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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

The role of the calpain proteases in skeletal muscle atrophy is poorly understood. One goal of these experiments was to clarify whether calpains act upstream of the ubiquitin–proteasome pathway (UPP). Calpain activation may also inhibit the anabolic signalling of Akt, since a molecular chaperone previously shown to mediate Akt activity, heat shock protein 90 (HSP 90), is a calpain substrate. Thus, an additional objective was to determine whether calpain activation affects the Akt signalling pathway. Ex vivo experiments were conducted using isolated rat diaphragm muscle. Calpain activation increased total protein degradation by 65%. Proteasome inhibition prevented this large rise in proteolysis, demonstrating that the proteasome was necessary for calpain-activated protein degradation. In addition, calpain activation increased proteasome-dependent proteolysis by 144%, further supporting the idea of sequential proteolytic pathways. Calpain reduced Akt and mammalian target of rapamycin (mTOR) phosphorylation by 35 and 50%, respectively, and activated glycogen synthase kinase-3 beta (GSK-3β) by 40%. Additionally, calpain activation reduced HSP 90β and mTOR protein content by 33 and 50%, respectively. These data suggest that calpains play a dual role in protein metabolism by concomitantly activating proteasome-dependent proteolysis and inhibiting the Akt pathway of protein synthesis.

Skeletal muscle atrophy is a clinically important problem because muscle mass is directly related to muscle function (Ibebunjo & Martyn, 1999). The maintenance of muscle mass requires a balance between the rates of protein degradation and protein synthesis, and catabolic conditions have been consistently linked with increased muscle proteolysis and reduced protein synthesis (Tisdale, 2005). Therefore, delineating the mechanisms of altered protein metabolism during catabolic conditions is critical to developing interventions to prevent muscle atrophy.

The ubiquitin–proteasome pathway (UPP) is the primary proteolytic system in skeletal muscle. This system accounts for the majority of proteolysis under normal physiological conditions as well as elevated proteolysis in a variety of catabolic states (see Lecker et al. 1999 for review). Catabolic conditions often activate multiple proteolytic pathways, however, and it now appears that other proteases initiate the degradation of muscle proteins and thereby activate the UPP (Du et al. 2004). Several catabolic states concomitantly activate the UPP and the calcium-activated proteases, or calpains. While recent research suggests that the calpains may also act ‘upstream’ of the UPP, data in the literature are equivocal.

The ubiquitous calpains are termed micro- and milli-calpain, nomenclature reflecting the calcium concentration required for their half-maximal proteolytic activation in vitro. Calpain substrates include proteins that are important to sarcomeric structural integrity. For instance, nebulin and titin, two proteins that connect myofilaments to the Z-disk, are excellent calpain substrates (Huang & Forsberg, 1998; Lim et al. 2004). Thus, calpains play a role in the degradation of myofibrillar proteins, which are critical to proper muscle function. Less is known, however, of the fate of calpain substrates following calpain digestion. Calpains cleave their protein substrates, rather than completely degrading them, thereby generating polypeptide fragments (see Goll et al. 2003 for calpain review). This limited proteolytic action of the calpains has led to speculation that the peptide fragments resulting from calpain cleavage become substrates for the UPP (Huang & Forsberg, 1998; Goll et al. 2003). In fact, some data support this hypothesis. For instance, calpain-3, the muscle-specific calpain, contributes to protein ubiquitination (Kramerova et al. 2005) and calpain activation in myotubes increases proteasome enzyme activity (Menconi et al. 2004). In contrast, Fareed et al. (2006) recently reported that calpain inhibition following a 16 h sepsis infection in rats failed to reduce proteasome-dependent protein degradation, which is indicative of independent proteolytic pathways. Elucidation of the interactions between these proteolytic pathways may have important implications for the development of interventions to prevent muscle atrophy. Thus, the primary goal of these experiments was to clarify whether calpain proteases act upstream of the UPP. To address this issue, we used an ex vivo experimental model of calpain activation and two different approaches to study the interactions between the pathways in isolated diaphragm muscle. We first activated calpain-dependent protein degradation while concomitantly inhibiting the proteasome to determine whether the proteasome was necessary for calpain-dependent proteolysis. Second, using the same experimental model, we assessed whether calpain activation was sufficient to activate the UPP.

