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1Department of Physiology and the Pennsylvania Muscle Institute and 2Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Submitted 27 December 2004 ; accepted in final form 20 June 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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hindlimb unloading; protease inhibitors; Bowman-Birk inhibitor; protein degradation
Much recent work has focused on the IGF-I/phosphatidylinositol 3-kinase/Akt signaling pathway and identified its role as a critical regulator of muscle cell size, capable of stimulating muscle hypertrophy (3) or inhibiting muscle atrophy (8, 40). Although transgenic IGF-I overexpression produced muscle hypertrophy, it did not attenuate muscle atrophy associated with hindlimb unloading (HU) (11); however, direct intramuscular injection of IGF-I was recently shown to reduce muscle atrophy in a denervation model (40). Expression of constitutively active Akt, a signaling protein downstream of IGF-I, was able to stimulate hypertrophy and to ameliorate denervation atrophy (8). Also, the levels and activities of other key signaling proteins involved in muscle growth have been shown to significantly change during modified muscle use (17, 18, 36). Together, these results suggest that maintaining growth signaling via gene therapy or pharmacological approaches likely could lead to an amelioration of muscle atrophy.
An alternative and perhaps complementary therapy to counter muscle atrophy could be to target the accelerated protein degradation rate associated with decreased muscle use. The removal of muscle protein, most notably myofibrillar proteins, occurs primarily through activation of the ubiquitin-proteasome pathway (38, 41, 42). However, this pathway is not involved in initial myofibrillar protein cleavage. Calcium-dependent proteinases (calpains), lysosomal-related proteolysis (cathepsins B+L), and apoptosis (caspases) are all likely involved, although the specific roles and the extent of involvement of each are unclear (1, 13, 19, 38, 43).
Other degradative processes that may be involved in muscle atrophy include intracellular and extracellular protease cascades, such as serine proteases (16, 37, 39) and matrix metalloproteinases (MMPs) (35). Serine proteases are widely expressed and play important roles in many cellular processes requiring regulated protein turnover. In muscle, serine proteases have been identified that are capable of cleaving myofibrillar proteins (16, 37), and chymase, a serine protease, can cleave soluble muscle proteins (12). Serine proteases have also been linked to extracellular matrix remodeling processes (15, 28) and activation of MMPs (10, 26, 35). Serine protease cascades potentially could act as a first step in the initiation of protein degradation. Thus blocking the initiation of such a cascade would likely provide significant benefit in reducing muscle loss.
The Bowman-Birk inhibitor (BBI) is a well-characterized, nontoxic serine protease inhibitor (22, 23), with the ability to inhibit the activity of numerous proteases, such as chymotrypsin, trypsin, cathepsin G, elastase, and chymase (6, 27, 46). BBI has been investigated in a number of anticarcinogenic and anti-inflammatory studies as purified BBI or as an extract enriched for BBI, the BBI concentrate (BBIC) (reviewed in Ref. 22). BBI inhibits proteolytic activity in lung, kidney, and liver tissue following intraperitoneal injections in mice, with similar dose-dependent inhibition of proteolytic activity observed both in vitro and in vivo (5, 34). Mice provided a diet of 1.0% BBIC exhibited no growth abnormalities and had a significantly extended life span (25) and minimized the overall loss of body weight in an animal model of leukemia (24).
To determine whether inhibition of serine protease activity could attenuate disuse atrophy of skeletal muscle, mice were provided food supplemented with BBIC and subjected to a period of HU. The muscles were then analyzed to determine whether BBIC attenuated the muscle mass and strength losses associated with HU. The muscles were assayed to measure serine endopeptidase and the chymotrypsin-like proteasome activity under both weight-bearing and HU conditions and to determine whether these activities were inhibited by BBI. As it has been suggested that serine proteases are able to activate MMP activity, we also tested whether supplementation with BBIC was able to blunt any increased MMP activity.
