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Characteristics of diaphragm muscle fibre types in hibernating squirrels
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ELSEVIER
Respiration Physiology101 (1995) 301-309
Characteristics of diaphragm muscle fibre types in hibernating squirrels W. Darlene Reid a,*, Anna Ng a, R. Keith Wilton a, William K. Milsom b a
School of Rehabilitation Sciences, T325-2211 WesbrookMall, Universityof British Columbia, Vancouver, B.C., Canada, V6T2B5 b Department of Zoology, 6270 UniversityBlvd., Universityof British Columbia, Vancouver, B.C., Canada, V6T 1Z4
Accepted 3 April 1995
Abstract
Muscle samples obtained from the diaphragm of 7 fall-awake (FA), 10 winter-awake (WA), and 8 hibernating (H) squirrels (Spermophilus lateralis) were quick frozen, sectioned and processed for NADH-TR reaction end-product and myofibrillar-ATPase. Both WA and H squirrels showed small increases in diaphragm weight, reductions in body weight, and hence, significant increases in the diaphragm weight to body weight ratio compared to FA squirrels. They also showed increases in muscle fibre type cross-sectional areas and in the oxidative capacity of type 2b fibres as well as a reduction in capillary density. Furthermore, there also was an increase in the proportion of type 2b fibres in the diaphragm of H squirrels. Thus, despite the dramatically reduced ventilation associated with hibernation, H squirrels exhibited (1) hypertrophy of the diaphragm which may represent an adaptive response that enables them to work against a stiffer chest wall, and (2) an increased oxidative capacity which enables them to fuel this with fat. Keywords: Hibernation; Mammals, squirrel (Spermophilus lateralis); Muscle, fibre type
1. Introduction
The hibernating mammal would appear to be a good model to study disuse atrophy of skeletal muscle. There is a profound decrease in the activity of limb muscles, and ventilatory levels decrease to approximately 8% of normal levels during hibernation. Even though the basal metabolic rate is reduced, an energy supply must be maintained during long periods of hibernation. Much of the energy appears to be derived from fat, however additional energy demands are met by protein. Skeletal muscle is by far
* Corresponding author, Tel: (604) 822-7402 FAX: (604) 8227624 Email: [email protected].
the largest protein reserve and the most important source of gluconeogenic precursors during prolonged fasting in normothermic mammals (Yacoe, 1983a). Catabolism of skeletal muscle in the hibernator would result in muscle fibre atrophy, and could result in loss of function and decreased survival, Although skeletal muscle atrophy of limb muscles following hibernation has been found in several species (Steffen et al., 1991; Wickler et al., 1987; Wickler et al., 1991; Yacoe, 1983a), reports are inconsistent, and decreased muscle size has not always been found (Vyskocil and Gutmann, 1977). Careful investigation of the diaphragm has not yet been performed. The diaphragm may respond differently to hibernation than other skeletal muscles for a couple of
0034-5687/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0034-5687(95)00036-4
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reasons. We found a decrease in specific lung compliance and a significant increase in both the elastic and total work required to ventilate hibernating animals at normal tidal volumes at mechanical ventilation frequencies of 100 and 150 breaths per min (Milsom and Reid, 1995). This decreased compliance at low lung volumes and increased work may provide a stimulus to maintain or increase inspiratory muscle mass even though respiration frequency is reduced in hibernators. Further, significant atrophy of the diaphragm during hibernation could have a very deleterious effect on survival if the atrophy prevented the animal from producing sufficient inspiratory muscle force to cope with the immediate and continuous loads of ventilation during and after arousal. Significant atrophy of the diaphragm in mechanically ventilated patients leads to difficulty in
weaning, and even death due to respiratory complications because of failure to wean. A better understanding of the fibre characteristics of the diaphragm in hibernating animals may be helpful in designing interventions to prevent the disuse atrophy that occurs in patients maintained on prolonged mechanical ventilation. The purpose of this study was to examine the effects of hibernation on muscle mass and fibre type sizes, fibre type proportions, and oxidative capacity of the squirrel diaphragm. We hypothesized that the diaphragm would undergo atrophy to a lesser extent than limb muscles in response to disuse during hibernation, in part because of continued respirations (albeit less frequent) and in part, because of the increased work required to overcome the decreased compliance of the respiratory system.
Fig. 1. Serial cross-sectionsof costal diaphragmprocessedfor M-ATPaseat preincubationpH's of 4.2 (A), 4.45 (B), 9.4(C), and NADH-TR (D). Scalebar is 25 /xm.
W.D. Reid et al. / Respiration Physiology 101 (1995) 301-309
2. Methods
Animals. Three groups of squirrels (Spermophilus lateralis) were studied: (1) seven fall-awake animals (FA) that had been captured at the end of August and held in captivity for three weeks; (2) ten winter awake animals (WA) that had been held in captivity for five months at 23°C.; and (3) eight hibernators (H) that had been held for 3 to 4 months at 5°C. The body temperature of H squirrels just before sacrifice was between 5 and 7°C.
Diaphragm muscle samples.. Following anesthesia with an intraperitoneal injection of sodium pentobarbital (Somnotol) (6.5 rag/100 g body weight), the animal was tracheotomized and placed in a pressure plethysmograph (Milsom and Reid, 1995). Following measurements of pulmonary mechanics, the squirrel was removed from the pressure plethysmograph. Through a midline abdominal incision, the entire diaphragm was excised and placed upon phosphatebuffered saline-soaked gauze. The diaphragm was cut in half and the right half was weighed. For histochemical analysis, muscle samples were cut from the anterior lateral costal region of the left hemi-diaphragm, mounted on gum tragacanth and frozen in isopentane cooled to the temperature of liquid nitrogen. Given the size of the diaphragm, these samples utilized most of the costal region. Muscle samples were stored at -70°C. until subsequent biochemical or histochemical processing.
Muscle Histochemistry.. Serial cross-sections were cut at 10 and 16 /zm thickness on a cryostat-microtome (Reichart-Jung). The 16 /zm sections were processed for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) (Reid et al., 1992) and the 10 /zm sections were processed for myosin adenosine-triphosphatase (M-ATPase) using preincubation pH's of 4.2, 4.45 and 9.4 (Reid et al., 1989) (Fig. 1). All cross-sections were cut and stained on the same day in the same batch to avoid possible artifacts that could arise from interassay variation.
