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MUSCLE CELL BIOLOGY AND CELL MOTILITY
1Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin; and 2Department of Surgery, The University of Texas Medical Branch, and Shriners Hospitals for Children, Galveston, Texas
Submitted 10 November 2006 ; accepted in final form 27 March 2007
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ABSTRACT |
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isotonic contractile properties; peak force and power; calcium sensitivity; essential amino acids
In the case of microgravity, the loss of muscle volume and torque appears to be exponential with the largest decline in the first month and a new steady state reached by 120 days. This is supported by the observation that 110 days of microgravity reduced ankle plantar flexion strength as much as 237 days (6). Observations following short and prolonged spaceflight suggest that while ankle extensors (plantar flexors) atrophy and lose function faster than ankle flexors, the difference by 237 days between ankle extensors and flexors is less apparent (6). Another consistent observation is that regardless of the type of unloading (BR or spaceflight) considerable variability exists between subjects with some demonstrating more muscle atrophy and functional decline than others (20, 22).
Muscle wasting, whether induced by spaceflight or clinically mandated BR, results from an imbalance between protein synthesis and breakdown (4), and it is also generally associated with elevated plasma concentrations of the stress hormone cortisol (6, 14). With prolonged space flights on MIR (>3 mo), astronauts and cosmonauts showed a 45% drop in whole body protein synthesis rates, and a reduced protein breakdown of a somewhat lesser magnitude (16). This suggests that the primary problem contributing to muscle wasting was the reduced protein synthetic rate. This also appears to be the dominate factor producing muscle atrophy with periods of BR. Ferrando et al. (4) observed a 50% drop in protein synthesis with no change in protein breakdown in the vastus lateralis (VL) of subjects following a 14-day BR. In many space crew members and patients confined to BR, the extent of muscle protein breakdown was exacerbated by an inadequate energy intake, which resulted in a negative caloric balance (14, 16, 17). In patients, the incidence of undernutrition during hospitalization has been estimated to be as high as 50% (1). The importance of proper nutrition in the maintenance of muscle mass was recently demonstrated by Paddon-Jones et al. (14). These investigators showed that the catabolic effects of prolonged bed rest with or without acute hypercortisolemia could be ameliorated by an essential amino acid and carbohydrate (AA/CHO) supplement through the maintenance of muscle protein synthesis. In contrast, the ingestion of a typical mixed clinical meal had minimal or no effect on protein synthesis (14).
The goal of this study was to assess the effect of chronic hypercortisolemia or AA/CHO supplement during BR on single muscle fiber function. While the former accelerates and the latter protects against BR-induced loss of muscle mass and strength (14), the cellular changes responsible for the limb muscle responses are unknown. The observed impact on muscle mass suggests that at least a portion of the effects of these interventions are likely mediated through differences in fiber atrophy. However, the fiber-type dependence (i.e., effects on slow and fast fiber types) of hypercortisolemia or AA/CHO supplements and whether or not changes in single fiber force and power independent of cell atrophy contribute to the altered muscle strength induced by these modalities are unknown. Given that hypercortisolemia and AA/CHO supplements are known to decrease and increase muscle protein (12, 14), respectively, we hypothesize that independent effects on force and power will exist. These questions will be studied by determining the extent to which hypercortisolemia exacerbates and AA/CHO supplement protects against the bed rest induced atrophy and functional decline of individual slow- and fast-twitch fibers of the soleus and VL muscles.
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METHODS |
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To achieve a stable plasma cortisol concentration of 
20–25 µg/dl, subjects received 5 daily doses of 10–15 mg of oral hydrocortisone sodium succinate throughout bedrest (days 2–27: 0800, 1200, 1600, 2000, 2400). On days 1 and 28 of bedrest, a continuous infusion of hydrocortisone sodium succinate (60 µg·kg–1·h–1; Pharmacia), was administered intravenously to induce/maintain hypercortisolemia and reduce potential minor fluctuations in plasma cortisol concentrations during collection of muscle biopsy samples.
Throughout bed rest, subjects in the BRAA group received three daily supplements (1100, 1600, 2100), each containing 16.5 g of essential amino acids and 30 g of sucrose (Table 2). The amino acids and sucrose were dissolved in 250 ml of a noncaloric, noncaffeinated soft drink. The additional daily caloric/nutrient intake (558 kcal) provided by the AA/CHO drinks represented a true dietary supplement and not a caloric replacement or substitution. Subjects in the BR group received only the diet soft drink.