In addition to playing a role in proteolysis, calpains may reduce protein synthesis signalling via Akt inhibition. The insulin-like growth factor-1 (IGF-1) pathway intermediate Akt activates downstream targets known to mediate protein synthesis (Hajduch et al. 1998; Kimball et al. 1999), and previous investigations have convincingly demonstrated a key role for Akt signalling in regulating skeletal muscle size. For example, the Akt signalling pathway is activated during skeletal muscle overload, and muscle disuse reduces Akt activation below control levels (Bodine et al. 2001b). Further, atrophy is attenuated in denervated skeletal muscle concomitantly treated with an active form of Akt (Bodine et al. 2001b). The molecular chaperone heat shock protein 90 (HSP 90) and Akt form a chaperone–client protein complex in non-skeletal muscle cells (Sato et al. 2000). This association appears to be essential to proper Akt function, since reduced HSP 90–Akt binding causes Akt inactivation (Sato et al. 2000). Importantly, HSP 90 is a calpain substrate, and calpain activation reduces HSP 90–client protein binding in non-skeletal muscle cells (Stalker et al. 2003). This suggests that calpain activation may diminish HSP 90–Akt binding in skeletal muscle, and consequently inactivate a signalling protein known to play a critical role in protein synthesis and the maintenance of muscle mass. Thus, additional goals of the present experiments were to determine the effect of calpain activation on the HSP 90–Akt complex and the Akt signalling pathway in skeletal muscle.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Animals

Forty Female Sprague–Dawley rats were acclimated for 1 week in a temperature- and humidity-controlled room maintained on a 12 h–12 h light–dark cycle. Animals weighed 230–250 g and had free access to a standard rat chow and water and were handled daily to reduce contact stress. The Institutional Animal Care and Use Committee approved the experimental procedures.

Experimental model and experimental design

An ex vivo experimental model was used in the present experiments because this model was previously shown to cause a calpain-dependent increase in total protein degradation (Zeman et al. 1985; Furuno & Goldberg, 1986). Diaphragm muscle was used because the thinness of the muscle facilitates oxygen, nutrient and ion delivery. The diaphragm is a highly oxidative skeletal muscle, even when compared with slow/oxidative locomotor skeletal muscle (Halseth et al. 1995). Nonetheless, alterations in protein metabolism are similar in the diaphragm and locomotor skeletal muscle during certain catabolic states. For instance, muscle unloading activates calpain proteases (Tidball & Spencer, 2002), increases proteolysis (Thomason et al. 1989) and reduces protein synthesis (Loughna et al. 1986) in locomotor muscle as well as the diaphragm (Shanely et al. 2002, 2004). Additionally, determination of the consequence of calpain activation in diaphragm is of practical significance because diaphragm injury (Reid & Belcastro, 2000) as well as diaphragm unloading (Shanely et al. 2002) activate the calpain proteases.

Animals were anesthetized via intraperitoneal injection of sodium pentobarbital (60 mg/kg bodyweight), and the entire diaphragm muscle was removed and submerged in ice-cold Krebs-Ringer physiological solution (Sigma: K4002, pH 7.4, supplemented with: 25 mm sodium bicarbonate, 0.1 mm isoleucine, 0.17 mm leucine, 0.2 mm valine, 1 mU/mL insulin, and 0.5 mm cycloheximide) saturated with 95% O2-5% CO2 gas. Cessation of heartbeat following diaphragm removal confirmed animal euthanasia. Strips of costal diaphragm muscles were fixed to a Plexiglass support, and randomly assigned to one of four groups: (1) Krebs–Ringer physiological solution described above (CON); (2) Krebs solution containing 3.5 mm calcium (Ca2+); (3) Krebs solution with 3.5 mm Ca2+ and the calpain inhibitor calpeptin (1 μm) (Ca2+/CI); or (4) Krebs solution with 3.5 mm Ca2+ and the proteasome inhibitor epoxomicin (30 μm) (Ca2+/PI). While calpeptin also inhibits the lysosomal proteases, previous investigations indicated that this experimental model of calcium overload does not activate the lysosomal proteases (Zeman et al. 1985; Baracos et al. 1986; Furuno & Goldberg, 1986). Incubation solutions were saturated with 95% O2–5% CO2 and maintained at 37°C in a shaking water bath throughout the experimental protocol. The muscles were allowed to shorten since this has been shown to maximally activate Ca2+ mediated protein degradation.

Muscles were pre-incubated for 30 min in Ca2+-free Krebs solution containing the appropriate protease inhibitor dissolved in DMSO or an equivalent volume of DMSO. A 1 h treatment period was initiated by replacing the pre-incubation medium with either fresh Ca2+-free medium (CON group), fresh medium containing Ca2+ (Ca2+ group), or fresh medium containing Ca2+ and the appropriate inhibitor (Ca2+/CI and Ca2+/PI groups). An equivalent volume of DMSO was included in the CON and Ca2+ groups during incubation. A 1 h incubation time was selected because preliminary studies suggested that protein degradation and proteasome activity were elevated in the calcium-treated group at this time point. Following incubation, muscles were washed in control medium to remove excess inhibitor, blotted dry, weighed, frozen in liquid nitrogen, and stored at –80°C. The incubation medium was then stored at –20°C for subsequent analysis. Of note, the purpose of the Ca2+/PI group was to determine the role of the proteasome in calpain-mediated protein degradation. Since the Ca2+/PI group provides no information concerning calpain activation and Akt signalling, the Ca2+/PI group was excluded from the Akt experiments.