Cross-sectional Area and Optical Density Measurements.. The cross-sectional area and optical density of the histochemical reaction for NADH-TR of muscle fibres were quantified using a computer-based
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image processing system (Kontron SEM-IPS, Munich) that consisted of a video monitor and a computer for the processing of images. The monitor was connected to a video camera (Model 68, Dage MTI) attached to a light microscope (Zeiss Universal research). A filter on the light source of the microscope was used to produce a narrow band of transmitted light with a wavelength spectrum that peaked at 546 nm which corresponded closely to the maximal absorbance of NADH-TR reaction end product. The video image of the muscle section was composed of a matrix of 480 × 512 pixels. Using a microscope magnification of × 20, each pixel had an area of 0.5568 /~m 2. A stage micrometer (Zeiss) delineated in microns was used to check the calibration of the vertical and horizontal axes of the video image daily. The image processing system had the ability to discern 255 grey levels. Calibration for optical densities or grey levels was performed at the beginning of each session following standard technique (Wied, 1966; Zimmer, 1973). For measurement of cross-sectional area and optical densities of each muscle fibre, the perimeter of the cross-sectional profile of the fibre was outlined using a mouse on a digitizing pad connected to the video monitor. The optical density of each pixel within the outlined fibre cross-section was determined and the mean optical density of all the pixels within the fibre was determined. Mitochondria (identified by NADH-TR) are distributed non-homogeneously, especially within the type 1 fibre which can have a large subsarcolemmal population of mitochondria (Fig. 1, panel D). Determining mean optical density by averaging the optical densities of each pixel within the cross-sectional area of the muscle fiber ensured the maximal inclusion of any deeply stained sub-sarcolemmal matter. Cross-sectional areas and optical density measures were determined for 200 fibers in the costal diaphragm from each squirrel. Muscle fibres were then typed as type 1, 2a, 2b (Reid et al., 1989) or 2c (Dubowitz, 1985) by identifying the same fibres in serial sections stained for M-ATPase at the three different pre-incubation pH's. Using this fibre typing nomenclature, type 1 fibres are dark when stained for M-ATPase after pre-incubation at pH of 4.2 and pH of 4.45 but intermediate after pre-incubation at a pH of 9.4; type 2a fibres are light when stained for M-ATPase after
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pre-incubation at pH of 4.2 and pH of 4.45 but dark after pre-incubation at a pH of 9.4; type 2b fibres are light when stained for M-ATPase after pre-incubation at pH of 4.2, intermediate in coloration after preincubation at a pH of 4.45 but dark after pre-incubation at a pH of 9.4; and type 2c fibres are intermediate when stained for M-ATPase after preincubation at pH of 4.2, dark in coloration after preincubation at a pH of 4.45 and dark after pre-incubation at a pH of 9.4, (Dubowitz, 1985). Fibre type proportions were determined by counting the number of fibres in each of the four fibre type categories and then dividing the number by the total number of fibres examined (200 fibres/biopsy).
2.2. Capillary Density Capillaries were visualized from sections stained for M-ATPase after acid (pH 4.2) preincubation as described by Sillau and Banchero (1977). Using the computer-based image processing system (Kontron SEM-IPS, Munich), a computer program was designed to determine: (1) capillaries per fibre; (2) capillaries surrounding type 1 or type 2 fibres; and (3) capillaries per cross-sectional area. At a microscope magnification of 310 × , the image was projected onto a monitor with a counting frame (190 X 230 microns). The number of capillaries per fibre was defined from the counts of muscle fibres and capillaries within and touching two sides of the counting frame. Any fibres or capillaries that were touching the other two sides of the counting frame were excluded from the counts. Capillaries surrounding type I and type 2 fibres were defined as all capillaries surrounding a specific fibre type. Since a capillary may lie between 2 or more fibres, capillaries may be counted more than once for this variable. Capillaries per cross-sectional area was defined as the number of capillaries within and touching two sides of the counting frame divided by the cross-sectional area of the counting frame.
2.3. Statistical analysis To test for differences among animal groups, a multivariate analysis was performed on the measures of diaphragm and body weight (diaphragm mass, body weight and diaphragm mass to body weight
ratio), and measures of capillary density. To test for differences among groups in fibre type proportions, the square root of the arcsin was taken in order to achieve a normal distribution and then an ANOVA was performed. To test for differences among groups in muscle fibre type cross-sectional areas, and optical densities, a multivariate analysis of variance was used (MANOVA) (Fleiss, 1986). Because the distributions of cross-sectional areas and optical densities were not normally distributed and in order to examine a broader distribution than shown by the median alone, each animal's optical density and cross-sectional area values were reduced to three values (the lower quartile, the median, and the upper quartile) (Zar, 1984). MANOVAs were performed on these three values in order to examine for interaction between group and fiber type, and to examine for differences among groups Interaction between animal group and fibre type was considered significant at p < 0.15 and no significant interaction between these two factors was present. Differences among groups were only considered to be different if all three values (the upper quartile, lower quartile, and median value) were different among groups at a P < 0.05.
3. Results WA squirrels weighed less ( P < 0.02) and H squirrels tended to weigh less than the FA squirrels (Table 1). All three groups had a similar diaphragm mass but the WA ( P < 0.001) and H ( P < 0.02) had a higher diaphragm weight to body weight ratio than FA squirrels (Table 1). Regarding muscle fibre type
Table 1 Diaphragm weight and body weight Group DiaphragmB o d y weight (g) weight(g) Fall Awake 0.63 + 0.13 219 + 33 Winter Awake 0.76+ 0.20 173 + 31 b Hibernating 0.75+0.17 193+30
Diaphragmweight/ Bodyweight ratio a 0.29 +_0.04 0.44± 0.09 c 0.39+0.05 b
a The diaphragmweight/body weight ratio is multiplied by 100 b significantly different than Fall Awake at P < 0.02. e significantlydifferent than Fall Awake at P < 0.001.
W.D. Reid et aL /Respiration Physiology 101 (1995) 301-309
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response to disuse during hibernation, in part because of continued respirations and in part, because of increased work required to overcome the decreased compliance of the respiratory system. The data show that the diaphragm in fact undergoes hypertrophy (in both WA and H squirrels), and further shows alterations in fibre type proportions and sizes, and an increased oxidative capacity.
0 Type 1
Tyloe 2a
Type 2b
Fig. 2. Muscle fibre type proportions of costal diaphragm in FA, WA and H squirrels. Means+ SD are shown. *H significantly different than WA at P < 0.05.
proportions, H squirrels had more type 2b fibres and fewer type 2a fibres in the costal diaphragm than those in the WA squirrels (Fig. 2). Of interest, less than 1% of fibres were type 2c fibres in the costal diaphragm of all three groups. Regarding muscle fibre size, H squirrels had larger type 1 and type 2a fibres in the costal diaphragm than those fibres in the FA and WA squirrels (Fig. 3). H squirrels also had larger type 2b fibres in the diaphragm than those fibres in the FA squirrels (Fig. 3). When the optical densities of all fibres regardless of type were grouped, analysis of optical densities of NADH-TR reaction end product showed similar optical densities of diaphragm muscle fibres in H and WA squirrels, but optical densities of these two groups were higher than those in the FA group indicating that the diaphragms of H and WA squirrels, in general, were more oxidative than those for FA squirrels. When individual fibre types were analyzed, the optical densities of NADH-TR reaction end-product of the type 2b fibres in the diaphragm of the WA group were more oxidative than those fibres in the FA squirrels (Fig. 4). Capillary densities expressed per fibre or per type 1 or 2 fibre did not differ among groups (Table 2). However, capillaries per cross-sectional area (expressed per mm:) were less in the diaphragm of the WA and H than in the FA squirrels (Table 2).
4. Discussion
We hypothesized that the diaphragm would undergo atrophy to a lesser extent than limb muscles in
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Fig. 3. Muscle fibre type cross-sectional areas in FA (O), WA ( • ) and H (El) squirrels. Vertical bars indicate SE of lower quartiles, medians and upper quartiles of animals in each group. Values for H animals are significantly different than those for FA animals for all fibre types ( P < 0.05) and than those for WA animals for type I and type 2a fibres ( P ___0.05).