The present results demonstrate that supplementation with BBIC significantly attenuates skeletal muscle atrophy during HU. An increase in serine protease activity was observed following HU, and the activity was reduced below basal levels in the presence of BBIC. Thus dietary intake of BBI represents a potential new therapy to limit the degree of skeletal muscle atrophy that arises from disuse.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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BBIC was purified as previously described (25). This purification procedure maintains the chymotrypsin inhibitor activity but reduces the level of trypsin inhibitory activity, as high levels of trypsin inhibitory activity have been shown to cause a potentially deleterious pancreatic feedback response in rats (22, 25). BBIC has been shown to have the same inhibitory profile as the purified protein BBI (22). BBIC food (1.0%) was prepared by Central Soya (Ft. Wayne, IN) and mixed with Rodent Diet AIN-93G (Bio-Serv, Frenchtown, NJ) to produce the food pellets. The activity of BBI is defined as chymotrypsin inhibitory units (CI units) (25), with the batch of BBIC used in this study containing 
100 CI units/g. Therefore, the food supplemented with 1.0% BBIC had a potency of 1 CI unit/g. Control food (Ctrl) was prepared similarly without the addition of BBIC. Before mixing, a quantity of BBIC was repeatedly autoclaved and then mixed with the powdered rodent diet to produce an inactive isocaloric Ctrl food (aBBIC). Repeated autoclaving has been shown to destroy the protease inhibitor activity of BBIC (21). All animals were provided food ad libitum.
Animals
The experiments in this study were approved by the University of Pennsylvania's Institutional Animal Care and Use Committee. Six-month-old male C57/Bl6 mice were used for this study. The animals were randomly separated into one of three feed groups: BBIC, aBBIC, or Ctrl. The mice were switched to the experimental diets, containing 1.0% BBIC, 1.0% aBBIC, or no additional supplementation (Ctrl) 5–7 days before the beginning of the experimental period for acclimation to the new food. One-half of the animals were hindlimb suspended in individual suspension cages, while the others were placed in individual cages to be used as nonsuspended controls. Thus the animals were randomly assigned to one of six groups: 1) control, nonsuspended (Ctrl-Non); 2) control, hindlimb suspended (Ctrl+HS); 3) BBIC, nonsuspended (BBIC-Non); 4) BBIC, hindlimb suspended (BBIC+HS); 5) aBBIC nonsuspended (aBBIC-Non); and 6) aBBIC, hindlimb suspended (aBBIC+HS).
Hindlimb Suspension
The animals were suspended by using a modified tail suspension technique originally described for rats (33) and adapted in-house for mice. The animals were anesthetized with an intraperitoneal injection of a mixture of ketamine and xylazine, and the body weight was measured. The tails were cleaned and attached to a stainless steel chain by using a strip of adhesive tape (Skin Trac; Zimmer, Warsaw, IN). The chain was attached loosely to a track at the top of the cage until the animal had recovered from the anesthetic. When the mice began to exhibit normal activity, the chain was lifted sufficiently to raise the hindlimbs off the floor of the cage. The suspension system enabled the mice to move freely around the cage while preventing the hindlimbs from touching the floor or walls.
Muscle Mechanical Measurements
Following the experimental period, the mice were anesthetized, body weight was measured, and the soleus and gastrocnemius (Gast) muscles were removed. One soleus muscle was prepared for mechanical muscle force measurements. The other soleus muscle was weighed and frozen immediately for subsequent biochemical analysis without any ex vivo stimulation. The Gast muscles were weighed and immediately frozen for biochemical analyses. Force measurements were performed as previously described (3). Briefly, the resting length was obtained by adjusting muscle length until achieving maximal twitch tension. Maximal tetanic force was measured by stimulating the soleus muscles with a 100-Hz, 500-ms pulse at supramaximal voltage. Following the tension measurements, the muscle was blotted, weighed, and then rapidly frozen in melting isopentane and stored at –80°C for subsequent histological analysis.
Immunohistochemical Analysis/Fiber Size Determination
Frozen muscle cross sections (10 µm) were cut from the midbelly on a cryostat and stored at –20°C. To determine fiber size, the frozen sections were stained with an antibody against laminin (NeoMarkers, Fremont, CA). The slides were washed in PBS and blocked in 5% BSA in PBS for 1 h at room temperature and then incubated overnight in 5% BSA/PBS containing the laminin antibody at 4°C. A rhodamine-conjugated anti-rabbit IgG (Jackson Immunoresearch Laboratories) was used as a secondary antibody to visualize staining. The slides were mounted with Vectashield (Vector Laboratories, Burlingame, CA) to slow photobleaching. Microscopy was performed on a Leitz DMR microscope (Leica, Bannockburn, IL), and image acquisition was performed by using a MicroMAX digital camera system (Princeton Instruments). The fiber sizes were calculated by using OpenLab imaging software (Improvision, Waltham, MA).