Muscle preparation: cytosolic and myofibrillar fractions

Proteasome enzyme activity, cleaved talin and cytosolic ubiquitinated protein content were assessed in the cytosolic fraction, while the myofibrillar fraction was collected to determine the amount of myofibrillar ubiquitinated proteins. The cytosolic fractions were isolated as described by Vigouroux et al. (2003). Approximately 15 mg of frozen muscle was homogenized in ice-cold buffer (50 mm Tris-HCl, 1 mm EDTA, 1 mm EGTA, 2.5 μmtrans-Epoxysuccinyl-L-leucylamido(4-guanidino) butane (E64), 2.5 μm pepstatin A and 10% glycerol, pH was adjusted to 8 with Tris-base) and centrifuged at 100 000g for 1 h at 4°C. The supernatant (cytosolic fraction) was collected and stored at –80°C. The myofibrillar fraction was isolated as previously described (Yimlamai et al. 2005), with slight modification. Approximately 15 mg of frozen muscle was homogenized as described above, and centrifuged at 1500g for 10 min at 4°C to separate the myofibrillar (pellet) fraction. The myofibrillar fraction was washed three times with homogenizing buffer containing 1% Triton X-100 and stored at –80°C.

Protein concentration

Protein concentration was determined using bicinchoninic acid (BCA) or Bradford method, depending on the contents of the homogenization buffer. Samples were run in triplicate, and protein concentration was determined using bovine serum albumin as a standard.

General SDS-PAGE and Western blotting protocol

Equal amounts of protein (∼20 μg) were loaded onto a 4–20% gradient gel, separated, and then transferred to a nitrocellulose membrane (0.45 μm, Millipore, Bedford, MA, USA). The membranes were then blocked for 1 h at room temperature with TBS-T blocking buffer (50 mm Tris base, pH 7.4, 150 mm NaCl and 1% Tween 20) and 5% non-fat dry milk. Next, the blots were incubated overnight at 4°C with the primary antibody. The blots were then washed with TBS-T wash buffer and subsequently incubated for 1 h with the secondary antibody. After another wash, blots were exposed to an enhanced chemiluminescence system (Amersham Pharmica Biotech, UK) for 1 min and to X-ray film for approximately 10 s. This general procedure was followed for all SDS-PAGE–Western blot experiments. The density of the bands was analysed using Kodak ID Image Analysis Software (Eastman Kodak Scientific Imaging Systems, Rochester, NY, USA). Membranes were stained with Ponceau S staining solution to confirm equivalent protein loading and transfer.

Antibodies

Primary antibodies were: talin, 1:1000 (Sigma-Aldrich, St Louis, MO, USA); ubiquitin, 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and Cell Signalling Technologies (Beverly, MA, USA) for Akt (phospho-Akt Ser473, Akt, 1:1000), mammalian target of rapamycin (phospho-mTOR Ser2448, mTOR, 1:250) and glycogen synthase kinase-3 beta (GSK-3β) (phospho-GSK-3β Ser9, GSK-3β, 1:1000). Antibodies to HSP 90α and HSP 90β (1:500) were purchased from Affinity Bioreagents (Golden, CO, USA). Secondary antibodies were antimouse or antirabbit IgG horseradish peroxidase conjugate, 1:1000 (Amersham, UK).

Confirmation of calpain activation

Since calpain cleaves talin to a 190 kDa fragment (Hayashi et al. 1999), the appearance of the 190 kDa talin fragment was used as a measure of calpain proteolytic activation.

Ubiquitin-conjugated protein content

The content of ubiquitin-conjugated proteins was determined in the cytosolic and myofibrillar fractions via SDS-PAGE–Western blot. Ubiquitin-conjugated proteins were quantified by measuring the integrated density of the entire lane > 37 kDa. Lane density was assessed three times and the average intensity was reported.

Proteasome enzyme activity

N-Succinyl-Leu-Leu-Val-Tyr 7-amido-4-methylcoumarin (Suc-LLVY-AMC), N-tert-Boc-Leu-Arg-Arg 7-amido-4-methylcoumarin (Boc-LRR-AMC) and Cbz-Leu-Leu-Leu-al (MG132) were purchased from Sigma. Cbz-Leu-Leu-Glu 7-amido-4-methylcoumarin (Z-LLE-AMC) was purchased from CalBiochem (San Diego, CA, USA) and lactacystin was purchased from Boston Biochem (Cambridge, MA, USA). Suc-LLVY-AMC, Boc-LRR-AMC and Z-LLE-AMC were used to measure chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolysing (PGPH) proteasome activity, respectively. Proteasome enzymatic activity was determined according to Vigouroux et al. (2003), with slight modifications. Protein concentration was determined and sample protein concentration standardized. Five micrograms of cytosolic fraction was incubated with the reaction buffer (50 mm Tris-HCl and 1 mm dithiothreitol (DTT), pH was adjusted with Tris-base) and 40 μm of substrate with 100 μm proteasome inhibitor or an equivalent volume of DMSO (< 5% of the total volume), for a total assay volume of 50 μl. After 30 min incubation at 37°C, the reaction was stopped with ice-cold methanol. Enzyme activity was calculated as the difference in free AMC in the absence or presence of the proteasome inhibitor lactacystin or MG132 (PGPH). The released free AMC was measured fluorometrically and quantified by a free AMC standard curve, and expressed as nanomoles per milligram per minute.