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4.1. Diaphragm mass, a r e a s a n d proportions
TYPE 1
1.0
and fibre type cross-sectional
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0.9 0.8 0.7 0.6 ~i
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Fig. 4. Muscle fibre type optical densities in F A ( O ) , W A ( v ) and H ( [ ] ) squirrels, Vertical bars indicate SE of lower quartiles,
medians and upper quartiles of animals in each group. Values for W A animals are significantly different than those of FA animals for type 2b fibres ( P < 0.05).
The maintenance of muscle weight, increase in muscle fibre type cross-sectional areas, and increased proportion of type 2b fibres in the diaphragm of H squirrels relative to FA squirrels are all indicative of increased stress being placed on the diaphragm in hibernating animals. We hypothesize that this increased stress arises primarily from the increased tension which must have to be generated against the less compliant respiratory system of these hibernating animals (specific compliance = 0.27, 0.32, and 0.12 m l / c m H 2 0 / m l FRC for FA, WA and H squirrels, respectively [Milsom and Reid, 1995]). Since tidal volume is maintained and compliance decreases during hibernation, each breath requires more work (2.75, 2.25, and 6.54 ml. cmH20 were required to pump 1 ml of air at a rate of 100 per min into the respiratory systems of FA, WA, and H animals, respectively). Hibernators still continued to contract their diaphragm but on average, this was only three times per min, approximately 8% of normal ventilatory levels. So although the frequency of diaphragm contractions and the power output of the diaphragm are greatly reduced during hibernation, the work required per breath is increased and this could have induced the increase in proportion of 2b fibers and the increase in cross-sectional area of all fiber types in the hibernating squirrels. The maintenance of diaphragm weight and increase in muscle fibre cross-sectional area of all fibre types of the diaphragm in hibernating squirrels is in sharp contrast to observations of limb skeletal mus-
Table 2 Capillary densities in the squirrel diaphragm Group
Capillaries per fiber
Capillaries per Type 1 fiber
Capillaries per Type 2 fiber
Capillaries per mm z
Fall Awake Winter Awake Hibernating
2.04 + 0.24 2.12 + 0,40 2.18 5:0,31
4.08 + 0.60 4.07 5:0.77 4.08 5:0.56
3.00 + 0.41 3.20 5:0.52 3.38 5:0.51
1547 + 229 1230 5:246 a 1059 + 146 b
a significantly different than Fall Awake at P < 0.015. b significantly different than Fall Awake at P < 0,001.
W.D. Reidet al./Respiration Physiology101 (1995)301-309 cle in hibernating species. Even in the same species, Steffen et al. (1991) found decreased weights of both soleus and plantaris (15-20%) in WA and H compared to summer awake squirrels (Spermophilus lateralis). They also found that the extensor digitorum longus of H squirrels was smaller than that of summer awake squirrels. Regarding muscle fibre crosssectional area, Steffen et al. (1991) found a 30% decrease in fibre size of soleus in WA compared to H and summer awake squirrels. Furthermore, WA and H squirrels had a 25 and 35% reduction in cross-sectional area of extensor digitorum longus, respectively, compared to control summer awake squirrels. Decreased weight of limb muscles has also been found in other species during hibernation. Yacoe (1983a) found a significant reduction in pectoralis muscle weight and protein content of hibernating bats. Further, Wickler et al. (1987) found a 60% decrease in muscle weight of the semitendinosus of hibernating hamsters. The different changes in muscle mass and fiber cross-sectional area in the limb muscles compared to the diaphragm in hibernators is likely due to the different changes in their activity levels; the limb muscles exhibit atonia during hibernation while the diaphragm performed more work with each breath. Hypertrophy of limb muscles in response to resistance training is well-documented however, transformation of slow to fast fibre type has not received as much study and is less consistently found (Booth and Thomason, 1991). Jansson et al. (1990) showed that sprint training using a lower-extremity cycle ergometer resulted in an increased proportion of fast-twitch muscle fibres. Lomo et al. (1980) showed that direct stimulation using a high frequency phasic pattern transformed slow-twitch muscle to faster contracting muscle in the denervated soleus muscle of the rat. It is quite possible that the stiffer chest wall in H squirrels resulted in a higher firing frequency of motorneurons during each breath, to overcome greater tension, and more phasic type of recruitment pattern, due to the decreased breathing frequency, contributed to the transformation of slow to fast fibres in the diaphragm of H squirrels.
4.2. Oxidatiue capacity of muscle fibres There was an increased oxidative capacity in the diaphragm of H and WA squirrels that was most
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prominent in the type 2b fibres of the WA group. We postulate that the increase in oxidative enzymes in the diaphragm of both hibernating and winter awake squirrels was due to the increased dependence on fat metabolism as an energy source and could have been further potentiated by increased plasma thyroxine levels. Lipids constitute an important aerobic energy source for animals during hibernation and arousal (Krilowicz, 1985). Although not measured in the species of squirrels used in this study, plasma levels of thyroxine and triiodothyronine are higher during the hibernation period in the Arctic ground squirrel (Citellus parryi) (Nevretdinova, 1989), and are higher during winter and spring but lower during the summer and fall in the Richardson's ground squirrel (Spermophilus richardsoni) (Wang, 1982). Thyroxine promotes mitochondrial biogenesis in skeletal muscle (Hood et al., 1992) and has been shown to increase histochemical reactivity of NADH-TR, the same marker of oxidative capacity used in this study, in limb muscles of fetal pigs (Hausman and Watson, 1994). Other possible explanations for the increased oxidative capacity in the hibernating group could include the increased oxidative demands required for shivering during arousal a n d / o r the increased mitochondrial density needed to compensate for the decreased blood flow. Other studies examining skeletal muscle from hibernating animals also found an increased oxidative capacity associated with increased storage of triglycerides, increases in enzymes involved in /3-oxidation, and increases in oxidative enzymes involved in the Kreb's cycle. Thus, Steffen et al. (1991) found a trend towards an increased muscle storage of triglycerides in the plantaris of hibernating squirrels while Wickler et al. (1987) found an increase in levels of enzymes involved in fl-oxidation in limb muscles of hibernating hamsters. Both groups found increases in citrate synthase activity in the hibernating group compared with summer awake squirrels (Steffen et al., 1991; Wickler et al, 1991). Increases in oxidative enzymes in limb muscles (Wickler et al., 1987; Vyskocil and Gutmann, 1977) and the diaphragm (Vyskocil and Gutmann, 1977) during hibernation have also been reported in hamsters. The increase in oxidative enzymes in both the diaphragm and limb muscles, in spite of differing activity levels, provides further support that dependence on lipid substrate
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utilization and/or an increase in plasma thyroxine level rather that changes in activity induces the increased oxidative capacity of skeletal muscle in hibernators. We postulate that some of the enzymatic changes related to disuse may be apparent in WA squirrels because, although the WA squirrels are more active than H squirrels, they spend much of their time in sleep and some in hibernation at 22°C. These animals were very inactive (including decreased feeding) and their body temperatures frequently dropped to room temperature (22°C). Thus, decreased activity and decreased food intake, as well as circannual rhythms of thyroxine levels (Wang, 1982) or of other factors (Mrosovsky, 1976) may promote a greater dependence on fat metabolism for energy metabolism in skeletal muscle. Because body temperatures of WA squirrels were higher than those found in the H squirrels, the reduced activity in WA squirrels may result in more profound changes in the diaphragm of the WA squirrels compared to the H squirrels. Hibernators may have a very limited time period for enzymatic adaptation to metabolic changes to occur because of their low body temperature. This has been shown by Yacoe (1983b) who found significant reductions in muscle protein synthesis and degradation rates during hibernation. These rates only returned to normothermic values during periodic arousals. Therefore, the time period available for muscle to undergo transformation in hibernators may be primarily during the arousal periods when their body temperatures are at euthermic values (37°C. vs. 5°C. during hibernation). In contrast, the inactivity in winter awake squirrels, which includes decreased feeding and thus, greater dependence on body fat stores, occurs at a higher body temperature and metabolic rate. This should enable a greater shift in energy metabolism and transformation of muscle metabolic characteristics to occur in WA animals than in hibernators. Even if the body temperature of winter awake squirrels drops to room temperature (22°C) for significant periods of time, this would still be the case.
