INTRODUCTION

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SKELETAL MUSCLE ATROPHY occurs in a variety of situations but is most commonly associated with removal of mechanical load from the musculoskeletal system. Examples of situations in which skeletal muscle atrophy occurs include the aging population (7) and patients who have undergone bed-rest immobilization after traumatic injury and/or surgery (2, 28) or after exposure to microgravity during spaceflight (38, 58). In all of these cases, the removal of mechanical load from skeletal muscle has been directly implicated in the initiation of the muscle atrophic response. In humans, these responses involve a reduction in overall muscle volume and myofiber cross-sectional area (3, 18, 38), alterations in muscle-specific protein synthesis and/or degradation (20, 40, 60, 63), disruption of neuromuscular function (17, 25, 32, 35), decreases in muscle strength (15, 37, 56), and changes in muscle capillarity (27). These changes are paralleled by similar changes in animal models of disuse or unloading, such as the rodent hindlimb suspension (5, 57) and cast immobilization (6) models.

The mechanism(s) whereby a reduction in mechanical load results in the initiation of an atrophic response (i.e., a negative growth status) is as yet unclear. However, it is certain that full-scale remodeling of skeletal muscle occurs as a result of the removal of mechanical load, as evidenced by changes not only in the contractile elements of the muscle (i.e., the myofiber) but also in the neuronal and vascular components of the tissue (47). Tissue remodeling, such as occurs during wound healing and during development, is under tight control by a number of highly potent growth factors, which can act either independently or in concert to bring about tissue restructuring. It is therefore logical to postulate that any tissue remodeling that occurs as a direct consequence of mechanical unloading must involve either the initiation of novel growth factor signaling pathways or the disruption of existing ones. It is the investigation of the latter of the two possibilities that concerns this report.

Our laboratory has previously described a mechanical load-induced plasma membrane phenomenon termed membrane wounding (8-11). This process, which involves the transient disruption of the plasma membrane of a mechanically loaded cell, is responsible for release of unbound cytosolic molecules from, and entry of freely diffusible extracellular molecules into, the cytoplasmic compartment of the wounded cell. This transient, survivable event has been reported in a variety of different mechanically active tissues in vivo (8, 10, 41, 42, 62) and in a broad range of different cell types exposed to mechanical force in vitro (11-13, 44). Membrane wounding has been shown to be capable of allowing the release of several different cytosolic molecules from wounded cells, including lactate dehydrogenase (43), creatine kinase (CK) (42), and fibroblast growth factor (FGF) (8, 9, 12, 30, 43). Because FGF does not contain a signal transduction peptide sequence (1), the mechanism by which FGF exits its producer cells to act at extracellular receptor sites has been unclear. It has become increasingly apparent from both our own experimental work and that of other researchers that one mechanism by which FGF [both the acidic (aFGF) and basic isoforms (bFGF)] is released from its producer cells is via a membrane wound-mediated mechanism.

In the context of skeletal muscle, our laboratory has previously shown that membrane wound-mediated FGF release occurs in both skeletal (bFGF) (10) and cardiac muscle tissue (both aFGF and bFGF) (8) in vivo. These studies have shown a clear linear relationship among the amount of mechanical load placed on the muscle, the amount of skeletal myofiber and/or cardiac myocyte wounding that occurred, and the amount of FGF released from the cytoplasm of the wounded muscle cells. In addition, using a mechanically active tissue culture model, we have shown that specific blockade of bFGF, released via mechanically induced wounds into the extracellular environment from differentiated skeletal myotubes, results in the inhibition of the usual mechanical load-induced growth response of these cells (9). A similar observation has recently been reported in the case of electrically stimulated cardiac myocytes (30). Furthermore, in animal exercise models, increased mechanical loading of skeletal muscle results in upregulation of muscle bFGF mRNA and bFGF protein synthesis (26, 46). These experimental observations have led us to suggest that mechanical load-induced, membrane wound-mediated myofiber FGF release is a signaling pathway whereby an increase in mechanical load can be transduced into a muscle hypertrophic response, in a fashion that also localizes the biological response in time and space.