Total protein degradation

Skeletal muscle can neither synthesize nor degrade the amino acid tyrosine. Therefore, the release of tyrosine into the muscle incubation medium, in the presence of the protein synthesis inhibitor cycloheximide (0.5 mm), represents the degradation of muscle proteins. Protein degradation was measured as previously described (Yimlamai et al. 2005). Briefly, 0.5 ml of the incubation medium was added to 2 ml dH2O and 0.5 ml of 30% trichloroacetic acid (TCA). The mixture was then incubated (10 minutes) and centrifuged (10 minutes, 4°C, 400 g), and 1 ml of the supernatant was transferred to a tube containing 0.5 ml of 1% nitrosonapthol (w/v) and 0.5 ml of nitric acid reagent (20% nitric acid, 2.5% of NaN2, w/v). The sample was then incubated and cooled; then 5 ml of ethylene dichloride was added and the solution was vortexed and then centrifuged as described above. The supernatant was collected and the amount of tyrosine was determined fluorometrically, quantified by a free AMC standard curve, and expressed as nanomoles per milligram wet weight per hour.

Akt–mTOR–GSK-3β signalling

The Akt signalling pathway was assessed using SDS-PAGE and Western blot analysis as described by Sakamoto et al. (2004), with slight modification. Approximately 15 mg of muscle was homogenized in ice-cold lysis buffer (Cell Signalling Technology, Beverly, MA, USA, catalogue no. 9803) containing 20 mm Tris pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mmβ-glycerophosphate, 1 mm Na3VO4, 10 μg ml−1 leupeptin and 1 mm phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 14 000g for 10 min at 4°C. The supernatant was collected and analysed for Akt, mTOR and GSK-3β phosphorylation and content using the general procedure outlined above.

HSP 90 content

Calpain activation reduces HSP 90 content in endothelial cells (Su & Block, 2000), and a reduction in protein content could limit the amount of HSP 90 available to chaperone Akt. Therefore, we also determined the effect of calpain activation on HSP 90α and HSP 90β content via SDS-PAGE–Western blot.

HSP 90–Akt binding

To determine the amount of HSP 90 associated with Akt, Akt was immunoprecipitated using the Akt antibody described in the Antibodies subsection. Immunoprecipitation was conducted according to manufacturer's instructions, with slight modification. Fifteen milligrams of muscle was homogenized (1:20, w/v) in lysis buffer (Cell Signalling Technology, catalogue no. 9803). The supernatant (cell lysate) was collected, and protein concentration was determined using the Pierce BCA protein assay (Pierce, Rockford, IL, USA) and standardized. The primary antibody was diluted 10-fold, and 20 μl of the antibody was added to 700 μg of the cell lysate and incubated. Next, 20 μl of Protein A Agrose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added and gently rocked overnight. The lysates were then centrifuged (1000 g, 5 min, 4°C). The supernatant was discarded and the pelleted immunoprecipitates were washed by adding 500 μl of lysis buffer and gently rotating the lysates 10 times. Next the lysates were centrifuged as described above, the pelleted immunoprecipitates were collected and washed two additional times using the same procedure. The lysates then underwent a series of centrifugations and washings and, ultimately, 15 μl of the supernatant was loaded onto a 4–20% gel, resolved by SDS-PAGE and transferred to a nitrocellulose membrane for immunoblot analysis. While the two HSP 90 isoforms, HSP 90α and HSP 90β, are 86% homologous, the isoforms may have different functions (Moore et al. 1989). Since no data are available regarding HSP 90–Akt binding in skeletal muscle, we investigated the effect of calpain activation on the binding of both HSP 90 isoforms to Akt.

Statistics

Data were analysed using a one-way analysis of variance (ANOVA). Where indicated, Tukey's multiple comparison test was used to assess differences among the means. The level of significance was set at P < 0.05. Values reported are means ±s.e.m.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Effect of muscle incubation on calpain activation

Since calpain cleaves intact talin to a 190 kDa fragment, calpain activation was assessed by measuring the amount of 190 kDa talin fragment. Incubation of muscle with calcium alone (Ca2+ group) increased the level of the 190 kDa talin fragment by almost 135% (Fig. 1) compared with control values. Talin cleavage was calpain dependent, since addition of the calpain inhibitor calpeptin to the Ca2+ treatment (Ca2+/CI group) prevented the appearance of fragmented talin. There was also a significant increase in cleaved talin in the Ca2+/proteasome inhibitor (Ca2+/PI group) group compared with CON and Ca2+/CI groups (+130 and +150%, respectively). These data indicate that talin cleavage was calpain mediated and that calpain activation was unaffected by the proteasome inhibitor.

image

Figure 1. Effect of diaphragm muscle incubation on proteolytic cleavage of talin

A, densitometric analysis of cleaved talin in control (CON; n= 12), calcium (Ca2+; n= 10), calcium + calpain inhibitor (Ca2+/CI; n= 9), and calcium + proteasome inhibitor (Ca2+/PI; n= 9)-treated muscles. B, representative Western blot of 190 kDa talin fragment. *P < 0.05 significantly different from CON; #P < 0.05 significantly different from Ca2+/CI.