fall awake squirrels. This is in contrast to the increase in capillary densities found in the extensor digitorum longus and soleus muscles of the hibernating squirrels (Steffen et al., 1991). The opposite changes of the capillary densities in the diaphragm versus the limb muscles is likely, in large part, to be due to the opposite changes in cross-sectional areas of muscle fibres during hibernation; the diaphragm has an increase in cross-sectional area of fibres whereas limb muscles show atrophy (Steffen et al., 1991). The lack of an increase in capillary densities of the diaphragm in H and WA squirrels further supports the hypothesis that an increased dependence on lipid metabolism and not an increased energy demand due to increased activity resulted in the increase in oxidative enzymes in the diaphragm of H and WA squirrels.
5. Conclusion In summary, we found no diaphragm atrophy in H and WA squirrels but rather maintenance of diaphragm weight and an increase in diaphragm weight to body weight and fibre cross-sectional area in spite of body weight loss and atrophy of limb muscles (Steffen et al., 1991). This may in part be related to the decreased compliance of the chest wall in the hibernators at reduced body temperature such that the greater force needed to generate each breath, despite fewer contractions, resulted in muscle hypertrophy and not disuse atrophy.
Acknowledgements The authors would like to acknowledge the support of funding from NSERC and B.C. Health Research Foundation. Dr. Reid was a B.C. Health Research Foundation Scholar during the performance of this study. The authors would also like to acknowledge Mr. Frank Chung for the performance of the statistical analysis.
4.3. Capillary densities of diaphragm
References
Capillary densities decreased in the diaphragm of hibernating and winter awake squirrels compared to
Booth, F.W. and D.B.Thomason (1991). Molecular and cellular adaptation of muscle in response to exercise: perspectives of various models. Physiol. Rev. 71: 541-585.
W.D. Reid et al. / Respiration Physiology 101 (1995) 301-309 Dubowitz, V. (1985). Muscle Biopsy: A Practical Approach. Second Ed. Philadelphia, PA: Bailliere Tindall, pp. 63-65. Fleiss, J.L. (1986). The Design and Analysis of Clinical Experiments. New York: John Wiley and Sons, pp. 68-73. Hausman, G.J. and R. Watson (1994). Regulation of fetal muscle development by thyroxine. Acta Anat. 149(1):21-30. Hood, D.A., J.-A. Simoneau, A.M. Kelly and D. Pette (1992). Effect of thyroid status on the expression of metabolic enzymes during chronic stimulation. Am. J. Physiol. 263 (Cell Physiol. 32):C788-793. Jansson, E., M. Esbjornsson, I. Holm and I. Jacobs (1990). Increase in the proportion of fast-twitch muscle fibres by sprint training in males. Acta Physiol. Scand. 140: 359-363. Krilowicz, B.L. (1985). Ketone body metabolism in a ground squirrel during hibernation and fasting. Am. J. Physiol. 249:R462-R470. Lomo, T., R.H. Westgaard and L. Engebretsen (1980). Different stimulation patterns affect contractile properties of denervated rat soleus muscle. In: Plasticity of Muscle, edited by D. Pette. Berlin: De Gruyter, pp. 297-309. Milsom, W.K. and W.D. Reid (1995). Pulmonary mechanics of hibernating squirrels (Spermophilus lateralis). Respir. Physiol. 101: 311-320. Mrosovsky, N. (1976). Lipid programmes and life strategies in hibernators. Am. Zool. 16: 685-697. Nevretdinova, Z.G. (1989). Seasonal rhythms in the functioning of the hypophyseo-thyroid system of the suslik Citellus parryi. Zhurnal Evolyutsionnoi Biokhimii i Fiziologii. 25(3):310-317. Reid, W.D., J.M. Hards, B.R. Wiggs, E.N. Wood, P.V. Wright and R.L. Pardy (1989). Proportions and sizes of muscle fibre types in the hamster diaphragm. Muscle and Nerve 12: 108118. Reid, W.D., B.R. Wiggs, P.D. Pare and R.L. Pardy (1992). Fibre type and regiona differences in oxidative capacity and glycogen content in the hamster diaphragm. Am. Rev. Respir. Dis. 146: 1266-1271.
309
Sillau, A.H. and N. Banchero (1977). Visualization of capillaries in skeletal muscle by the ATPase reaction. Pfliigers Arch. 369: 269-271. Steffen J.M., D.A. Koebel, X.J. Musacchia and W.K. Milsom (1991). Morphometric and metabolic indices of disuse in muscles of hibernating ground squirrels. Comp. Biochem. Physiol. 99B:815-819. Vyskocil, F. and E. Gutmann (1977). Contractile and histochemical properties of skeletal muscles in hibernating and awake golden hamsters. J. Comp. Physiol. 122: 385-390. Wang, L.C.H. (1982). Hibernation and the endocrines. In: Hibernation and Torpor in Mammals and Birds, edited by C.P. Lyman, J.S. Willis, A. Malan and L.C.H. Wang. New York: Academic Press, pp. 206-236. Wickler, S.J., B.A. Horowitz and K.S. Kott (1987). Muscle function in hibernating hamsters: a natural analog to bed rest? J. Therm. Biol. 12: 163-166. Wickler, S.J., D.F. Hoyt and F. Van Breukelen (1991). Disuse atrophy in the hibernating golden-mantled ground squirrel, Spermophilus lateralis. Am. J. Physiol. 261 (Regulatory Integrative Comp. Physiol. 30):R1214-1217. Wied, G.L. (1966). Introduction to Quantitative Cytochemistry. New York and London: Academic Press. 718 p. Yacoe, M.E. (1983a). Maintenance of the pectoralis muscle during hibernation in the big brown bat, Eptesicus fuscus. J. Comp. Physiol. 152: 97-104. Yacoe, M.E. (1983b). Protein metabolism in the pectoralis muscle and liver of hibernating bats, Eptesicus fuscus. J. Comp. Physiol. 152: 137-144. Zar, J.H. (1984). Biostatistical Analysis. Second Edition. Englewood Cliffs, New Jersey: Prentice-Hall, 623 p. Zimmer, H.G. (1973). Microphometry. In: Micromethods in Molecular Biology. Berlin, Heidelberg, New York: Springer. pp. 297-328.