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Test solutions and single fiber isolation procedures. The total and free concentrations of each metal, ligand, and metal ligand complex in the relaxing and activating solutions were calculated using an iterative computer program (3). Stability constants used in these calculations (8) were adjusted for the temperature, pH, and ionic strength conditions of the present experiments. Each solution contained (in mM) 7 EGTA, 20 imidazole, 14.5 creatine phosphate, 1 free Mg2+, 4 free MgATP, sufficient KCl and KOH to produce a total ionic strength of 180 mM and a pH of 7.0. In addition, the relaxing and activating solutions had a free [Ca2+] of pCa 9.0 and pCa 4.5, respectively (where pCa = –log Ca2+ concentration). Creatine phosphokinase was not added as we and others have shown that the endogenous levels of this enzyme are adequate (9).
The single fiber isolation and test procedures were exactly as described previously (21, 22) and briefly reviewed here. On the day of an experiment, a single fiber segment was isolated from the muscle bundle and transferred to the stainless steel experimental chamber that contained three solution wells. While submerged under relaxing solution, the fiber was mounted between a force transducer (model 400; Cambridge Technology, Watertown MA) and a direct current position motor (model 300B; Cambridge Technology). The mounting procedure utilized 4-0 monofilament pins and 10-0 suture to fasten the fiber ends securely into small stainless steel troughs extending from the transducer and motor (22). The experimental chamber was attached to the stage of an inverted microscope so that the fiber could be viewed at x800 during data collection. Sarcomere length was adjusted to 2.5 µm using an eyepiece micrometer and this sarcomere length was maintained throughout the experiement. The length of the fiber (FL) suspended between the transducer and motor was measured with a micrometer that advanced the plate across the field of view. A charge-coupled device camera (model Provideo CVC-140; Sci/Speco) relayed a video of the fiber while it was briefly suspended in air (<5 s). A frame grabber card (model All-in-Wonder 128; ATI) captured a still image of the fiber, and diameter was measured at three points along the fiber using SCION Image software (Scion). Fiber diameter was determined from the average of the three measurements. Fiber cross-sectional area was calculated from fiber diameter under the assumption that the fiber forms a circular cross-section when suspended in air (11).
To initiate contraction, the fiber was rapidly transferred into an adjacent well filled with activating solution. Output from the force transducer and position motor were displayed on a digital oscilloscope before being amplified and interfaced to a personal computer via a LabMaster data-acquisition board. Custom software performed online analysis and stored data to disk. Relaxing and activating solutions were maintained at 15°C during all experiments.
Fibers were subjected to slack tests to measure unloaded maximal shortening velocity (V0), and the force-velocity relation was determined from a series of isotonic releases as described previously (21). P0 was defined as the peak force obtained during the experiment. Vmax was defined as the intercept of the force-velocity curve with the velocity axis. Peak absolute power was calculated from P0, Vmax, and a/P0, the parameter which specifies the curvature of the force-velocity relation. For graphical purposes, composite force-velocity-power relationships were plotted using the mean parameters.
A subgroup of fibers were activated with a series of solutions that had free Ca2+ concentrations ranging from pCa 6.8 to pCa 5.0. These solutions were made by mixing appropriate volumes of the activating (pCa 4.5) and relaxing (pCa 9.0) solutions. Peak force of each contraction was recorded and expressed as a fraction of the peak force obtained during maximal Ca2+ activation. The first and approximately every fourth subsequent contraction was performed at pCa 4.5. Hill plots were used to determine the Ca2+ concentration associated with Ca2+ activation threshold and with half-maximal activation (21). During these experiments, the fibers were subjected to sinusoidal length changes (frequency, 1.5 kHz; amplitude, 0.05% of FL), first in relaxing solution and then at the peak of the Ca2+ activated contraction. Changes in length (
length) and force (force) were used to calculate peak elastic modulus or E0 {E0 = [force in activating solution – force in relaxing solution/(length)] (FL/CSA)}, where CSA is cross-sectional area.
Following the contractile measurements, the fiber was removed from the transducer and motor, solubilized in 10 µl of 1% SDS sample buffer, and stored at –80°C. The myosin heavy chain content of each individual fiber was determined by 5% polyacrylamide gel electrophoresis (22).