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Effect of calpain activation on total protein degradation

As shown in Fig. 2A, total protein degradation was significantly elevated in the Ca2+ group compared with the CON group (+65%). Calpain inhibition prevented the increase in proteolysis, indicating that calpain activation was sufficient and necessary for elevated proteolysis in the present model. Therefore, in the present model, if calpains are activated, then protein degradation should be elevated. However, despite significant calpain activation (Fig. 1), calpain-mediated protein degradation was blocked by proteasome inhibition (Fig. 2A). Thus, the proteasome was necessary for calpain-mediated protein degradation.

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Figure 2. Effect of calpain and proteasome inhibition on calcium-stimulated muscle proteolysis

A, effect of calpain activation on total protein degradation. CON (n= 17), Ca2+ (n= 17), Ca2+/CI (n= 11) and Ca2+/PI (n= 12). *P < 0.01 significantly different from all other groups. B, effect of calpain activation on proteasome-mediated protein degradation. CON (n= 17), CON/PI (n= 9), Ca2+ (n= 17) and Ca2+/PI (n= 12). *P < 0.01 significantly different from both control groups; #P < 0.05 significantly different from calcium-incubated group.

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In a follow-up study, we incubated muscles in control solution containing the proteasome inhibitor to compare control proteasome-dependent protein degradation with calpain-activated, proteasome-dependent proteolysis. By calculating the difference in proteolysis between muscles incubated with or without proteasome inhibitor, it was determined that calpain activation increased proteasome-dependent proteolysis by 144% (0.050 versus 0.122 μmol (mg wet weight)−1 h−1; Fig. 2B). Therefore, calpain activation was sufficient for elevated proteasome-mediated protein degradation. Together, these data support the hypothesis that the calpain proteases act upstream of the proteasome.

Effect of calpain activation on protein–ubiquitin conjugates and proteasome enzyme activity

If calpain cleavage products provide substrates for the UPP, calpain activation may cause an increase in ubiquitin-conjugated proteins. As expected, Ca2+ treatment in combination with the proteasome inhibitor (Ca2+/PI group) significantly increased the content of cytosolic ubiquitin-conjugated proteins (+170%) compared with all other groups (Fig. 3A). However, the level of cytosolic ubiquitin conjugates was unaffected by Ca2+ or Ca2+/CI treatment (Fig. 3A). Further, there were no differences in myofibrillar protein–ubiquitin conjugates between the groups (Fig. 4A). Similarly, while the proteasome inhibitor epoxomicin significantly reduced CT-L, T-L and PGPH enzyme activity (–99, –85 and –95%, respectively), proteasome activity did not differ between CON, Ca2+, and Ca2+/CI groups at the end of the 1 h muscle incubation (Table 1).

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Figure 3. Effect of calpain activation on cytosolic ubiquitin-conjugated protein content

Densitometric analysis (A) and representative Western blot of cytosolic ubiquitin conjugates (B). CON (n= 8), Ca2+ (n= 6), Ca2+/CI (n= 7) and Ca2+/PI (n= 7). *P < 0.001 significantly different from all other groups.

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image

Figure 4. Effect of calpain activation on myofibrillar ubiquitin-conjugated protein content

Densitometric analysis (A) and representative Western blot of myofibrillar ubiquitin conjugates (B). CON (n= 7), Ca2+ (n= 8), Ca2+/CI (n= 8) and Ca2+/PI (n= 7).

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Table 1.  Effect of calpain activation on proteasome enzyme activity
GroupCT-LT-LPGPH
  1. Chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolysing (PGPH) enzyme activity. Values presented are means ±s.e.m. Activities are expressed in nmoles per milligram per minute. * Significantly different from all other groups (P < 0.001). For CT-L, n= 12, 14, 8 and 7, respectively; for T-L and PGPH, n= 10, 12, 8 and 6, respectively.

CON0.1653 ± 0.0080.02007 ± 0.0020.07227 ± 0.007
Ca2+0.1702 ± 0.0130.01938 ± 0.0020.06922 ± 0.006
Ca2+/CI0.1680 ± 0.0130.01926 ± 0.0030.07086 ± 0.004
Ca2+/PI  0.00025 ± 0.00005*  0.0034 ± 0.002*  0.0040 ± 0.003*

Effect of calpain activation on the Akt–mTOR–GSK-3β pathway

Since calpain cleaves a key Akt regulator protein, HSP 90, we hypothesized that calpain activation would adversely affect Akt and its downstream targets mTOR and GSK-3β. As shown in Fig. 5, treatment of muscle with Ca2+ did not alter Akt protein content (Fig. 5B), but significantly reduced the amount of phosphorylated Akt by 35% (Fig. 5A). This reduction in Akt phosphorylation was prevented in muscles concomitantly treated with Ca2+ and the calpain inhibitor, indicating that the Ca2+-induced Akt inhibition was calpain mediated.