ELSEVIER
Respiration Physiology101 (1995) 301-309
Characteristics of diaphragm muscle fibre types in hibernating squirrels W. Darlene Reid a,*, Anna Ng a, R. Keith Wilton a, William K. Milsom b a
School of Rehabilitation Sciences, T325-2211 WesbrookMall, Universityof British Columbia, Vancouver, B.C., Canada, V6T2B5 b Department of Zoology, 6270 UniversityBlvd., Universityof British Columbia, Vancouver, B.C., Canada, V6T 1Z4
Accepted 3 April 1995
Abstract
Muscle samples obtained from the diaphragm of 7 fall-awake (FA), 10 winter-awake (WA), and 8 hibernating (H) squirrels (Spermophilus lateralis) were quick frozen, sectioned and processed for NADH-TR reaction end-product and myofibrillar-ATPase. Both WA and H squirrels showed small increases in diaphragm weight, reductions in body weight, and hence, significant increases in the diaphragm weight to body weight ratio compared to FA squirrels. They also showed increases in muscle fibre type cross-sectional areas and in the oxidative capacity of type 2b fibres as well as a reduction in capillary density. Furthermore, there also was an increase in the proportion of type 2b fibres in the diaphragm of H squirrels. Thus, despite the dramatically reduced ventilation associated with hibernation, H squirrels exhibited (1) hypertrophy of the diaphragm which may represent an adaptive response that enables them to work against a stiffer chest wall, and (2) an increased oxidative capacity which enables them to fuel this with fat. Keywords: Hibernation; Mammals, squirrel (Spermophilus lateralis); Muscle, fibre type
1. Introduction
The hibernating mammal would appear to be a good model to study disuse atrophy of skeletal muscle. There is a profound decrease in the activity of limb muscles, and ventilatory levels decrease to approximately 8% of normal levels during hibernation. Even though the basal metabolic rate is reduced, an energy supply must be maintained during long periods of hibernation. Much of the energy appears to be derived from fat, however additional energy demands are met by protein. Skeletal muscle is by far
* Corresponding author, Tel: (604) 822-7402 FAX: (604) 8227624 Email: [email protected].
the largest protein reserve and the most important source of gluconeogenic precursors during prolonged fasting in normothermic mammals (Yacoe, 1983a). Catabolism of skeletal muscle in the hibernator would result in muscle fibre atrophy, and could result in loss of function and decreased survival, Although skeletal muscle atrophy of limb muscles following hibernation has been found in several species (Steffen et al., 1991; Wickler et al., 1987; Wickler et al., 1991; Yacoe, 1983a), reports are inconsistent, and decreased muscle size has not always been found (Vyskocil and Gutmann, 1977). Careful investigation of the diaphragm has not yet been performed. The diaphragm may respond differently to hibernation than other skeletal muscles for a couple of
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reasons. We found a decrease in specific lung compliance and a significant increase in both the elastic and total work required to ventilate hibernating animals at normal tidal volumes at mechanical ventilation frequencies of 100 and 150 breaths per min (Milsom and Reid, 1995). This decreased compliance at low lung volumes and increased work may provide a stimulus to maintain or increase inspiratory muscle mass even though respiration frequency is reduced in hibernators. Further, significant atrophy of the diaphragm during hibernation could have a very deleterious effect on survival if the atrophy prevented the animal from producing sufficient inspiratory muscle force to cope with the immediate and continuous loads of ventilation during and after arousal. Significant atrophy of the diaphragm in mechanically ventilated patients leads to difficulty in
weaning, and even death due to respiratory complications because of failure to wean. A better understanding of the fibre characteristics of the diaphragm in hibernating animals may be helpful in designing interventions to prevent the disuse atrophy that occurs in patients maintained on prolonged mechanical ventilation. The purpose of this study was to examine the effects of hibernation on muscle mass and fibre type sizes, fibre type proportions, and oxidative capacity of the squirrel diaphragm. We hypothesized that the diaphragm would undergo atrophy to a lesser extent than limb muscles in response to disuse during hibernation, in part because of continued respirations (albeit less frequent) and in part, because of the increased work required to overcome the decreased compliance of the respiratory system.
Fig. 1. Serial cross-sectionsof costal diaphragmprocessedfor M-ATPaseat preincubationpH's of 4.2 (A), 4.45 (B), 9.4(C), and NADH-TR (D). Scalebar is 25 /xm.
W.D. Reid et al. / Respiration Physiology 101 (1995) 301-309
2. Methods
Animals. Three groups of squirrels (Spermophilus lateralis) were studied: (1) seven fall-awake animals (FA) that had been captured at the end of August and held in captivity for three weeks; (2) ten winter awake animals (WA) that had been held in captivity for five months at 23°C.; and (3) eight hibernators (H) that had been held for 3 to 4 months at 5°C. The body temperature of H squirrels just before sacrifice was between 5 and 7°C.
Diaphragm muscle samples.. Following anesthesia with an intraperitoneal injection of sodium pentobarbital (Somnotol) (6.5 rag/100 g body weight), the animal was tracheotomized and placed in a pressure plethysmograph (Milsom and Reid, 1995). Following measurements of pulmonary mechanics, the squirrel was removed from the pressure plethysmograph. Through a midline abdominal incision, the entire diaphragm was excised and placed upon phosphatebuffered saline-soaked gauze. The diaphragm was cut in half and the right half was weighed. For histochemical analysis, muscle samples were cut from the anterior lateral costal region of the left hemi-diaphragm, mounted on gum tragacanth and frozen in isopentane cooled to the temperature of liquid nitrogen. Given the size of the diaphragm, these samples utilized most of the costal region. Muscle samples were stored at -70°C. until subsequent biochemical or histochemical processing.
Muscle Histochemistry.. Serial cross-sections were cut at 10 and 16 /zm thickness on a cryostat-microtome (Reichart-Jung). The 16 /zm sections were processed for nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR) (Reid et al., 1992) and the 10 /zm sections were processed for myosin adenosine-triphosphatase (M-ATPase) using preincubation pH's of 4.2, 4.45 and 9.4 (Reid et al., 1989) (Fig. 1). All cross-sections were cut and stained on the same day in the same batch to avoid possible artifacts that could arise from interassay variation.
