones and anabolic markers (e.g., HSP72, p-Akt1, p-S6K) in HT40, along with the lack of the training effect on proteolytic activity, is likely crucial for muscle mass catch-up in the unloaded muscle.

Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages.

Skeletal muscle is one of the most sensitive tissues to mechanical loading, and unloading inhibits the regeneration potential of skeletal muscle after injury. This study was designed to elucidate the specific effects of unloading stress on the function of immunocytes during muscle regeneration after injury. We examined immunocyte infiltration and muscle regeneration in cardiotoxin (CTX)-injected soleus muscles of tail-suspended (TS) mice. In CTX-injected TS mice, the cross-sectional area of regenerating myofibers was smaller than that of weight-bearing (WB) mice, indicating that unloading delays muscle regeneration following CTX-induced skeletal muscle damage. Delayed infiltration of macrophages into the injured skeletal muscle was observed in CTX-injected TS mice. Neutrophils and macrophages in CTX-injected TS muscle were presented over a longer period at the injury sites compared with those in CTX-injected WB muscle. Disturbance of activation and differentiation of satellite cells was also observed in CTX-injected TS mice. Further analysis showed that the macrophages in soleus muscles were mainly Ly-6C-positive proinflammatory macrophages, with high expression of tumor necrosis factor-α and interleukin-1β, indicating that unloading causes preferential accumulation and persistence of proinflammatory macrophages in the injured muscle. The phagocytic and myotube formation properties of macrophages from CTX-injected TS skeletal muscle were suppressed compared with those from CTX-injected WB skeletal muscle. We concluded that the disturbed muscle regeneration under unloading is due to impaired macrophage function, inhibition of satellite cell activation, and their cooperation.

Seasonal biochemical plasticity of a flight muscle in a bat, Murina leucogaster

Cellular and biochemical responses of the pectoral muscle to variation in seasonal activity were studied in the bat, Murina leucogaster ognevi. We collected bats in mid-hibernation (February), end-hibernation (April), and mid-summer (August) to track major activity periods in their annual cycle. Our findings indicated that myofiber cross-sectional area decreased to 68% between mid- and end-hibernation, but returned to the winter level in mid-summer. Total soluble protein and total RNA concentrations were not altered over these sampling periods. Oxidative potential gauged by citrate synthase activity increased 1.47-fold from mid- to end-hibernation and then remained at the similar level in mid-summer. Glycolytic potential gauged by lactate dehydrogenase activity changed little between mid- and end-hibernation but increased 1.42-fold in summer, compared with the winter level. Thus, the myofibers underwent disuse atrophy during hibernation, while enzymatic catalytic function recovered towards the level of mid-summer.