Our laboratory and others have previously shown that there is a constitutive level of myofiber wounding (10, 41) and consequent membrane wound-mediated bFGF release (10) in the skeletal muscle of normal ambulatory rodents. We hypothesize that the removal of mechanical load from the antigravity muscles of humans during spaceflight results in the disruption of this constitutive level of myofiber wounding, which results in the reduction of the amount of FGF (both aFGF and bFGF) released into the muscle microenvironment. Evidence to support this hypothesis stems from the observation that the MM isoform of CK (CKMM; released on myofiber wounding) is significantly reduced in astronauts after spaceflight (36). To fully test this hypothesis in a controlled situation, we have utilized a model of spaceflight-induced skeletal muscle atrophy, specifically a 14-day, 6° head-down-tilt bed-rest model (4). As this study involved human subjects, the experimental protocols used to assess the amount of myofiber wounding and consequent FGF release from unloaded skeletal muscle necessitated the least invasive measures possible, namely, monitoring of pre-bed-rest and post-bed-rest circulating levels of CKMM and FGF (both aFGF and bFGF) in an attempt to indirectly determine the amounts of CKMM and FGF released into the muscle microenvironment. In addition, a needle biopsy was performed in the same region of the antigravity vastus lateralis muscle before bed rest and after bed rest to assess the effects of mechanical unloading on the myofiber cross-sectional area. Furthermore, this study was also designed to determine the effectiveness of resistive exercise during bed rest in preventing unloading-induced skeletal muscle atrophy. As such, this study is unique in that experimental observations from three separate loading states (i.e., bed rest alone, bed rest plus resistive exercise, and ambulatory plus resistive exercise) have been compared within the same study of human subjects.

	MATERIALS AND METHODS

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Materials. The mouse monoclonal antibody to human aFGF was purchased from Biodesign International (Kennebunk, ME). The mouse monoclonal antibody to human bFGF and purified recombinant aFGF and bFGF were obtained from Oncogene Science (Cambridge, MA). The horseradish peroxidase-conjugated sheep anti-mouse IgG polyclonal antibody (preadsorbed against human serum proteins) was purchased from Sigma Chemical (St. Louis, MO). Immulon II 96-well microtiter plates were obtained from Dynatech Laboratories (Chantilly, VA). All other laboratory reagents were of reagent grade or higher and were purchased from Sigma Chemical.

Subject characteristics. All subjects were men and were recruited from the greater Houston (TX) metropolitan area. Subjects were randomly assigned to the following experimental groups: no exercise, 14 days of bed rest (NExB); resistance exercise, 14 days of bed rest (RExB); and resistance exercise, ambulatory (RExA). The average age, height, and weight of each experimental group were as follows: NExB, 28.1 ± 5.3 (SD) yr, 178.6 ± 5.9 cm, 74.2 ± 10.3 kg; RExB, 29.4 ± 6.9 yr, 178.4 ± 4.1 cm, 80.6 ± 15.1 kg; and RExA, 32.6 ± 6.4 yr, 175.3 ± 2.9 cm, 71.7 ± 5.9 kg, respectively. No statistical differences were detected among experimental groups with regard to age, height, or weight. Candidates were normotensive, nonsmoking, healthy, and ±1 SD of their ideal body weight. Subjects passed a comprehensive physical examination, including a maximal graded treadmill test with 12-lead electrocardiogram, which was reviewed by a physician. To ensure that the effects of resistance exercise were studied exclusive of the effects of detraining, only individuals who had not participated in a resistance training program for at least 1 yr before the start of the study were allowed to participate. All subjects were given an oral and written briefing before they gave their informed consent. Bed-rest subjects were housed in the General Clinical Research Center at The University of Texas Medical Branch-Galveston (UTMB) for 16 days (1 day ambulatory, 14 days of bed rest, 1 day of recovery). The protocol was approved by the Institutional Review Boards of the National Aeronautics and Space Administration (NASA)-Johnson Space Center and UTMB.

Bed-rest protocol. The bed-rest and resistive exercise protocols utilized in this study have been reported in detail elsewhere (4). Briefly, 16 healthy men (all of whom had passed an Air Force Class III physical examination), split into two equal groups at random, were bed rested for 14 days (NExB) or bed rested for 14 days with resistive exercise (RExB). Resistive exercise training was carried out by using a horizontal leg-training device (Cybex Strength Systems, Ronkonkoma, NY). The knee extensors and plantor flexors were exercised every other day during bed rest with the subject in the supine position. Exercises were performed at a volume and intensity known to effectively induce gains in strength and muscle mass in terrestrial conditions (33). To minimize muscle soreness at the onset of training, exercise volume and intensity were increased progressively during the first three exercise sessions. By session 3, five sets to volitional muscle failure were completed for each muscle group. A third ambulatory group of six men performed identical resistive exercise but were not bed rested (RExA).