Cross-sectional Area and Optical Density Measurements.. The cross-sectional area and optical density of the histochemical reaction for NADH-TR of muscle fibres were quantified using a computer-based
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image processing system (Kontron SEM-IPS, Munich) that consisted of a video monitor and a computer for the processing of images. The monitor was connected to a video camera (Model 68, Dage MTI) attached to a light microscope (Zeiss Universal research). A filter on the light source of the microscope was used to produce a narrow band of transmitted light with a wavelength spectrum that peaked at 546 nm which corresponded closely to the maximal absorbance of NADH-TR reaction end product. The video image of the muscle section was composed of a matrix of 480 × 512 pixels. Using a microscope magnification of × 20, each pixel had an area of 0.5568 /~m 2. A stage micrometer (Zeiss) delineated in microns was used to check the calibration of the vertical and horizontal axes of the video image daily. The image processing system had the ability to discern 255 grey levels. Calibration for optical densities or grey levels was performed at the beginning of each session following standard technique (Wied, 1966; Zimmer, 1973). For measurement of cross-sectional area and optical densities of each muscle fibre, the perimeter of the cross-sectional profile of the fibre was outlined using a mouse on a digitizing pad connected to the video monitor. The optical density of each pixel within the outlined fibre cross-section was determined and the mean optical density of all the pixels within the fibre was determined. Mitochondria (identified by NADH-TR) are distributed non-homogeneously, especially within the type 1 fibre which can have a large subsarcolemmal population of mitochondria (Fig. 1, panel D). Determining mean optical density by averaging the optical densities of each pixel within the cross-sectional area of the muscle fiber ensured the maximal inclusion of any deeply stained sub-sarcolemmal matter. Cross-sectional areas and optical density measures were determined for 200 fibers in the costal diaphragm from each squirrel. Muscle fibres were then typed as type 1, 2a, 2b (Reid et al., 1989) or 2c (Dubowitz, 1985) by identifying the same fibres in serial sections stained for M-ATPase at the three different pre-incubation pH's. Using this fibre typing nomenclature, type 1 fibres are dark when stained for M-ATPase after pre-incubation at pH of 4.2 and pH of 4.45 but intermediate after pre-incubation at a pH of 9.4; type 2a fibres are light when stained for M-ATPase after
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pre-incubation at pH of 4.2 and pH of 4.45 but dark after pre-incubation at a pH of 9.4; type 2b fibres are light when stained for M-ATPase after pre-incubation at pH of 4.2, intermediate in coloration after preincubation at a pH of 4.45 but dark after pre-incubation at a pH of 9.4; and type 2c fibres are intermediate when stained for M-ATPase after preincubation at pH of 4.2, dark in coloration after preincubation at a pH of 4.45 and dark after pre-incubation at a pH of 9.4, (Dubowitz, 1985). Fibre type proportions were determined by counting the number of fibres in each of the four fibre type categories and then dividing the number by the total number of fibres examined (200 fibres/biopsy).
2.2. Capillary Density Capillaries were visualized from sections stained for M-ATPase after acid (pH 4.2) preincubation as described by Sillau and Banchero (1977). Using the computer-based image processing system (Kontron SEM-IPS, Munich), a computer program was designed to determine: (1) capillaries per fibre; (2) capillaries surrounding type 1 or type 2 fibres; and (3) capillaries per cross-sectional area. At a microscope magnification of 310 × , the image was projected onto a monitor with a counting frame (190 X 230 microns). The number of capillaries per fibre was defined from the counts of muscle fibres and capillaries within and touching two sides of the counting frame. Any fibres or capillaries that were touching the other two sides of the counting frame were excluded from the counts. Capillaries surrounding type I and type 2 fibres were defined as all capillaries surrounding a specific fibre type. Since a capillary may lie between 2 or more fibres, capillaries may be counted more than once for this variable. Capillaries per cross-sectional area was defined as the number of capillaries within and touching two sides of the counting frame divided by the cross-sectional area of the counting frame.
2.3. Statistical analysis To test for differences among animal groups, a multivariate analysis was performed on the measures of diaphragm and body weight (diaphragm mass, body weight and diaphragm mass to body weight
ratio), and measures of capillary density. To test for differences among groups in fibre type proportions, the square root of the arcsin was taken in order to achieve a normal distribution and then an ANOVA was performed. To test for differences among groups in muscle fibre type cross-sectional areas, and optical densities, a multivariate analysis of variance was used (MANOVA) (Fleiss, 1986). Because the distributions of cross-sectional areas and optical densities were not normally distributed and in order to examine a broader distribution than shown by the median alone, each animal's optical density and cross-sectional area values were reduced to three values (the lower quartile, the median, and the upper quartile) (Zar, 1984). MANOVAs were performed on these three values in order to examine for interaction between group and fiber type, and to examine for differences among groups Interaction between animal group and fibre type was considered significant at p < 0.15 and no significant interaction between these two factors was present. Differences among groups were only considered to be different if all three values (the upper quartile, lower quartile, and median value) were different among groups at a P < 0.05.
3. Results WA squirrels weighed less ( P < 0.02) and H squirrels tended to weigh less than the FA squirrels (Table 1). All three groups had a similar diaphragm mass but the WA ( P < 0.001) and H ( P < 0.02) had a higher diaphragm weight to body weight ratio than FA squirrels (Table 1). Regarding muscle fibre type
Table 1 Diaphragm weight and body weight Group DiaphragmB o d y weight (g) weight(g) Fall Awake 0.63 + 0.13 219 + 33 Winter Awake 0.76+ 0.20 173 + 31 b Hibernating 0.75+0.17 193+30
Diaphragmweight/ Bodyweight ratio a 0.29 +_0.04 0.44± 0.09 c 0.39+0.05 b
a The diaphragmweight/body weight ratio is multiplied by 100 b significantly different than Fall Awake at P < 0.02. e significantlydifferent than Fall Awake at P < 0.001.
W.D. Reid et aL /Respiration Physiology 101 (1995) 301-309
60[ 5O
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l
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I
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VAv/'/AH
2o 10
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response to disuse during hibernation, in part because of continued respirations and in part, because of increased work required to overcome the decreased compliance of the respiratory system. The data show that the diaphragm in fact undergoes hypertrophy (in both WA and H squirrels), and further shows alterations in fibre type proportions and sizes, and an increased oxidative capacity.
0 Type 1
Tyloe 2a
Type 2b
Fig. 2. Muscle fibre type proportions of costal diaphragm in FA, WA and H squirrels. Means+ SD are shown. *H significantly different than WA at P < 0.05.
proportions, H squirrels had more type 2b fibres and fewer type 2a fibres in the costal diaphragm than those in the WA squirrels (Fig. 2). Of interest, less than 1% of fibres were type 2c fibres in the costal diaphragm of all three groups. Regarding muscle fibre size, H squirrels had larger type 1 and type 2a fibres in the costal diaphragm than those fibres in the FA and WA squirrels (Fig. 3). H squirrels also had larger type 2b fibres in the diaphragm than those fibres in the FA squirrels (Fig. 3). When the optical densities of all fibres regardless of type were grouped, analysis of optical densities of NADH-TR reaction end product showed similar optical densities of diaphragm muscle fibres in H and WA squirrels, but optical densities of these two groups were higher than those in the FA group indicating that the diaphragms of H and WA squirrels, in general, were more oxidative than those for FA squirrels. When individual fibre types were analyzed, the optical densities of NADH-TR reaction end-product of the type 2b fibres in the diaphragm of the WA group were more oxidative than those fibres in the FA squirrels (Fig. 4). Capillary densities expressed per fibre or per type 1 or 2 fibre did not differ among groups (Table 2). However, capillaries per cross-sectional area (expressed per mm:) were less in the diaphragm of the WA and H than in the FA squirrels (Table 2).
4. Discussion
We hypothesized that the diaphragm would undergo atrophy to a lesser extent than limb muscles in
25[ 20O0
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2000 f
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Lower 25th Percentile
Median
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Fig. 3. Muscle fibre type cross-sectional areas in FA (O), WA ( • ) and H (El) squirrels. Vertical bars indicate SE of lower quartiles, medians and upper quartiles of animals in each group. Values for H animals are significantly different than those for FA animals for all fibre types ( P < 0.05) and than those for WA animals for type I and type 2a fibres ( P ___0.05).
