Departments of 1 Molecular and Integrative Physiology and 2 Animal Science, The University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
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ABSTRACT |
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To determine the effect of plasma growth hormone (GH) on skeletal muscle function, we measured the free Ca2+ concentration-tension relationship of slow-twitch (soleus) and fast-twitch (peroneus longus) muscles isolated from rats undergoing acromegaly in response to implanted, GH-secreting tumors. Muscles from adult (9 mo) and aged rats (24 mo) were studied after the tumor-bearing rats weighed over 50% more than their age-matched controls. Ca2+-activated isometric tension was recorded from skinned muscle fibers. For soleus muscles, the free Ca2+ concentration producing 50% of maximal tension ([Ca2+]50) was 2.0 µM for rats with tumors and 3.4-3.6 µM for controls. For peroneus longus fibers, [Ca2+]50 shifted from 6.1-6.7 µM in controls to 3.5 µM after tumors were introduced into either adult or aged rats. Soleus muscle fibers from neonatal rats (14 days) were less sensitive to Ca2+ than those isolated from adult rats, having a [Ca2+]50 of 7.3 µM. The Ca2+ sensitivity of peroneus longus fibers did not change with age. We conclude that significant increases in myofibrillar Ca2+ sensitivity occur in skeletal muscles undergoing rapid growth induced by GH-secreting tumors.
skinned muscle fibers; isometric tension; GH3 cells
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INTRODUCTION |
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THE AVAILABILITY OF biosynthetic human growth hormone (GH) has increased its use in the treatment of certain growth disorders as well as in nonclassical applications (4, 12). GH is a critical determinant of postnatal growth in mammals. An increase in serum GH triggers the linear growth that accompanies sexual maturation in adolescent animals. In addition, GH has significant anabolic effects on adult animals as well as humans, which include the stimulation of skeletal muscle growth. Interestingly, even very old animals are able to respond to elevated GH levels with significant changes in lean body mass, which suggests that muscle tissue does not lose its ability to activate critical biochemical pathways associated with gene expression and protein synthesis (6, 24). Although the influence of GH on muscle mass in mature animals and humans is well documented, less is known about the physiological changes that accompany the increase of muscle mass, especially in skeletal muscles. Functional changes in cardiac papillary muscles have been found in rats in response to chronic GH stimulation (16).
In this study, we have investigated whether significant changes in the physiological function of skeletal muscle cells occur in conjunction with GH-induced hypertrophy by studying the Ca2+ sensitivity of the contractile apparatus of slow-twitch and fast-twitch skeletal muscle fibers isolated from rats with GH-secreting tumors. Contractile activation in skeletal muscle is initiated by the binding of Ca2+ to the regulatory protein troponin. Because the relationship between free Ca2+ concentration ([Ca2+]) and the extent of contractile activation is steep, relatively small changes in the Ca2+ sensitivity of activation might result in significant alterations of physiological function in GH-treated animals. A useful experimental model for the study of the effects of elevated GH on muscle growth and function is female Wistar-Furth rats with implanted GH-secreting tumors (24). The tumors, which are derived from GH3 cells, secrete GH that is indistinguishable from native GH as determined by its biological and immunologic activity. Rats with implanted GH-secreting tumors enter an active growth phase and increase their body weight by >50% in 8-10 wk as compared with age-matched controls. We have found that skeletal muscle fibers from animals with GH-secreting tumors are activated at significantly lower levels of Ca2+ than control animals, suggesting that GH alters muscle function in addition to increasing its mass. A preliminary report of these results has appeared (26).
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METHODS |
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Experimental animals. Adult (9 mo) and aged (24 mo) Female Wistar-Furth rats were injected with ~1 × 106 GH3 cells (CCL 82.1, American Type Culture Collection) in 1 ml Ham's F-10 medium subcutaneously in the right flank as previously described (25). Control animals received 1 ml Ham's F-10 medium only. A localized tumor could be palpated within 2-3 wk at the site of injection. The tumors secrete GH as well as other hormones (e.g., prolactin). According to previous reports, serum GH levels begin to increase 15-20 days after GH3 cell implantation, rising from typical control levels of 10-100 ng/ml to >2,000 ng/ml (24). In the present experiments, increases in body weight and muscle mass occurred in both adult and aged groups beginning ~3-4 wk after injection, although the increase was slower in the aged rats (Fig. 1, see also Ref. 25). Eight to ten weeks after GH3 cell injection, the rats with tumors weighed >50% more than age-matched controls.