Proteolytic Assays
Assays to measure the proteolytic activity of the muscle extracts were performed to determine whether 1) HU increased proteolytic activity and whether 2) BBI could attenuate any increased proteolytic activity. To determine whether there were any changes in serine protease activity, we utilized the fluorogenic substrate butoxycarbonyl-Val-Pro-Arg-(4-methyl)-coumarylamine (Boc-VPR-MCA) that has been used previously to monitor serine endopeptidase activity in other tissues in mice and humans (31, 34, 45). The chymotrypsin-like proteasome activity was measured using N-succinyl-Leu-Leu-Val-Tyr-(4-methyl)-coumarylamine (N-Suc-LLVY-MCA). Another assay, gelatin zymography, was used to determine whether supplementation with the BBIC was able to reduce the activation of MMPs previously observed following hindlimb immobilization (35) that may suggest serine protease involvement in increased extracellular remodeling.
For the proteolytic assays, the frozen muscles were homogenized, and the proteins were extracted (1:5 wt/vol) by using a buffer consisting of 50 mM Tris·HCl, pH 7.5, 5 mM EDTA, and 5 mM DTT. The homogenates were centrifuged (30,000 g for 30 min at 4°C), and the supernatant was collected and stored at –80°C until use. The protein concentration was determined by using the Bradford assay (Bio-Rad) with BSA as a standard.
Proteasome and Serine Protease Activity
To determine whether HU increased either the chymotrypsin-like proteasome or serine protease activity, the rate of hydrolysis of the fluorescent peptides N-Suc-LLVY-MCA and Boc-VPR-MCA (Sigma, St. Louis, MO) was measured in Gast muscle extracts from unloaded and weight-bearing, nonsuspended animals. The chymotrypsin-like proteosome activity in the muscle extracts (0.5 mg/ml total protein) was determined by measuring the rate of N-Suc-LLVY-MCA cleavage (0.2 mM) in the presence of 0.05% SDS, as described (19, 44). Serine endopeptidase activity was determined via measurement of the rate of Boc-VPR-MCA hydrolysis (100 µM) in the muscle extracts (0.1 mg/ml) in a buffer containing 50 mM Tris·HCl (pH 7.0) and 1 mM DTT, as described previously (45), with minor modifications. The proteolytic activity was measured by using three muscle samples from both suspended and nonsuspended animals. The protease inhibitors MG-132 or purified BBI (Sigma) were added before addition of the substrate, at a final concentration of 40 and 20 µM, respectively. The reaction was initiated by the addition of the fluorescent peptides with the release of free MCA monitored at excitation and emission wavelengths of 380 and 460 nm, respectively, in a plate-reading fluorimeter for 90 min at 37°C.
Gelatin Zymography
To determine whether there was increased extracellular matrix remodeling, as measured by MMP-2/9 activity, gelatin zymography of the muscle extracts, from Ctrl- and BBIC-fed mice, was performed following previously described methods (35) with modifications. The muscle samples (50 µg total protein) were mixed with 2x nonreducing sample buffer and loaded onto an 8% SDS-polyacrylamide gel containing 1 mg/ml gelatin. After electrophoresis, the gels were washed twice for 20 min in 2.5% Triton X-100 in PBS and equilibrated at room temperature in the developing buffer [50 mM Tris·HCl (pH 7.5), 200 mM NaCl, 5 mM CaCl2] for 30 min and then incubated overnight at 37°C in fresh developing buffer. Following incubation, the gels were stained with 0.5% Coomassie brilliant blue twice for 30 min and then destained in 50% methanol/7% acetic acid. Quantification of the in-gel protease activity was performed by using a scanner and densitometry software (Kodak 1D, Eastman-Kodak, Rochester, NY).
Western Blotting
Analyses of protein levels were determined by using standard SDS-PAGE and Western blotting methods on the muscle extracts. Antibodies against desmin (Santa Cruz Biotech, Santa Cruz, CA) and talin (Sigma) were used to determine whether changes in proteolytic activity promoted cleavage of these proteins.