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Figure 5. Effect of calpain activation on Akt

Densitometric analysis of Akt phosphorylation (A), with CON (n= 7), Ca2+ (n= 7) and Ca2+/CI (n= 8); and Akt content (B), with CON (n= 8), Ca2+ (n= 8) and Ca2+/CI (n= 7). *P < 0.05 significantly different from CON; #P < 0.05 significantly different from Ca2+/CI. C, representative Western blot of Akt phosphorylation and Akt content.

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The phosphorylation of Akt's downstream target, mammalian target of rapamycin (mTOR), was reduced by approximately 50% following Ca2+ incubation, and calpain inhibition prevented the Ca2+-mediated reduction in mTOR phosphorylation (Fig. 6A). Similar results were obtained for mTOR protein content, in that Ca2+ treatment reduced the level of mTOR by about 50% and calpain inhibition prevented this reduction (Fig. 6B).

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Figure 6. Effect of calpain activation on mTOR

Densitometric analysis of mTOR phosphorylation (A), with CON (n= 7), Ca2+ (n= 7) and Ca2+/CI (n= 8); and mTOR content (B), with CON (n= 7), Ca2+ (n= 8) and Ca2+/CI (n= 7). *P < 0.05, **P < 0.01 significantly different from CON; #P < 0.05 significantly different from Ca2+/CI. C, representative Western blot of mTOR phosphorylation and mTOR content.

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Akt promotes protein synthesis by phosphorylating (inactivating) GSK-3β, a negative regulator of protein synthesis. We found that GSK-3β phosphorylation was reduced (activated) by about 40% with Ca2+ incubation (Fig. 7A), and that calpain inhibition blocked GSK-3β activation. The amount of GSK-3β protein did not differ between groups (Fig. 7B). Given the prominent role of the Akt–mTOR–GSK-3β signalling pathway in protein synthesis, these data suggest that calpain activation may be detrimental to skeletal muscle protein synthesis.

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Figure 7. Effect of calpain activation on GSK-3β

Densitometric analysis of GSK-3β phosphorylation (A), with CON (n= 7), Ca2+ (n= 6) and Ca2+/CI (n= 8); and GSK-3β content (B), with CON (n= 9), Ca2+ (n= 14) and Ca2+/CI (n= 14). C, representative Western blot of GSK-3β phosphorylation and GSK-3β content. *P < 0.01 significantly different from CON; #P < 0.05 significantly different from Ca2+/CI.

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Effect of calpain activation on HSP 90 content

Since a reduction in HSP 90 content could affect the amount of HSP 90 available to chaperone Akt, we determined whether calpain activation affected the level of HSP 90. While calpain activation had no effect on the amount of HSP 90α (Fig. 8A), Ca2+ incubation significantly diminished HSP 90β content (by –33%; Fig. 8B), and the Ca2+-mediated reduction in HSP 90β was prevented by calpain inhibition.

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Figure 8. Effect of calpain activation on HSP 90 content

Densitometric analysis of HSP 90α content (A), with CON (n= 7), Ca2+ (n= 7) and Ca2+/CI (n= 8); and HSP 90β content (B); with CON (n= 6), Ca2+ (n= 6) and Ca2+/CI (n= 8). *P < 0.05 significantly different from CON; #P < 0.05 significantly different from Ca2+/CI. C, representative Western blot of HSP 90α and HSP 90β content.

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Effect of calpain activation on Akt–HSP 90 binding

To determine whether calpain activation affects the protein–protein interactions between Akt and HSP 90, Akt was immunoprecipitated and the amount of HSP 90 bound to Akt was determined. Experiments carried out to determine the levels of HSP 90α and HSP 90β bound to Akt were run concurrently, using the same cell lysates. The HSP 90β was found to be associated with Akt, whereas HSP 90α was not (data not shown). Calpain activation had no effect on the amount of HSP 90β associated with Akt (Fig. 9), suggesting that calpain-mediated Akt pathway inhibition was independent of Akt–HSP 90 binding.

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Figure 9. Effect of calpain activation on Akt–HSP 90β binding

A, representative Western blot of immunoprecipitated Akt and HSP 90β. B, densitometric analysis of HSP-90β co-precipitated with Akt. n= 10 per group.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

The broad goal of these experiments was to investigate the effect of calpain activation on pathways of protein degradation and protein synthesis in skeletal muscle. Data from these studies in diaphragm muscle indicate that: (1) the proteasome was necessary for calpain-activated protein degradation; and (2) calpain activation was sufficient to increase proteasome-mediated proteolysis. These results support the hypothesis that the calpains act ‘upstream’ of the proteasome. We also report the novel finding that calpain activation inhibits the Akt signalling pathway in skeletal muscle. Since this pathway plays a critical role in protein synthesis (Hajduch et al. 1998; Kimball et al. 1999), these data suggest that calpain-mediated inhibition of the Akt signalling pathway may reduce skeletal muscle protein synthesis. Together, these findings suggest that the calpains play a dual role in protein metabolism, a discovery that may have important implications for developing interventions to prevent skeletal muscle atrophy.