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Permeabilized fiber preparations. Skeletal muscles contain two broad classes of muscle fibers, slow-twitch (type I) and fast-twitch (type II), according to their contraction rate. Soleus was chosen for its high proportion of type I fibers and peroneus longus for its high content of type II fibers and ease of dissection (2). Whole muscles were removed from anesthetized rats and bathed in Ringer solution. A small bundle of fibers was cut off from the whole muscle and blotted dry. Single fibers were separated in mineral oil, and the sarcolemma was removed by microdissection. For rats younger than ~30 days, it is very difficult to separate and remove the sarcolemma from a single fiber. Instead, a small bundle of fibers was incubated in a relaxing solution ([Ca2+] = 0.01 µM) containing 0.5% Brij-35 detergent for 8-10 min to dissolve the surface membrane. Aluminum foil clips were attached to the ends of the skinned fibers, and they were then mounted in a photoelectric tension transducer. The diameter of each individual fiber was measured at slack fiber length. The diameter of the fiber bundle was not recorded, since the number of fibers per bundle was not known. A stretch of ~20% was applied before tension measurements were made. This was done by slowly stretching the fibers or fiber bundles until an increase in passive tension was observed and then relaxing the fibers or bundles slightly until passive tension declined to zero.
Bathing solutions.
Solutions of different
[Ca2+] were prepared
from recipes calculated by a computer program that solved the multiple
equilibrium reactions (5). The stability constants used were from a
paper by Godt and Lindley (10). Solutions contained (in mM) 100 K+, 5 ethylene
glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 2 MgATP, 1 Mg2+, and 15 creatine phosphate. The major anion in the solution was propionate (100 mM). 3-(N-morpholino)propanesulfonic acid was added as a
buffer and also to adjust the ionic strength to 0.15. pH was 7.0. The
relaxing solution had a
[Ca2+] of 0.01 µM,
whereas the solution used to induce maximal tension had a
[Ca2+] of 100 µM.
Creatine phosphokinase (15 U/ml) was added to the solutions just before
experiments.
Isometric tension measurements. Bathing solutions were contained in a series of 2.5-ml troughs in a spring-supported tray. During the course of experiments, a change of bathing solution was accomplished by compressing the springs, sliding a different solution-filled trough under the skinned fiber, and then releasing the tray to its original position.
Data were collected using a "stepping" protocol (Fig. 2). Baseline tension was recorded in a relaxing solution ([Ca2+] = 0.01 µM), and the fiber was then transferred to a test solution with various [Ca2+] that activated the fiber submaximally. When a steady-state plateau of submaximal tension was reached, the fiber was transferred to the maximally activating solution ([Ca2+] = 100 µM) to record maximal tension. Fibers were then relaxed before a new submaximally activating solution was tested. If the value of the maximum tension decayed to <80% of the original maximum tension, the fiber was discarded.
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Data analysis. The submaximal tension developed at various [Ca2+] was expressed as the percentage of maximum tension (T%). The relation between T% and [Ca2+] was fitted by the Hill equation, T% = 100 × [Ca2+]nH/{[Ca2+]nH + ([Ca2+]50)nH}, using a nonlinear least-squares regression program, where [Ca2+]50 is the [Ca2+] giving 50% of the maximum tension and nH is the Hill coefficient that reflects the steepness of the fitted curve. In these experiments, the Hill coefficient is not related to the degree of cooperativity between, or number of, Ca2+ binding sites on the contractile proteins due to the complex nature of the molecular events linking Ca2+ binding to force production.
Experiments were performed at room temperature. For each group, data were collected in total of 8-16 fibers from 2 or 3 rats and are reported as means ± SE. Error bars are not shown in figures when they are smaller than the size of symbols used to indicate the mean values. Student's t-test or one-way analysis of variance was used for statistical comparison of the mean between two groups or among three groups. P < 0.05 was considered statistically significant. ![]() |
RESULTS |
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The free Ca2+ sensitivity of slow-twitch and fast-twitch muscle fibers from 9-mo-old (adult) and 24-mo-old (aged) rats with and without induced, GH-secreting tumors was determined and compared with the normal developmental changes in Ca2+ sensitivity that occurred as rats matured from postnatal day 14 to 9 mo. The results are summarized in Fig. 4 for soleus and in Fig. 5 for peroneus longus muscles.