Needle biopsy protocol. Samples were removed from the same area of the vastus lateralis muscle of the dominant leg in accordance with the needle biopsy technique of Evans et al. (19) on day 1 and again on day 14 of bed rest. Standard sterile surgical techniques for minor surgery were used for all biopsies. Muscle samples were taken from the biopsy needle and immediately mounted on cork blocks by using Tissue-Tek OCT mounting compound (Miles, Elkhart, IN) and were oriented cross-sectionally by using a dissecting microscope. Mounted samples were then quickly frozen in isopentane cooled to 160°C with liquid nitrogen. Muscle biopsy samples were not obtained from the RExA experimental group.

Frozen sectioning. Snap-frozen muscle was placed on a sectioning stub, mounted in OCT embedding compound, and frozen in the vapor phase of liquid nitrogen. Frozen cross sections (10 µm in thickness) were cut by using a Zeiss Microm HM 500 OM microtome cryostat and were picked up onto three-well, Teflon-masked Superfrost Plus glass slides (Erie Scientific, Portsmouth, NH). Sections were then allowed to air dry for 90 min before they were stained.

Muscle staining procedures. Air-dried sections of snap-frozen vastus lateralis muscle were stained by using the metachromatic dye-ATPase myofibrillar stain and by using the method of Ogilvie and Feeback (49). With the use of this stain, myofiber types I, IIa, and IIb are colored turquoise, light violet, and dark violet, respectively.

Myofiber typing and histomorphometry. Digitized images of stained vastus lateralis muscle sections were analyzed by using the Global Lab Image Processing and Analysis Software Package (Data Translation, Marlboro, MA). Images from each different group in the study (i.e., pre-bed rest and post-bed rest from NExB and RExB groups) were acquired by using the same camera settings so that equivalent measurements were made in control and test sections. Measurement of myofiber dimensions was made in sections stained for myofibrillar ATPase as part of the image-analysis procedure required to identify myofiber type based on staining color. Data were archived in the Global Lab Image Spread Sheet format and analyzed by using the Excel spreadsheet program. Myofiber cross-sectional area was expressed in square micrometers. Three myofiber types were identified by using the metachromatic ATPase staining technique, namely type I, type IIa, and type IIb.

Serum sample collection. Serum sample collection was performed immediately before muscle biopsy by standard venipuncture technique by using a "red-top" Vacutainer tube. Blood was allowed to clot for 10 min at room temperature and then was centrifuged at 3,000 g for 20 min at 4°C. Serum was carefully removed from the packed red cell mass and aliquoted in 0.5-ml aliquots, and then it was immediately snap frozen in liquid nitrogen and stored at 80°C until analysis. As total blood volume decreases as a consequence of bed rest, both serum CKMM and FGF measurements after bed rest were normalized on an individual-subject basis to account for the reduction in blood volume. Blood volume measurements were carried out as part of a parallel study by using radioisotope labeling of erythrocytes (data provided courtesy of Dr. S. Fortney, NASA-Johnson Space Center).

Serum CKMM analysis. Total CK was determined as previously described (9). Briefly, serum samples were assayed for total CK activity by using a commercially available assay for CK based on the conversion of NADP to reduced nicotinamide adenine dinucleotide phosphate (NADPH) (Roche Diagnostic Systems), which was measured at 340 nm by using a Cobas Mira Chemical Analyzer (Roche Diagnostic Systems). The normal serum CK range (as determined by clinical standards provided by Roche Diagnostics), with the use of our procedure, was 21-510 IU/l. In addition, CK isoenzyme profiles were also determined from the same samples by using the commercially available automated Paragon Gel System (Beckman, Fullerton, CA), followed by densitometry, as described by the manufacturer. This system separates CK isoforms by using agarose gel electrophoresis followed by incubation of the gel in CK substrate buffer containing creatine phosphate, hexokinase, ATP, D-glucose, glucose-6-phosphate, and NADP as the major components. The chemical reaction of the CK isoenzymes with its substrate within the matrix of the gel results in the production of NADPH, which is then quantified by using fluorescent densitometry at an excitation wavelength of 340 nm and emission wavelength of 455 nm.