One specific aim of the present investigation was to clarify whether the calpain proteases act upstream of the ubiquitin–proteasome pathway (UPP). Previous studies indicated that ex vivo muscle incubation in the presence of calcium increased total protein degradation, and further demonstrated that calpain inhibitors prevented calcium-activated proteolysis (Zeman et al. 1985; Furuno & Goldberg, 1986). Based on these results, we reasoned that this experimental model would prove useful in delineating the interactions between the two proteolytic systems. We first determined whether the proteasome was necessary for calpain-dependent protein degradation, since a similar approach was used in the ex vivo studies described above. Results from our experiments indicate that the proteasome is indispensable to calpain-activated protein degradation, since proteasome inhibition prevented calpain-dependent proteolysis. Further, in a follow-up study we found that calpain activation was sufficient for increased proteasome-dependent proteolysis. Indeed, calpain activation increased proteasome-meditated protein degradation by 144%. These data indicate that the calpain proteases and the proteasome work in a co-ordinated manner to degrade muscle proteins, and further suggest that the calpain proteases act upstream of the proteasome. These findings are in agreement with recent studies suggesting that the calpain proteases and the UPP work in sequence (Menconi et al. 2004; Kramerova et al. 2005).

Another approach used to investigate whether calpain activation was sufficient to activate the UPP was to assess activation of the UPP following calpain activation. Data from those experiments suggest that calpain activation was not sufficient to activate the UPP, because calpain activation failed to increase ubiquitin conjugates or proteasome enzyme activity at the end of the 1 h experiment. These results are contrary to our findings above, as well as to recent reports linking calpains to protein–ubiquitin conjugation (Kramerova et al. 2005) and proteasome enzyme activity (Menconi et al. 2004). The cause of this discrepancy is unknown; however, one explanation may be that the UPP was activated at an earlier time point than measured in these experiments. The ‘build-up’ of ubiquitinated proteins observed during catabolic conditions reflects the inability of the proteasome to keep pace with the rate of protein ubiquitination. Perhaps, in our relatively brief experimental model, calpain cleavage products were ubiquitin conjugated and rapidly degraded by the 26S proteasome, thereby preventing an observable increase in ubiquitinated proteins at the end of the experiment. This could also explain why proteasome enzyme activity was not elevated at the end of the experiments. This idea of a temporal activation of the UPP is supported by earlier studies in which calcium treatment caused a transitory increase in proteasome enzyme activity, rather than a sustained increase in activity (Kawahara & Yokosawa, 1994; Aizawa et al. 1996). The 1 h incubation was selected because preliminary studies suggested that calcium treatment increased protein degradation and proteasome enzyme activity at this time point. Additional studies would be required to determine whether calpains activate the UPP at an earlier time point in this model. Nonetheless, we provide evidence in the first set of experiments that calpain activation was sufficient to increase proteasome-dependent proteolysis, as well as evidence that the proteasome was necessary for calpain-activated proteolysis. These findings support the notion that the calpains act upstream of the proteasome.

It was recently reported that calpain inhibition failed to reduce proteasome-dependent protein degradation following a 16 h sepsis insult (Fareed et al. 2006). However, previous research indicates that 16 h of sepsis increases protein–ubiquitin conjugation in skeletal muscle (Tiao et al. 1996; Solomon et al. 1998). Since ubiquitin conjugation appears to be the rate-limiting step in UPP-mediated protein degradation (Solomon et al. 1998), the presence of excessive proteasome substrates could potentially fuel elevated proteasome-dependent protein degradation in spite of calpain inhibition. Therefore, measuring the contribution of calpain to proteasome-dependent protein degradation while the UPP may be ‘primed’ for increased proteasome-dependent protein breakdown could potentially underestimate the contribution of calpain to proteasome-dependent proteolysis. Confirmation that there was no build-up in proteasome substrates and/or assessment of proteasome-mediated proteolysis from muscles treated with the calpain inhibitor throughout the catabolic insult would provide stronger evidence for independent proteolytic pathways.

Although purely speculative, a potential connection between calpain, Akt and the ubiquitin system is noteworthy, given the context of the present investigation. Our findings suggest that calpain activation may modulate gene expression of the ubiquitin system. Two muscle-specific, ubiquitin ligases, atrogin-1/MAFbx and MuRF1, have been identified as part of the so-called ‘atrophy programme’, a set of transcriptional adaptations common to various types of muscle atrophy (Bodine et al. 2001a). The forkhead box O (FOXO) family of transcription factors regulate atrogin-1/MAFbx and MuRF1 expression, and there is now strong evidence that Akt is a negative regulator of the FOXO transcription factors (Brunet et al. 1999; Sandri et al. 2004). The finding that calpain activation reduces Akt phosphorylation suggests that calpain activation may promote the transcription of two ubiquitin ligases known to play significant roles in skeletal muscle atrophy. A recent study reported that in vivo administration of the calpain inhibitor BN82270 throughout a 16 h sepsis insult failed to prevent elevated atrogin-1/MAFbx and MuRF1 mRNA expression in skeletal muscle (Fareed et al. 2006). However, since calpain activity from muscles of animals administered BN82270 in vivo was not reported, the efficacy of BN82270 as a calpain inhibitor when administered in vivo is not known. Additional experiments are required to determine unequivocally whether calpains transcriptionally activate the ubiquitin system.