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In control animals, fibers from soleus muscle, a predominantly slow-twitch muscle, of adult and aged rats are activated at lower [Ca2+] than fibers from peroneus longus muscle, a predominantly fast-twitch muscle. [Ca2+]50 values were 3.6 and 3.4 µM for soleus fibers from the adult and aged rats, respectively, lower than the value of 6.7 µM and 6.1 needed for 50% activation for peroneus longus fibers from the adult or aged animals. These results are consistent with a number of previous studies that have documented higher Ca2+ sensitivity of slow-twitch vs. fast-twitch muscle fibers (1, 13, 15, 23).
For both age groups studied, fibers isolated from rats with GH-secreting tumors were more sensitive to Ca2+ than those from controls. For fibers from soleus, [Ca2+]50 decreased from a control value of 3.6 to 2.0 µM in the adult tumor-bearing rats and from 3.4 to 2.0 µM in the aged tumor-bearing rats. A similar increase in Ca2+ sensitivity occurred in the peroneus longus fibers. Here the changes of [Ca2+]50 were from 6.7 to 3.5 µM and from 6.1 to 3.5 µM for the adult and aged rats, respectively. The steepness of the [Ca2+]-tension relationship measured by the nH value did not change much in fibers from the tumor-bearing rats except for peroneus longus fibers from aged tumor-bearing animals which showed an increase of nH from 1.6 to 2.6.
We also determined the Ca2+ sensitivity of muscle fibers from neonatal (14 days) and young rats (30 days) to compare the shifts of [Ca2+]-tension curve that occur during normal maturation to those that occur in response to the GH-secreting tumors. For fibers from soleus muscles, the [Ca2+]-tension relationship for neonatal rats was shifted to the right relative to the adult animals. [Ca2+]50 for soleus fibers from neonatal animals was 7.3 µM, about twice the value of 3.6 µM for fibers from the adult rats (Fig. 4). [Ca2+]-tension relationship of soleus muscle fibers from young rats was similar to that of the adult rats (data not shown). [Ca2+]50 for soleus muscle fibers from 30-day-old rats is 3.4 µM, which is comparable to 3.6 µM for those of 9-mo-old rats. The shift for peroneus longus fibers was very small, with [Ca2+]50 being 7.2 µM in the neonatal animals compared with 6.7 µM in the adult animals.
Tension per unit cross-sectional area during maximal activation was calculated for the aged rats only. Normalized tension was similar in fibers from control and tumor-bearing rats, 1.5 ± 0.2 kg/cm2 (n = 15) vs. 1.4 ± 0.2 kg/cm2 (n = 13) for soleus and 1.8 ± 0.4 kg/cm2 (n = 20) vs. 2.0 ± 0.3 kg/cm2 (n = 15) for peroneus longus, respectively. These values are very close to the results of McCarter and McGee (17), who reported normalized tension of 1.2-1.7 kg/cm2 at maximal activation for the soleus muscle from 6- to 30-mo-old rats.
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DISCUSSION |
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The major finding of this study is that both slow-twitch and fast-twitch muscle fibers from adult and aged rats with induced GH-secreting tumors show higher Ca2+ sensitivity than those from corresponding controls. For rats with GH-secreting tumors, [Ca2+]50 is reduced by ~44% with respect to that of the controls for both slow-twitch (soleus) and fast-twitch (peroneus longus) muscle fibers. Increases in myofibrillar Ca2+ sensitivity have also been reported in skinned cardiac papillary muscle fibers isolated from rats with GH-secreting tumors (16). However, the magnitude of [Ca2+]50 reduction observed in skinned papillary muscle fibers of tumor-bearing rats (9%) was much smaller than the [Ca2+]50 reduction we observed in skinned skeletal muscle fibers of tumor-bearing rats (44%). [Ca2+]50 was also found to be significantly lower in hypertrophied soleus (58%) and plantaris fibers (29%) induced by surgical ablation of the synergic muscles (14). Therefore, increases in myofilament sensitivity to Ca2+ seem to be a common phenomenon in hypertrophied muscles.
We also observed some changes in the nH value of [Ca2+]-tension relations. The biggest effect was seen with peroneus longus fibers in aged rats, which showed a larger nH in fibers isolated from the rats with tumors compared with the controls (2.6 vs. 1.6). However, the nH of the [Ca2+]-tension relation did not change in papillary muscle fibers from rats with GH-secreting tumors (16) or in hypertrophied skeletal muscle fibers induced by surgery (14). The nH value has been shown to be sarcomere length dependent, whereas [Ca2+]50 is much less sensitive to this parameter (19). The sarcomere length of fibers used in this study was not precisely determined.