Serum FGF analysis. Serum aFGF and bFGF levels were determined by using a modification of the methodology previously described (8, 9). Briefly, 50 µl of serum were dispensed into a single well of an Immulon II 96-well ELISA plate. Each sample was then diluted with 30 µl of calcium/magnesium-free PBS (pH 7.4). Serum samples from each subject were measured in triplicate. A standard curve was constructed on the same plate by using recombinant human aFGF or bFGF diluted in calcium/magnesium-free PBS containing 50 µg/ml of BSA. In addition, a pooled serum sample, which had previously been rendered free of FGF by heparin affinity chromatography, was used as a negative control serum sample. The plate was then sealed with plastic film and incubated at room temperature for 16 h. The wells were washed five times with Dulbecco's PBS containing 1 mM calcium and 1 mM magnesium (D-PBS; pH 7.4), which contained 0.1% Tween 20 (wash buffer) over a period of 20 min at 37°C. The wells were blocked by using D-PBS containing 4% BSA and 0.1% Tween 20 for 1 h at 37°C. Blocking buffer was removed by washing the wells once with wash buffer and was replaced with 50 µl of primary antibody solution (either 0.5 µg/ml of mouse anti-aFGF IgG monoclonal antibody or 0.25 µg/ml of mouse anti-bFGF IgG monoclonal antibody made up in D-PBS containing 0.1% BSA and 0.1% Tween 20) and incubated for 1 h at 37°C. Primary antibody solution was removed by washing the wells with five changes of wash buffer at 37°C. Fifty microliters of secondary antibody solution (2 µg/ml of horseradish peroxidase-conjugated sheep anti-mouse IgG preadsorbed against human serum proteins made up in D-PBS containing 0.1% BSA and 0.1% Tween 20) were then dispensed into each well and incubated for 1 h at 37°C. The wells were then washed with five changes of wash buffer over 20 min at 37°C, and antibody binding was disclosed by using a hydrogen peroxide-catalyzed o-phenylenediamine reaction carried out in a citrate buffer (pH 6.0) as previously described. Optical density was determined by using a Spectromax 96-well plate reader at 490 nm. Wells containing the FGF-free pooled serum sample were used as the blank. Serum aFGF and bFGF levels were calculated relative to their respective standard curves constructed on the same plate.

Data analysis. All group scores on dependent variables were reported as the mean and its SD. Statistical analysis of pre-bed-rest and post-bed-rest serum CKMM, aFGF, and bFGF levels in individual subjects within experimental groups was carried out by using a paired t-test. Statistical analysis of the relationship between overall myofiber wounding (determined by serum CKMM levels) and myofiber cross-sectional area by fiber type in the vastus lateralis muscle of individual subjects was carried out by calculating a Pearson correlation coefficient after linear regression. The null hypothesis in all cases was rejected if P < 0.05.

	RESULTS

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The effect of mechanical loading level on myofiber cross-sectional area. Fourteen days of 6° head-down-tilt bed rest resulted in a significant (P < 0.05) reduction (16.8%) in mean myofiber cross-sectional area, assessed after morphometric analysis of metachromatic ATPase-stained frozen sections of the vastus lateralis muscle. Significant (P < 0.05) decreases in type I (14.6%) and type IIa (17.7%), but not type IIb, myofiber cross-sectional areas were observed in the NExB group. No significant changes in myofiber cross-sectional area were observed in the RExB group. As muscle biopsies were not performed on the RExA group, no myofiber cross-sectional area data were available for these subjects. A complete description and analysis of the muscle biopsy data obtained from this bed-rest study, presented in brief in this report, can be found in Bamman et al. (4).

The effect of mechanical loading level on serum CKMM levels. Circulating levels of the myofiber wound marker CKMM were determined in serum samples obtained from subjects in the NExB, RExB, and RExA groups. There was a significant decrease in circulating levels of CKMM in the NExB group, whereas there was a significant increase in circulating levels of CKMM in the RExB group (Fig. 1). In the RExA group, circulating CKMM was also significantly increased (Fig. 1). However, in the RExB group the increase in circulating CKMM was much greater (i.e., a 381% increase) than that observed in the RExA group (i.e., a 26% increase) (Fig. 1).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Effect of bed rest, bed rest plus resistive exercise, or resistive exercise alone on circulating levels of myofiber wound marker creatine kinase MM (CKMM); n = 7 subjects per experimental group. Pre, before bed rest; post, after bed rest. * P < 0.05 vs. pre-bed-rest condition.