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4.1. Diaphragm mass, a r e a s a n d proportions
TYPE 1
1.0
and fibre type cross-sectional
7
0.9 0.8 0.7 0.6 ~i
0.5 TYPE 2a
1.0 r~
0.9 0.8 0.7 0.6 0.5 TYPE 2b
1.0 0.9 0.8 0.7 0.6
-
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Lower
Median
Upper
25th
25th
Percentile
Percentile
Fig. 4. Muscle fibre type optical densities in F A ( O ) , W A ( v ) and H ( [ ] ) squirrels, Vertical bars indicate SE of lower quartiles,
medians and upper quartiles of animals in each group. Values for W A animals are significantly different than those of FA animals for type 2b fibres ( P < 0.05).
The maintenance of muscle weight, increase in muscle fibre type cross-sectional areas, and increased proportion of type 2b fibres in the diaphragm of H squirrels relative to FA squirrels are all indicative of increased stress being placed on the diaphragm in hibernating animals. We hypothesize that this increased stress arises primarily from the increased tension which must have to be generated against the less compliant respiratory system of these hibernating animals (specific compliance = 0.27, 0.32, and 0.12 m l / c m H 2 0 / m l FRC for FA, WA and H squirrels, respectively [Milsom and Reid, 1995]). Since tidal volume is maintained and compliance decreases during hibernation, each breath requires more work (2.75, 2.25, and 6.54 ml. cmH20 were required to pump 1 ml of air at a rate of 100 per min into the respiratory systems of FA, WA, and H animals, respectively). Hibernators still continued to contract their diaphragm but on average, this was only three times per min, approximately 8% of normal ventilatory levels. So although the frequency of diaphragm contractions and the power output of the diaphragm are greatly reduced during hibernation, the work required per breath is increased and this could have induced the increase in proportion of 2b fibers and the increase in cross-sectional area of all fiber types in the hibernating squirrels. The maintenance of diaphragm weight and increase in muscle fibre cross-sectional area of all fibre types of the diaphragm in hibernating squirrels is in sharp contrast to observations of limb skeletal mus-
Table 2 Capillary densities in the squirrel diaphragm Group
Capillaries per fiber
Capillaries per Type 1 fiber
Capillaries per Type 2 fiber
Capillaries per mm z
Fall Awake Winter Awake Hibernating
2.04 + 0.24 2.12 + 0,40 2.18 5:0,31
4.08 + 0.60 4.07 5:0.77 4.08 5:0.56
3.00 + 0.41 3.20 5:0.52 3.38 5:0.51
1547 + 229 1230 5:246 a 1059 + 146 b
a significantly different than Fall Awake at P < 0.015. b significantly different than Fall Awake at P < 0,001.
W.D. Reidet al./Respiration Physiology101 (1995)301-309 cle in hibernating species. Even in the same species, Steffen et al. (1991) found decreased weights of both soleus and plantaris (15-20%) in WA and H compared to summer awake squirrels (Spermophilus lateralis). They also found that the extensor digitorum longus of H squirrels was smaller than that of summer awake squirrels. Regarding muscle fibre crosssectional area, Steffen et al. (1991) found a 30% decrease in fibre size of soleus in WA compared to H and summer awake squirrels. Furthermore, WA and H squirrels had a 25 and 35% reduction in cross-sectional area of extensor digitorum longus, respectively, compared to control summer awake squirrels. Decreased weight of limb muscles has also been found in other species during hibernation. Yacoe (1983a) found a significant reduction in pectoralis muscle weight and protein content of hibernating bats. Further, Wickler et al. (1987) found a 60% decrease in muscle weight of the semitendinosus of hibernating hamsters. The different changes in muscle mass and fiber cross-sectional area in the limb muscles compared to the diaphragm in hibernators is likely due to the different changes in their activity levels; the limb muscles exhibit atonia during hibernation while the diaphragm performed more work with each breath. Hypertrophy of limb muscles in response to resistance training is well-documented however, transformation of slow to fast fibre type has not received as much study and is less consistently found (Booth and Thomason, 1991). Jansson et al. (1990) showed that sprint training using a lower-extremity cycle ergometer resulted in an increased proportion of fast-twitch muscle fibres. Lomo et al. (1980) showed that direct stimulation using a high frequency phasic pattern transformed slow-twitch muscle to faster contracting muscle in the denervated soleus muscle of the rat. It is quite possible that the stiffer chest wall in H squirrels resulted in a higher firing frequency of motorneurons during each breath, to overcome greater tension, and more phasic type of recruitment pattern, due to the decreased breathing frequency, contributed to the transformation of slow to fast fibres in the diaphragm of H squirrels.
4.2. Oxidatiue capacity of muscle fibres There was an increased oxidative capacity in the diaphragm of H and WA squirrels that was most
307
prominent in the type 2b fibres of the WA group. We postulate that the increase in oxidative enzymes in the diaphragm of both hibernating and winter awake squirrels was due to the increased dependence on fat metabolism as an energy source and could have been further potentiated by increased plasma thyroxine levels. Lipids constitute an important aerobic energy source for animals during hibernation and arousal (Krilowicz, 1985). Although not measured in the species of squirrels used in this study, plasma levels of thyroxine and triiodothyronine are higher during the hibernation period in the Arctic ground squirrel (Citellus parryi) (Nevretdinova, 1989), and are higher during winter and spring but lower during the summer and fall in the Richardson's ground squirrel (Spermophilus richardsoni) (Wang, 1982). Thyroxine promotes mitochondrial biogenesis in skeletal muscle (Hood et al., 1992) and has been shown to increase histochemical reactivity of NADH-TR, the same marker of oxidative capacity used in this study, in limb muscles of fetal pigs (Hausman and Watson, 1994). Other possible explanations for the increased oxidative capacity in the hibernating group could include the increased oxidative demands required for shivering during arousal a n d / o r the increased mitochondrial density needed to compensate for the decreased blood flow. Other studies examining skeletal muscle from hibernating animals also found an increased oxidative capacity associated with increased storage of triglycerides, increases in enzymes involved in /3-oxidation, and increases in oxidative enzymes involved in the Kreb's cycle. Thus, Steffen et al. (1991) found a trend towards an increased muscle storage of triglycerides in the plantaris of hibernating squirrels while Wickler et al. (1987) found an increase in levels of enzymes involved in fl-oxidation in limb muscles of hibernating hamsters. Both groups found increases in citrate synthase activity in the hibernating group compared with summer awake squirrels (Steffen et al., 1991; Wickler et al, 1991). Increases in oxidative enzymes in limb muscles (Wickler et al., 1987; Vyskocil and Gutmann, 1977) and the diaphragm (Vyskocil and Gutmann, 1977) during hibernation have also been reported in hamsters. The increase in oxidative enzymes in both the diaphragm and limb muscles, in spite of differing activity levels, provides further support that dependence on lipid substrate
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utilization and/or an increase in plasma thyroxine level rather that changes in activity induces the increased oxidative capacity of skeletal muscle in hibernators. We postulate that some of the enzymatic changes related to disuse may be apparent in WA squirrels because, although the WA squirrels are more active than H squirrels, they spend much of their time in sleep and some in hibernation at 22°C. These animals were very inactive (including decreased feeding) and their body temperatures frequently dropped to room temperature (22°C). Thus, decreased activity and decreased food intake, as well as circannual rhythms of thyroxine levels (Wang, 1982) or of other factors (Mrosovsky, 1976) may promote a greater dependence on fat metabolism for energy metabolism in skeletal muscle. Because body temperatures of WA squirrels were higher than those found in the H squirrels, the reduced activity in WA squirrels may result in more profound changes in the diaphragm of the WA squirrels compared to the H squirrels. Hibernators may have a very limited time period for enzymatic adaptation to metabolic changes to occur because of their low body temperature. This has been shown by Yacoe (1983b) who found significant reductions in muscle protein synthesis and degradation rates during hibernation. These rates only returned to normothermic values during periodic arousals. Therefore, the time period available for muscle to undergo transformation in hibernators may be primarily during the arousal periods when their body temperatures are at euthermic values (37°C. vs. 5°C. during hibernation). In contrast, the inactivity in winter awake squirrels, which includes decreased feeding and thus, greater dependence on body fat stores, occurs at a higher body temperature and metabolic rate. This should enable a greater shift in energy metabolism and transformation of muscle metabolic characteristics to occur in WA animals than in hibernators. Even if the body temperature of winter awake squirrels drops to room temperature (22°C) for significant periods of time, this would still be the case.