Pre- and post-bed rest means between groups were analyzed using a one-way ANOVA, and a post hoc two-tailed t-test. Statistical significance was accepted at P < 0.05. All data are presented as means ± SE.
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RESULTS |
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2-fold greater in the soleus compared with fibers (Table 3). For the slow type I fibers of both muscles, P0 was reduced more than could be attributable to fiber atrophy such that the relative force (kN/m2) was depressed post-bed rest (Tables 3). For fast fibers, all of the force loss with BRHC was explained by fiber atrophy, such that, the specific force (kN/m2) was unaltered. In comparison, Bed rest alone produced a 7% and 8% decline in peak force in the type I fibers of the soleus (0.98 ± 0.03 to 0.91 ± 0.03 mN) and VL (0.83 ± 0.03 to 0.76 ± 0.03), respectively, and no change in the VL type II fiber P0. Unlike BRHC, bed rest alone had no effect on specific force in any fiber type. The mean pre-bed rest peak fiber force for the type I fibers of both muscles was significantly higher than the pre-bed rest BRHC subjects and this was in large part due to a greater mean fiber size. The mean soleus type I fiber diameter for 101 fibers was 101 ± 2 compared with the pre-bed rest value of 87 ± 1 for the 140 soleus type I fibers in the BRHC study (Table 3).
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The force velocity and power data for the BRHC group are shown in Table 4. It is clear that the primary effect of BRHC was to depress the peak power of the slow type I fibers in the soleus (19%) and VL (15%). The peak power of the VL type II fibers showed an 11% decline, but this drop was not significant. In contrast, the peak power of the VL type II fiber in the BR group showed a significant 17% depression. In the soleus, there were too few fast type II fibers to assess peak power. The reduced power in the type I and type II fibers of the BRHC group can be entirely explained by the 20% drop in force at peak power as the velocity at peak power was unaltered (Table 4).
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DISCUSSION |
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In this study, the administration of cortisol was designed to mimic the plasma levels reached during hospitalization associated with injury or illness, and the plasma cortisol concentrations frequently observed with prolonged space flight (16). The primary effect of excess cortisol is to increase muscle protein catabolism such that breakdown exceeds synthesis resulting in a loss of lean body mass (5). In a companion study of these same subjects, Paddon-Jones et al. (14) observed that a hypercortisolemic challenge contributed to a reduction in post-absorptive and post-meal net muscle protein synthesis. The reduced muscle protein balance compared with the eucortisolemia condition likely caused the greater atrophy and loss of force in the soleus fibers of the BRHC group. In addition, while bedrest alone had no effect on fiber force per cross-sectional area, this parameter was depressed by 7% in the soleus and VL type I fibers of the BRHC subjects. This suggests that the loss of contractile protein exceeded cell atrophy in the BRHC but not the BR subjects, and implies that the inhibition of net muscle protein synthesis (in part by accelerating protein degradation) by hypercortisolemia selectively affects myofibrillar proteins.
BRHC but not bed rest alone caused a greater atrophy and loss of force and power in type I fibers of the soleus compared with the VL. It is not clear why cortisol would have a preferential affect on one muscle over another. However, a comparison of the results of Yamashita-Goto et al. (23) with Trappe et al. (19) suggests that longer periods of bed rest (>2 mo) may elicit preferential atrophy of the soleus without cortisol injections. At 84 days of bed rest, the latter group observed 15, 47, and 28% decline in the fiber size, force (mN) and force/CSA of VL type I fibers, while after 4 mo of bed rest, the former found a 36, 76, and 42% decrease for these properties in soleus type I fibers. The differences could be attributed to: the greater duration of bed rest in the Yamashita-Goto et al. (23) study; a greater susceptibility of the soleus compared with the VL to bed rest induced atrophy; and/or a faster time course of the fiber atrophy and functional loss in the soleus compared with the VL. The latter was shown to be the case for ankle plantar and dorsal flexors during microgravity-induced loss of muscle strength (6).
While the data suggests that the type I fibers from both the soleus and VL of the BRHC subjects selectively lost contractile protein, the remaining fibrils appeared to function normally. The rate of cross-bridge binding as reflected by Ktr (Table 5) and the pCa-force relationship (Table 6 and Fig. 4) were unaltered. The slope of the Hill plot for the pCa-force relationship below one-half maximal force (n2) is thought to reflect the degree of cooperativity; a parameter influenced by both thin and thick filament proteins, and it was not affected by the bed rest plus hypercortisolemia condition.