The effect of mechanical loading level on serum FGF levels. Circulating levels of the muscle growth factors aFGF and bFGF were determined in serum samples obtained from subjects in the NExB, RExB, and RExA groups. There was a significant decrease in circulating levels of aFGF in the NExB group, whereas there was a significant increase in circulating levels of aFGF in the RExB group (Fig. 2). In the RExA group, circulating aFGF remained unchanged (Fig. 2). No significant changes in circulating levels of bFGF were detected in any of the experimental groups (data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effect of bed rest, bed rest plus resistive exercise, or resistive exercise alone on circulating levels of acidic fibroblast growth factor (aFGF); n = 7 subjects per experimental group. * P < 0.05 vs. pre-bed-rest condition.

Relationship between myofiber wounding and myofiber cross-sectional area. The percent change in mean myofiber cross-sectional area, or percent change in myofiber cross-sectional area by myofiber type (i.e., type I or type II), was plotted against the percent change in circulating CKMM in both the NExB and RExA groups (Fig. 3). A significant correlation between changes in myofiber cross-sectional area and the amount of myofiber wound marker (CKMM) present in the serum on an individual-subject basis was disclosed between mean myofiber cross-sectional area (Fig. 3A) and type II myofiber cross-sectional area (Fig. 3B), but not type I cross-sectional area (Fig. 3C).

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Relationship between overall myofiber wounding and myofiber cross-sectional area in vastus lateralis muscle. %Change in circulating CKMM vs. %change in mean cross-sectional area for type I and type II myofibers (A), type II myofibers only (B), and type I myofibers only (C). Data obtained from a total of 14 subjects (7 bed rest alone, 7 bed rest plus resistive exercise). ns, Not significant.

	DISCUSSION

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A period of 14 days of 6° head-down-tilt bed rest resulted in significant myofiber atrophy in the vastus lateralis muscle. The atrophic response observed in this muscle was paralleled by a significant reduction in circulating levels of the myofiber wound marker CKMM. Although the vastus lateralis muscle is not the only source of CKMM released into the circulation, these data suggest that mechanical unloading of skeletal muscle tissue during bed rest, including the vastus lateralis muscle, results in a significant decrease in the incidence of mechanically induced myofiber wounding. It has been shown previously that resistive exercise is a highly efficient means of inducing myofiber wounds in skeletal muscle (10, 42, 48). Resistive exercise in ambulatory subjects (RExA) resulted in a significant increase in circulating CKMM, an observation previously reported in an animal exercise model (42) and one directly correlated with mechanical load-induced sarcolemma wounding (30). However, in bed-rest subjects who performed resistive exercise (RExB), circulating CKMM levels increased dramatically compared with those detected in the ambulatory exercise control group (RExA). This observation indicates that the sarcolemma of unloaded skeletal muscle is much more susceptible to mechanical load-induced wounding than is the sarcolemma of normal muscle. Such an observation may explain the large-scale overt myofiber damage detected after reambulation in the skeletal muscle of rodents that have undergone muscle unloading due to hindlimb suspension (29, 34) or spaceflight (51-53). It must be stated that skeletal muscle is not the only potential source of circulating CKMM. Cardiac muscle is also known to contain the MM isoform of CK. However, as no significant amounts of the cardiac-specific MB isoform of CK were detected in the serum of any of our bed-rest subjects, with or without the performance of resistive exercise, or in our ambulatory resistive exercise control group (data not shown), the possibility of cardiac myocyte wounding contributing to the alterations observed in circulating CKMM levels as a consequence of bed rest or resistive exercise appears unlikely.