In contrast to the submaximal Ca2+ sensitivity, the specific tension per fiber cross-sectional area at maximal activation (Po) was not significantly different between aged control and tumor-bearing rats for either soleus or peroneus longus fibers. Inconsistent results have been reported for Po in hypertrophied muscles. Po of skinned papillary muscle fibers was markedly higher (39%) in rats with GH-secreting tumors than in control rats (16), whereas Po was slightly depressed (3-8%) in hypertrophied plantaris and soleus fibers when compared with the corresponding control (14).
Our results showed that slow-twitch (soleus) muscle fibers had a significant increase in Ca2+ sensitivity during early postnatal development, whereas fast-twitch (peroneus longus) muscle fibers did not. For neonatal rats, both slow-twitch and fast-twitch muscle fibers had very similar Ca2+ sensitivity, which is comparable to that of the adult fast-twitch muscle fibers. As rats matured, the Ca2+ sensitivity of slow-twitch muscle fibers increased significantly and reached the level of adults by the end of the first month. Similar results have been reported for rabbit muscles during development (15). At the molecular level, the synthesis of fast troponin and myosin light-chain isoforms is dominant in all skeletal muscles of the rat fetus. In slow-twitch muscle fibers, the amount of fast troponin and myosin light-chain isoforms progressively decreases after birth, and at the same time, the synthesis of slow isoforms increases. However, no transformation in synthesis of troponin and myosin occurs in fast-twitch muscle fibers (8, 21). The developmental changes in synthesis of contractile and regulatory proteins are probably the underlying mechanisms for the changed Ca2+ sensitivity of contractile activation during postnatal growth.
The sensitivity of the contractile apparatus to Ca2+ activation is determined to a great extent by the number and the affinity of Ca2+ binding sites on troponin (3, 20). Different muscle types have characteristically different sensitivity to Ca2+ activation, which results from the different isoforms of troponin found in the different muscle tissues (7). Changes in the expressed isoforms of troponin occur during development (8), in cross-innervated muscles (7), and in some forms of muscular dystrophy (9), leading to changes in Ca2+ sensitivity of muscle cells. It has been shown that the genes coding for certain isoforms of troponin and myosin that are silent under normal conditions can be turned on due to a change in the pattern of muscular activity, a change in thyroid hormone levels, or overload-induced myocardial hypertrophy (7, 18, 22). In all these reports, the genes turned on by different induction factors coded either for slow or for fast isoforms of troponin and myosin. The leftward shift of [Ca2+]-tension relation in peroneus longus fibers we found in rats with GH-secreting tumors may partially be explained by possible contractile protein isoform switching (from fast to slow). Interestingly, our results demonstrated that the Ca2+ sensitivity of slow-twitch muscle fibers isolated from tumor-bearing rats was higher than the Ca2+ sensitivity of both slow-twitch and fast-twitch muscle fibers of the control rats, which may indicate that novel isoforms of troponin and/or myosin are being synthesized. Another possible mechanism for the increased Ca2+ sensitivity is that although the specific isoforms of troponin and myosin being expressed are not altered, their relative distributions are. Guba et al. (11) found that muscle disuse changed the ratios of myosin light chains (LC1:LC2:LC3) and of troponin (Tn) subunits (TnC:TnI:TnT) in both fast-twitch and slow-twitch muscles.
In summary, significant changes in the physiological function of muscle fibers accompanied the skeletal muscle hypertrophy induced by chronic GH stimulation. In rats with GH-secreting tumors, the functional changes are mainly reflected by marked increases in myofibrillar Ca2+ sensitivity in both soleus and peroneus longus fibers without significant change in specific tension at maximal activation. Further studies are needed to understand the molecular mechanisms responsible for the increased Ca2+ sensitivity of skeletal muscles in GH-stimulated rats.
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ACKNOWLEDGEMENTS |
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Current address of P. J. Bechtel: Dept. of Food Science and Human Nutrition, Colorado State University, Fort Collins, CO 80523.
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FOOTNOTES |
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Current address and address for reprint requests: X. Xu, Cardiology Foundation of Lankenau, Suite 558, Medical Office Bldg. East, 100 Lancaster Ave., Wynnewood, PA 19096.
Received 7 August 1997; accepted in final form 12 December 1997.
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