Previously, we observed 3 wk of bed rest to significantly increase soleus type I fiber V0, a change associated with and perhaps caused by a selective loss in the actin (thin) relative to the myosin (thick) filament (22). Here, soleus type I fiber V0 was unaltered by any of the bed rest conditions suggesting that the filament spacing was normal. Bed rest has been shown to either have no effect or depress V0 in both slow and fast fibers of the VL (10, 19). In agreement, we observed a significant decline in VL type I fiber V0 following BR an effect not prevented by the AA/CHO supplement. The apparent slowing of this fiber type by bed rest in the VL was also reflected in the Ca2+ sensitivity as the activation at lower free Ca2+ and reduced n2 post bed rest are characteristic of slower fibers. In contrast, the AA/CHO supplement increased the cooperativity of force development compared with bed rest alone. The mechanism for this effect is unknown, but could be related to the supplements stimulation of myofibrillar protein synthesis (14).
AA/CHO supplementation prevented the bed rest-induced loss of force in the slow type I fibers of the soleus, but not the slow fibers of the VL. This result is surprising because the study by Paddon-Jones et al. (12), which focused on the same subjects, showed the supplement to preserve lean leg mass and ameliorate the loss of single-leg, one-repetition maximum (1RM) leg extension strength compared with the BR group. Therefore, one would expect the protective effects of the supplement to preserve contractile protein and force in all fibers and leg muscles. Paddon-Jones et al. (12) showed that the protective effect of the AA/CHO supplement was associated with higher mixed muscle protein synthetic rates and not mediated by changes in plasma cortisol which were unaltered by the supplement. It seems that the protective effect of the 1RM leg extension was at least in part caused by the AA/CHO amelioration of the bed rest-induced decline in the peak power of the fast type II fibers of the VL. Fiber V0 for the VL type II fiber in the AA/CHO supplement group was higher than the pre-bed rest value and significantly higher (P < 0.1) than the post bed rest control group. This increase in type II fiber velocity was entirely responsible for the increased peak power in this group.
Paddon-Jones et al. (12) used stable isotope methodology and DEXA to infer that the BR subjects were in a mild catabolic state throughout the 28 days of bed rest. The AA/CHO supplement preserved muscle protein synthesis and muscle mass. While the molecular mechanisms for the AA/CHO protection of single fiber function observed in this study are unknown, they are most likely linked to the ability of the supplement to stimulate muscle protein synthesis. Thus it seems reasonable to speculate that this supplement would reduce the deleterious changes associated with space flight as well as bed rest. Since the microgravity-induced loss of fiber function is primarily caused by inhibition of muscle protein synthesis leading to a selective loss of myofibrillar protein, fiber atrophy and a loss of fiber force and power (6, 20), an AA/CHO supplement that stimulates muscle protein synthesis should prove to be an effective countermeasure. This hypothesis needs to be tested in controlled studies aboard the International Space Station.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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2. Edgerton VR, Zhou MY, Ohira Y, Klitgaard H, Jiang B, Bell G, Harris B, Saltin B, Gollnick PD, Roy RR, Day MK, Greenisen M. Human fiber size and enzymatic properties after 5 and 11 days of spaceflight. J Appl Physiol 78: 1733–1739, 1995.
3. Fabiato A, Fibiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol 75: 463–505, 1979.
4. Ferrando AA, Lane HW, Stuart CA, Wolfe RR. Prolonged bed rest decreases skeletal muscle and whole body protein synthesis. Am J Physiol Endocrinol Metab 270: E627–E633, 1996.
5. Ferrando AA, Stuart CA, Sheffield-Moore M, Wolfe RR. Inactivity amplifies the catabolic response of skeletal muscle to cortisol. J Clin Endocrinol Metab 84: 3515–3521, 1999.
6. Fitts RH, Riley DA, Widrick JJ. Physiology of a microgravity environment invited review: microgravity and skeletal muscle. J Appl Physiol 89: 823–839, 2000.
7. Fitts RH, Riley DA, Widrick JJ. Functional and structural adaptations if skeletal muscle to microgravity. J Exp Physiol 204: 3201–3208, 2001.