The reduction in circulating CKMM detected in bed-rest subjects was also paralleled by a decrease in circulating levels of aFGF, but not bFGF. Our laboratory has reported previously that myofiber wounding results in the release of bFGF into the muscle microenvironment (10), from which it can enter the circulation. The transfer of both aFGF and bFGF from the muscle microenvironment into the circulation has been demonstrated in the isolated rat heart, where both aFGF and bFGF, released as a consequence of cardiac myocyte wounding, can be detected and quantified in the perfusion solution (8). aFGF and bFGF, both small, freely diffusible cytosolic molecules, lack a signal transduction peptide sequence and hence are not released via the classic vesicular exocytotic pathway associated with other growth factors produced within cells (1). We, and others, have shown previously that mechanically induced plasma membrane wounds are an efficient mechanism for the release of cytosolic FGF from a variety of cells, including skeletal myofibers (10), cardiac myocytes (8, 30), endothelial cells (12, 43), smooth muscle cells (39), and epithelial cells (50). On the basis of these experimental observations and the relationship between myofiber wounding and CKMM release, it is logical to assume that the changes in circulating aFGF may be due to alterations in the level of myofiber wounding. Again, it must be stated that skeletal muscle is not the only potential source for circulating FGF. The two major sources of FGF in the body on a per-unit-of-protein basis are cardiac and skeletal muscle (54). However, the lack of any significant amount of the cardiac-specific MB isoform of CK in the serum of any of our subjects suggests that the possibility of cardiac myocyte involvement in the alterations observed in circulating aFGF levels as a consequence of bed rest or resistive exercise appears unlikely. A third potential source of FGF in the body that may have an impact on circulating levels of FGF is endothelial cells. Our laboratory has shown previously that fluid shear stress can induce plasma membrane wound-mediated bFGF release from endothelial cells in vitro (12). Similar mechanically induced endothelial wounding has been shown to be a common event in vivo (62). During resistive exercise, it is entirely possible that increased blood flow to the muscle microvascular bed, or indeed the compressive mechanical forces placed on the muscle microvascular bed during muscle contraction, may lead to wound-mediated release of endothelial-derived FGF into the circulation. However, the relative amount of FGF available for release from mature endothelial cells on a per-unit-microgram-of-protein basis is very small compared with the amounts available for release from skeletal and cardiac muscle. Hence, even if a substantial number of endothelial cells were wounded in contracting muscle, the overall contribution of endothelial-derived FGF to the circulating FGF pool would be negligible.

Our laboratory has shown previously in a tissue culture model of the mechanically active human skeletal muscle myotube that specific antibody neutralization of FGF released into the extracellular environment via mechanically induced sarcolemmal wounds resulted in the inhibition of the usual mechanical load-induced muscle cell growth response (9). On the basis of this direct experimental observation, we have suggested previously that mechanical load-induced, membrane wound-mediated FGF release is a signaling mechanism capable of transducing mechanical load into a biological skeletal muscle growth response. To determine whether there was a correlation in our bed-rest model between changes in the mechanical loading level (and consequent myofiber wounding) and the modulation of myofiber cross-sectional size in the vastus lateralis muscle, we plotted percent change in CKMM vs. percent change in myofiber cross-sectional area on an individual-subject basis (pre-bed-rest to post-bed-rest levels). When the percent change in mean myofiber cross-sectional area (i.e., type I and II myofibers) was plotted against the percent change in serum CKMM, a significant positive correlation between myofiber wounding (as reported by circulating CKMM) and mean myofiber cross-sectional area in the vastus lateralis muscle was detected. If percent change in myofiber cross-sectional area, expressed on a myofiber-type basis, was plotted against percent change in serum CKMM, a significant positive correlation between myofiber wounding and type II myofiber cross-sectional area, but not type I myofiber cross-sectional area, was detected in the vastus lateralis muscle. These data suggest that a decrease or an increase in mechanically induced myofiber wounding is paralleled by a decrease or increase in myofiber cross-sectional area, respectively. It is interesting to note that the highest correlation between myofiber wounding and alteration in myofiber cross-sectional area was found in type II rather than type I myofibers. In exercised human muscle, type II myofibers appear to undergo selective hypertrophy (32), whereas, in unloaded human muscle, type II myofibers appear to undergo selective atrophy (4). These observations are consistent with the possibility that type II myofibers have an increased susceptibility to mechanical load-induced sarcolemma wounding and consequent myofiber FGF release than do type I myofibers. Such a result may provide an explanation for the observations made by numerous investigators that type II myofibers are much more plastic in their growth responses to mechanical unloading than are type I myofibers (4, 18, 24, 33, 55). However, the observation that smaller myofibers (consistent with a type I phenotype in human but not necessarily in rat muscle) are more prone to myofiber wounding on reloading of rat muscle after hindlimb suspension (29) raises a second possibility, namely, that type I myofibers are more susceptible to mechanical load-induced sarcolemma wounding than are type II myofibers. However, as neither the previous study (29) nor our own study directly correlates myofiber type with wounding incidence on a myofiber-by-myofiber basis, further investigation is required before a definitive answer to this question will be available.