fall awake squirrels. This is in contrast to the increase in capillary densities found in the extensor digitorum longus and soleus muscles of the hibernating squirrels (Steffen et al., 1991). The opposite changes of the capillary densities in the diaphragm versus the limb muscles is likely, in large part, to be due to the opposite changes in cross-sectional areas of muscle fibres during hibernation; the diaphragm has an increase in cross-sectional area of fibres whereas limb muscles show atrophy (Steffen et al., 1991). The lack of an increase in capillary densities of the diaphragm in H and WA squirrels further supports the hypothesis that an increased dependence on lipid metabolism and not an increased energy demand due to increased activity resulted in the increase in oxidative enzymes in the diaphragm of H and WA squirrels.
5. Conclusion In summary, we found no diaphragm atrophy in H and WA squirrels but rather maintenance of diaphragm weight and an increase in diaphragm weight to body weight and fibre cross-sectional area in spite of body weight loss and atrophy of limb muscles (Steffen et al., 1991). This may in part be related to the decreased compliance of the chest wall in the hibernators at reduced body temperature such that the greater force needed to generate each breath, despite fewer contractions, resulted in muscle hypertrophy and not disuse atrophy.
Acknowledgements The authors would like to acknowledge the support of funding from NSERC and B.C. Health Research Foundation. Dr. Reid was a B.C. Health Research Foundation Scholar during the performance of this study. The authors would also like to acknowledge Mr. Frank Chung for the performance of the statistical analysis.
4.3. Capillary densities of diaphragm
References
Capillary densities decreased in the diaphragm of hibernating and winter awake squirrels compared to
Booth, F.W. and D.B.Thomason (1991). Molecular and cellular adaptation of muscle in response to exercise: perspectives of various models. Physiol. Rev. 71: 541-585.
W.D. Reid et al. / Respiration Physiology 101 (1995) 301-309 Dubowitz, V. (1985). Muscle Biopsy: A Practical Approach. Second Ed. Philadelphia, PA: Bailliere Tindall, pp. 63-65. Fleiss, J.L. (1986). The Design and Analysis of Clinical Experiments. New York: John Wiley and Sons, pp. 68-73. Hausman, G.J. and R. Watson (1994). Regulation of fetal muscle development by thyroxine. Acta Anat. 149(1):21-30. Hood, D.A., J.-A. Simoneau, A.M. Kelly and D. Pette (1992). Effect of thyroid status on the expression of metabolic enzymes during chronic stimulation. Am. J. Physiol. 263 (Cell Physiol. 32):C788-793. Jansson, E., M. Esbjornsson, I. Holm and I. Jacobs (1990). Increase in the proportion of fast-twitch muscle fibres by sprint training in males. Acta Physiol. Scand. 140: 359-363. Krilowicz, B.L. (1985). Ketone body metabolism in a ground squirrel during hibernation and fasting. Am. J. Physiol. 249:R462-R470. Lomo, T., R.H. Westgaard and L. Engebretsen (1980). Different stimulation patterns affect contractile properties of denervated rat soleus muscle. In: Plasticity of Muscle, edited by D. Pette. Berlin: De Gruyter, pp. 297-309. Milsom, W.K. and W.D. Reid (1995). Pulmonary mechanics of hibernating squirrels (Spermophilus lateralis). Respir. Physiol. 101: 311-320. Mrosovsky, N. (1976). Lipid programmes and life strategies in hibernators. Am. Zool. 16: 685-697. Nevretdinova, Z.G. (1989). Seasonal rhythms in the functioning of the hypophyseo-thyroid system of the suslik Citellus parryi. Zhurnal Evolyutsionnoi Biokhimii i Fiziologii. 25(3):310-317. Reid, W.D., J.M. Hards, B.R. Wiggs, E.N. Wood, P.V. Wright and R.L. Pardy (1989). Proportions and sizes of muscle fibre types in the hamster diaphragm. Muscle and Nerve 12: 108118. Reid, W.D., B.R. Wiggs, P.D. Pare and R.L. Pardy (1992). Fibre type and regiona differences in oxidative capacity and glycogen content in the hamster diaphragm. Am. Rev. Respir. Dis. 146: 1266-1271.
309
Sillau, A.H. and N. Banchero (1977). Visualization of capillaries in skeletal muscle by the ATPase reaction. Pfliigers Arch. 369: 269-271. Steffen J.M., D.A. Koebel, X.J. Musacchia and W.K. Milsom (1991). Morphometric and metabolic indices of disuse in muscles of hibernating ground squirrels. Comp. Biochem. Physiol. 99B:815-819. Vyskocil, F. and E. Gutmann (1977). Contractile and histochemical properties of skeletal muscles in hibernating and awake golden hamsters. J. Comp. Physiol. 122: 385-390. Wang, L.C.H. (1982). Hibernation and the endocrines. In: Hibernation and Torpor in Mammals and Birds, edited by C.P. Lyman, J.S. Willis, A. Malan and L.C.H. Wang. New York: Academic Press, pp. 206-236. Wickler, S.J., B.A. Horowitz and K.S. Kott (1987). Muscle function in hibernating hamsters: a natural analog to bed rest? J. Therm. Biol. 12: 163-166. Wickler, S.J., D.F. Hoyt and F. Van Breukelen (1991). Disuse atrophy in the hibernating golden-mantled ground squirrel, Spermophilus lateralis. Am. J. Physiol. 261 (Regulatory Integrative Comp. Physiol. 30):R1214-1217. Wied, G.L. (1966). Introduction to Quantitative Cytochemistry. New York and London: Academic Press. 718 p. Yacoe, M.E. (1983a). Maintenance of the pectoralis muscle during hibernation in the big brown bat, Eptesicus fuscus. J. Comp. Physiol. 152: 97-104. Yacoe, M.E. (1983b). Protein metabolism in the pectoralis muscle and liver of hibernating bats, Eptesicus fuscus. J. Comp. Physiol. 152: 137-144. Zar, J.H. (1984). Biostatistical Analysis. Second Edition. Englewood Cliffs, New Jersey: Prentice-Hall, 623 p. Zimmer, H.G. (1973). Microphometry. In: Micromethods in Molecular Biology. Berlin, Heidelberg, New York: Springer. pp. 297-328.