Statistical Analysis
Statistical significance was determined by applying the raw data to either an unpaired t-test or a one-way ANOVA, with Bonferroni post hoc analysis, where applicable. The data are shown as means ± SE, unless otherwise noted. P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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To determine whether consumption of BBIC was able to attenuate muscle loss during HU, we first compared the soleus muscle mass of Ctrl mice to a group of BBIC-treated mice following 3, 7, and 14 days of HU (Fig. 1A). Following 3 days of hindlimb suspension, no significant difference between the Ctrl+HS and BBIC+HS soleus mass was observed [8.0 ± 0.3 mg (n = 4) and 8.9 ± 0.4 mg (n = 4), respectively]. However, after 7-day HU, the soleus mass of the BBIC-treated mice was significantly greater than that of the Ctrl mice [8.6 ± 0.4 mg (n = 4) vs. 7.2 ± 0.3 mg (n = 4); P < 0.05], and, following 14-day HU, the muscle weights were BBIC+HS, 7.9 ± 0.3 mg (n = 4) vs. Ctrl+HS, 6.6 ± 0.3 mg (n = 4); P < 0.05.
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0.5 g/day less (2.7 ± 0.2 g/day) than the other three groups. The Gast and soleus muscle weights of the weight-bearing, nonsuspended animals were similar for all treatment groups (Table 1), suggesting that BBIC supplementation does not produce any observable hypertrophy in unperturbed muscles. However, dietary supplementation with BBIC slowed the loss of muscle mass, with significant reductions observed in both the Gast and soleus muscles (Fig. 1B; Table 1). The soleus muscle weights of the BBIC+HS mice (7.9 ± 0.2 mg, n = 9) were significantly greater than in both Ctrl+HS and aBBIC+HS mice (6.4 ± 0.4 mg, n = 7; and 7.2 ± 0.1 mg, n = 8, respectively), with no difference determined between the Crtl+HS and aBBIC+HS groups following the 14 days of hindlimb suspension. The soleus muscle weights of the Ctrl+HS and aBBIC+HS animals decreased by 39 ± 5 and 35 ± 3% (Fig. 1B), compared with the Ctrl-Non and aBBIC-Non, respectively. The percent atrophy in the BBIC+HS soleus muscles was limited to 26 ± 4%, significantly attenuating mass loss by 25–35%.
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As body weight changes and fasting can greatly affect muscle mass, the individual muscle weights relative to the body weight were determined (Table 1). A significant attenuation in muscle atrophy of the soleus, normalized to body weight, was determined when the BBIC-fed mice (11 ± 1% loss) were compared with both the Ctrl mice (22 ± 2%) and aBBIC-fed mice (20 ± 3%). In the Gast muscles, the normalized percent atrophy was reduced from 7.5 ± 1.0% in the Ctrl+HS and aBBIC+HS mice to no observable loss in the BBIC+HS animals.
If an active compound within the BBIC-supplemented food is an inhibitor of muscle atrophy during unloading, then a correlation between BBIC intake and muscle weight should be apparent. As described above, the daily food intake of the BBIC- and aBBIC-treated mice was monitored. A significant correlation between the muscle weights and daily food intake was determined for the BBIC+HS animals (r = 0.785; P = 0.036; n = 7) but not for the aBBIC+HS group (r = 0.131; P = 0.81; n = 6), suggesting the active BBIC is responsible for limiting the loss of muscle mass. Mice eating >3.0 g/day of BBIC food lost only 7.2 ± 0.6% of their muscle mass, compared with 19.4 ± 1.8% in the aBBIC-fed mice, when normalized to body weight.
Fiber Size in BBIC- and aBBIC-treated Mice
BBIC was found to significantly attenuate mass loss associated with HU (Fig. 1). To determine whether the maintained muscle mass was due to maintenance of muscle fiber size, the individual fiber areas were measured in BBIC- and aBBIC-treated mice. For the measurements, soleus muscle cross sections were stained with laminin and used to calculate the fiber cross-sectional area (CSA). Two nonoverlapping regions were visualized on individual muscle sections from three different mice, resulting in a total of >600 individual fiber areas being measured for both BBIC+HS and aBBIC+HS mice. As shown in Fig. 2, BBIC treatment significantly attenuated the decrease in fiber size following unloading. The mean fiber size of aBBIC+HS mice (578 ± 33 µm2) was reduced by 37% compared with the aBBIC-Non (920 ± 22 µm2). BBIC treatment limited the reduction in fiber area to 26% (weight bearing, 933 ± 51 µm2 vs. unloaded, 689 ± 36 µm2). The fiber size decreases shown compare well with the muscle mass data (Fig. 1; Table 1), suggesting BBIC is inhibiting the loss of muscle fibers rather than other nonmuscle components.