Our data suggest that a reduction in the amount of myofiber wound-mediated release of sarcoplasmic FGF may be in part responsible for the initiation of a muscle atrophic response. Therefore, it may be expected that, if this hypothesis is correct, then the amount of FGF detected in the serum would also be correlated with the percent change in myofiber cross-sectional area. However, when similar plots were prepared for the relationship between the changes in circulating aFGF and the changes in myofiber cross-sectional area, no statistically significant correlation was detected. However, unlike CKMM, muscle tissue has a very specific extracellular "sink" for FGF, namely extracellular matrix heparin sulfate. The presence of this extracellular sink, which has a higher affinity for bFGF than aFGF (31), may explain why no significant differences in circulating bFGF were detected. In addition, our preliminary results in exercising men have shown that serum bFGF levels appear to reach a maximum within 2-3 h after exercise, whereas aFGF appears to remain in the circulation for a longer period of time and have similar kinetics to CKMM (data not shown). As post-bed-rest blood samples from resistive exercised subjects were collected 20 h after the last bout of resistive exercise, coupled with the presence of a large extracellular sink for FGF, it is entirely possible that much larger changes in circulating aFGF and bFGF may have been detected if blood samples had been obtained within 4 h of cessation of resistive exercise in both bed-rest and ambulatory subjects.

One unexpected observation made in this study was that the skeletal muscle of subjects in the RExB group appeared to be much more susceptible to mechanical load-induced sarcolemma damage as a consequence of resistive exercise than that of the RExA subjects. This observation has previously been shown to be true on reloading of muscle from hindlimb-suspended (29, 34) and space-flown rats (52). The molecular basis of this difference is as yet unclear. However, our laboratory has demonstrated previously that modulation of the physical properties of the plasma membrane (namely, the molecular ordering parameter of the lipid bilayer), by using structural lipids such as cholesterol or synthetic ordering and disordering agents, results in alterations in the susceptibility of the plasma membrane to mechanical load-induced wounding (11, 12). On the basis of preliminary results obtained from hindlimb-suspended rat muscle that indicate an approximate increase of 60% in sarcolemmal cholesterol content compared with control muscle (data not shown), we postulate that the increased susceptibility to mechanical load-induced myofiber wounding observed after bed rest may be due to alterations in the structural lipid composition of the sarcolemma.

In summary, the data presented in this study indicate that mechanical unloading of skeletal muscle in the whole human results in a decrease in the incidence of mechanical load-induced myofiber wounding and consequent release of FGF into the muscle microenvironment. A decrease in this signaling mechanism appears to be positively correlated with a reduction in cross-sectional area of myofibers in the antigravity vastus lateralis muscle. The performance of resistive exercise during bed rest abolishes vastus lateralis muscle myofiber atrophy, and this abolition is positively correlated with an increase in myofiber wounding and consequent FGF release into the muscle microenvironment. In addition, the correlation appears to be stronger in the case of type II myofibers than for type I myofibers. Furthermore, bed rest appears to cause remodeling of not only the contractile apparatus of muscle but also that of the sarcolemma. FGF is involved in a number of other signaling cascades within tissues. These include the regulation of insulin-like growth factor-I binding protein production, the modulation of the growth effects of insulin-like growth factor-1 on muscle cells (21, 45, 59, 61), the production of nerve growth factor, and the expression of nerve growth factor receptors by neuronal cells (14, 16, 23) and as a direct angiogenic stimulus to capillary endothelial cells (22). As such, it is entirely possible that a decrease in the amount of membrane wound-mediated FGF release into the muscle microenvironment not only may play a key role in the disruption of the maintenance of muscle mass (i.e., myofibrilar protein) but also may contribute to the disruption of neuronal input and the decrease in capillary density commonly observed in unloaded skeletal muscle.