8. Godt RE, Lindley BD. Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog. J Gen Physiol 80: 279–297, 1982.
9. Knuth ST, Dave H, Peters JR, Fitts RH. Low cell pH depresses peak power in rat skeletal muscle fibres at both 30 and 15 degrees centigrade: Implications for muscle fatigue. J Physiol 575: 887–899, 2006.
10. Larsson L, Xiaopeng L, Berg WE, Frontera WR. Effects of removal of weight bearing function on contractility and myosin isoform composition in single human skeletal muscle cells. Pflügers Arch 432: 320–328, 1996.[CrossRef][ISI][Medline]
11. Metzger JM, Moss RL. Shortening velocity in skinned single muscle fibers. Influence of filament lattice spacing. Biophys J 52: 127–131, 1987.[ISI][Medline]
12. Paddon-Jones D, Sheffield-Moore M, Urban RJ, Sanford AP, Aarsland A, Wolfe RR, Ferrando AA. Essential amino acid and carbohydrate supplementation ameliorates muscle protein loss in humans during 28 days bedrest. J Clin Endocrinol Metab 89: 4351–4358, 2004.
13. Paddon-Jones D, Sheffield-Moore M, Urban RJ, Sanford AP, Aarsland A, Wolfe RR, Ferrando AA. Exogenous amino acids stimulate human muscle anabolism without interfering with the response to mixed meal ingestion. Am J Physiol Endocrinol Metab 288: E761–E767, 2005.
14. Paddon-Jones D, Sheffield-Moore M, Urban RJ, Aarsland A, Wolfe RR, Ferrando AA. The catabolic effects of prolonged inactivity and acute hypercortisolemia are offset by dietary supplementation. J Clin Endocrinol Metab 90: 1453–1459, 2005.
15. Riley DA, Bain JLW, Thompson JL, Fitts RH, Widrick JJ, Trappe SW, Trappe TA, Costill DL. Disproportionate loss of thin filament in human soleus muscle after 17-day bed rest. Muscle Nerve 21: 1280–1289, 1998.[CrossRef][ISI][Medline]
16. Stein TP, Leskiw MJ, Schluter MD, Donaldson MR, Larina I. Protein kinetics during and after long-duration spaceflight on MIR. Am J Physiol Endocrinol Metab 276: E1014–E1021, 1999.
17. Stein TP, Leskiw MJ, Schluter MD, Hoyt RW, Lane HW, Gretebeck RE, LeBlanc AD. Energy expenditure and balance during spaceflight on the space shuttle. Am J Physiol Regul Integr Comp Physiol 276: R1739–R1748, 1999.
18. Stein TP, Schluter MD, Moldawer LL. Endocrine relationships during human spaceflight. Am J Physiol Endocrinol Metab 276: E155–E162, 1999.
19. Trappe S, Trappe T, Gallagher P, Harber M, Alkner B, Per Tesch B. Human single muscle fibre function with 84 day bed-rest and resistance exercise. J Physiol 557: 501–513, 2004.
20. Widrick JJ, Knuth ST, Norenberg KM, Romatowski JG, Bain JLW, Riley DA, Karhanek M, Trappe SW, Trappe TA, Costill DL, Fitts RH. Effect of a 17 day spaceflight on contractile properties of human soleus muscle fibres. J Physiol 516: 915–930, 1999.
21. Widrick JJ, Norenberg KM, Romatowski JG, Blaser CA, Karhanek M, Sherwood J, Trappe SW, Trappe TA, Costill DL, Fitts RH. Force-velocity-power and force-pCa relationships of human soleus fibres after 17 days of bed rest. J Appl Physiol 85: 1949–1956, 1998.
22. Widrick JJ, Romatowski JG, Bain JLW, Trappe SW, Trappe TA, Thompson JL, Costill DL, Riley DA, Fitts RH. Effect of 17 days of bed rest on peak isometric force and unloaded shortening velocity of human soleus fibers. Am J Physiol Cell Physiol 273: C1690–C1699, 1997.
23. Yamashita-Goto K, Okuyama R, Honda M, Kawasaki K, Fujita K, Yamada T, Nonaka I, Ohira Y, Yoshioka T. Maximal and submaximal forces of slow fibers in human soleus after bed rest. J Appl Physiol 91: 417–424, 2001.
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