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It is possible that the maintenance of fiber size and mass could lead to reduced force output by inhibiting critical protein clearance pathways and accumulation of nonfunctional proteins. Thus we performed contractile measurements to determine whether BBIC treatment preserved muscle strength as well as mass and fiber area. The calculated CSA (9) of the Ctrl+HS and aBBIC+HS mice was reduced by 36 and 34%, respectively (Table 2). As shown for muscle mass, the addition of BBIC to the food attenuated the HU-induced reduction in CSA to 26%.
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Western Blots
Western blots of desmin and talin were performed to determine whether calpain-dependent protein cleavage was reduced in the BBIC+HS mice. Analysis of 3-, 7-, and 14-day HU, BBIC-treated mice exhibited no difference in either desmin or talin cleavage compared with weight-bearing Ctrl mice, suggesting BBIC does not influence calpain-mediated protein degradation (data not shown).
Inhibition of Proteolytic Activity by BBIC
Proteolytic activity. To determine whether BBI was capable of directly interfering with the chymotrypsin-like activity of the proteasome, the fluorescent peptide, N-Suc-LLVY-MCA, was mixed with Gast muscle extracts from either weight-bearing or HU mice. As shown in Fig. 3A, BBI does not directly inhibit the chymotrypsin-like activity of the proteasome in muscle extracts. Also, we did not observe an increase in the rate of chymotrypsin-like proteolytic activity following 14 days of tail suspension. The presence of BBI did not affect the proteolytic activity, either in weight-bearing or HU muscles (Fig. 3B), while the proteasome inhibitor MG-132, as expected, significantly reduced proteosome activity in both the suspended and nonsuspended muscle homogenates by >80%.
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Zymography. To determine whether BBIC supplementation could influence extracellular matrix remodeling, MMP-2/9 activity was measured in Gast muscle extracts of nonsuspended and hindlimb-suspended mice given either Ctrl or BBIC-supplemented food. The muscle extracts from these mice were homogenized and run on nondenaturing gels containing 1.0% gelatin. Figure 4 shows the quantification of gelatinase activity. The proteolytic activity was measured, and the band intensities from the BBIC-Non, BBIC+HS, and Ctrl+HS homogenates were compared against Ctrl-Non mice. Comparison of the groups, relative to Ctrl-Non, revealed increased gelatinase activity in the Ctrl+HS animals only. The BBIC+HS muscles did not exhibit elevated levels of proteolytic activity compared with the Ctrl-Non animals. Gels developed in an EDTA-containing buffer displayed no bands, suggesting that the observed gelatinase activity was due to MMPs (data not shown).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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An alternative strategy to slow disuse atrophy is to inhibit the elevated protein degradation pathways, rather than boost the diminished growth signaling. This approach is more immediately tractable from a therapeutic standpoint, as a number of pharmacological agents exist that target various classes of proteases. In this study, we specifically examined whether dietary supplementation with a well-characterized serine protease inhibitor was sufficient to modulate the degree of atrophy associated with muscle unloading. Our results indicate that dietary supplementation with BBIC significantly attenuated the degree of muscle atrophy following HU in mice. The maintenance of muscle mass was functional, with the specific force in BBIC-treated animals similar to those treated with inactive aBBIC or standard mouse feed. Also, we have identified a novel proteolytic activity that is elevated following a period of hindlimb muscle unloading and inhibited by a well-characterized serine protease inhibitor. The results suggest that BBIC attenuates the observed muscle atrophy by reducing the activity of unloading-induced serine protease activity, slowing the decrease in fiber size and thereby maintaining the overall mass of the muscle.
It is important to note that the amelioration of atrophy in the BBIC+HS animals was not due to any initial hypertrophy of the muscles, as the BBIC animals exposed to normal loading showed no increase in muscle mass compared with either Ctrl-Non or aBBIC-Non animals. This enabled direct comparison of the BBIC+HS muscle weights, both absolute and relative to body weight, with those of the aBBIC+HS and Ctrl+HS. Following 3 days of HU, there was no difference between the Ctrl-fed and BBIC-fed muscle weights. However, after 7 and 14 days, the muscle weights were significantly greater in the BBIC-treated HU animals. The maintenance of muscle mass was paralleled by maintenance of force production. The specific force, measured as force per CSA and force per milligram muscle wet weight, was similar for all HU groups.
Investigation into the role of protein degradation during disuse atrophy has focused on three primary proteolytic pathways: the Ca2+-dependent proteolysis (calpains), ATP-dependent proteolysis (ubiquitin-proteasome degradation), and lysosomal proteolysis (cathepsin B+L). The ubiquitin-proteasome pathway appears responsible for the majority of muscle protein degradation (19, 36, 41, 42). However, intact myofibrillar proteins are not substrates of the proteasome (19, 38), so the initial proteolysis of myofibrillar proteins requires other proteases. Because these pathways can be independently targeted by specific inhibitors, potentially providing additive effects, we asked whether the activity of BBI affects these or other degradation pathways.
Following HU, components of the ATP-dependent ubiquitin-proteosome system have been shown to be upregulated (19, 39, 41). The level of chymotrypsin-like proteosome activity was previously shown to be minimally elevated after 14 days of hindlimb suspension with significant increases only observed following 21 days of unloading (19). We measured the proteosome-dependent cleavage of the fluorogenic substrate N-Suc-LLVY-MCA and did not observe an increase in the chymotrypsin-like proteasome activity following 14 days of suspension. However, as expected, the proteosome inhibitor MG-132 significantly reduced the peptidase activity. The proteosome-dependent activity was not inhibited by purified BBI in either nonsuspended or suspended muscle extracts. BBI does not directly inhibit proteosome activity, but it is not clear whether or not BBI inhibition influences flux through the ubiquitin-proteosome pathway by inhibiting upstream protein degradation pathways, thus decreasing proteosome substrates. Also, neither talin nor desmin degradation was influenced by the presence of BBIC, suggesting little to no effect on the calcium-dependent proteolytic pathway by the serine protease inhibitor. It would thus be of interest to examine calpain inhibitors in combination with BBI, since a calpain inhibitor (leupeptin) has been reported to attenuate muscle loss associated with denervation (2).
Although the three well-characterized degradative pathways described above clearly are major sources of observed muscle atrophy, our data suggest that there are other serine proteases that make important contributions to the atrophy process. These may include previously described serine protease cascades (39), as others have identified myofibrillar serine proteases capable of directly degrading myosin and actin (16, 37). In the present study, we have identified serine endopeptidase activity that is significantly elevated in HU muscle extracts (Fig. 3).
While we have not elucidated the specific serine proteases responsible for the component of the atrophy response that we have attenuated, Stevenson et al. (39) identified differential expression of several serine proteases and serine protease inhibitors following HU. A potential target of BBI could be the serine protease, mast cell chymase, which is usually released with tryptase and tumor necrosis factor-
during activation and degranulation of mast cells. BBI/BBIC is a potent inhibitor of chymase (46), which has been shown to degrade soluble muscle proteins (12), to directly cleave components of the extracellular matrix (e.g., fibronectin) and to activate MMPs (28). Also, a chymase-like serine protease has been found within cardiac muscle cells (15) and associated with increased MMP-9 activity during cardiac remodeling (10). Increased muscle MMP-2/9 activity following periods of immobilization and during muscle regeneration was observed previously (26, 35). In agreement, our results indicated an increase in MMP activity following 14 days of hindlimb suspension in the Ctrl+HS muscle extracts, which was reduced with BBIC treatment (Fig. 4). In vitro studies have found that BBI is capable of inhibiting activation of pro-MMP-9 (4), suggesting BBI/BBIC could reduce protease activity in the extracellular space. Our results suggest that some component of this serine protease activity must be an initiating event in the stimulation of MMP activity to bring about extracellular matrix changes (Fig. 4).
It is currently unclear if these serine protease activities that are elevated in disuse atrophy function upstream of, or in parallel with, the three primary degradative pathways. What is clear is that BBI significantly inhibited the elevated serine endopeptidase activity, leading to an amelioration of muscle loss and preservation of function during a period of disuse. Importantly, while there was a significant reduction in the serine protease activity of BBIC-treated, nonsuspended mice muscle extracts, there is no evidence that the inhibitory activity affects normal function. As BBIC is nontoxic, orally bioavailable, and is currently being evaluated in human trials, it is likely that human trials on the effects of BBIC in disuse atrophy of skeletal muscle could be performed in the near future. If functional muscle loss can be prevented via simple dietary supplementation, this could lead to more rapid recovery from prolonged bed rest or limb immobilization (casting).
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Current address of C. A. Morris: Department of Medicine, Boston University School of Medicine, 88 East Newton St. E201, Boston, MA 02118.
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DISCLOSURES |
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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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, Change. **Significantly different (P < 0.05) from Ctrl+HS and autoclaved BBIC (aBBIC) + HS muscle weights.