The goal of the second set of experiments was to investigate whether calpain activation affected a prominent pathway in protein synthesis, the Akt–mTOR–GSK-3β pathway. To our knowledge, this is the first study to demonstrate that calpains inhibit Akt signalling in skeletal muscle. Akt has numerous cellular functions; therefore, the consequences of calpain-mediated Akt inhibition will require further investigation. However, given the well-established role of the Akt signalling pathway in protein synthesis (Hajduch et al. 1998; Kimball et al. 1999), the observation that calpain adversely affects this signalling network effectively implicates calpain in reduced protein synthesis in skeletal muscle. Akt activates at least two signalling pathways known to mediate the translation phase of protein synthesis. Akt phosphorylates and inactivates GSK-3 (Rommel et al. 2001), a negative regulator of eukaryotic initiation factor 2B (eIF2B). This, in turn, allows eIF2B to exchange eIF2 GDP for GTP, a step required for continued translation initiation (Price & Proud, 1994). Akt also activates the mammalian target of rapamycin (mTOR; Rommel et al. 2001), which in turn inhibits eIF4E binding protein (4E-BP), a negative regulator of eIF4E. Inhibition of 4E-BP allows eIF4E to carry out its function of recognizing and binding the 5′ cap structure of mRNA during translation initiation (Pause et al. 1994). Therefore, the altered GSK-3β and mTOR activation observed in the present experiments suggests that calpain activation may subsequently reduce translation initiation.

mTOR also regulates the production of translational machinery necessary to carry out mRNA translation. mTOR stimulates p70S6K, which subsequently activates ribosomal S6. The S6 promotes selective synthesis of proteins involved in mRNA translation, such as ribosomes (Kawasome et al. 1998). Calpain-mediated mTOR inhibition could potentially diminish global rates of protein synthesis by reducing the amount of translational machinery available to maintain protein synthesis. Taken together, these data suggest that calpain activation may inhibit proteins key to the maintenance of protein synthesis. Additional research is necessary to determine whether calpain activation affects GSK-3β and/or mTOR downstream targets and protein synthesis.

To investigate potential mechanisms of calpain-mediated Akt inhibition, we next determined whether calpain activation affected HSP 90 protein content and/or the protein interaction between HSP 90 and Akt. The finding that calpain diminished HSP 90β content suggests that this isoform may be a preferential calpain substrate in skeletal muscle. However, interpretation of the significance of reduced HSP 90β content is difficult, since there are no data available regarding the relative abundance and the functions of HSP 90α and HSP 90β in skeletal muscle. Additional experiments are necessary to determine the proportion and functions of the HSP 90 isoforms in skeletal muscle and the significance of reduced HSP 90β content.

The molecular complex formed between HSP 90 and Akt is critical to Akt phosphorylation and activation (Sato et al. 2000). Since calpain activation was shown previously to reduce HSP 90–endothelial nitric oxide synthase (eNOS) binding in mesenteric tissue (Stalker et al. 2003), we hypothesized that calpain activation would reduce HSP 90–Akt binding in skeletal muscle. Co-precipitation experiments revealed that no HSP 90α was associated with Akt. Though HSP 90β was detected in the immunoprecipitates, there were no differences in the association of HSP 90β and Akt between the groups. Thus, calpain-mediated inhibition of Akt appears to be unrelated to the binding of the two proteins.

Diaphragm muscle differs from locomotor skeletal muscle in its histology, frequency of use, contractile properties and metabolic characteristics. Therefore, calpain activation may affect locomotor muscle in a different manner. However, since the proteins studied in the present experiments using diaphragm are also present in locomotor skeletal muscle, calpain activation probably affects pathways of protein synthesis and protein degradation in locomotor skeletal muscle as well. Additional experiments should be conducted to confirm the results of this study in locomotor skeletal muscle.

While the calcium dependence of the ubiquitous calpains is well appreciated, a recent report suggests that calpain-3 may also be calcium sensitive (Murphy et al. 2006). Therefore, it is possible that calpain-3 was activated in the present experiments. It should be noted that the calpain substrate used to confirm calpain activation in these experiments, talin, is also a calpain-3 substrate (Taveau et al. 2003). In addition, an alternatively spliced variant of calpain-3, a variant used to study calpain-3 proteolytic activity, is inhibited by calpeptin (Ono et al. 2004), the calpain inhibitor used in the present studies. Therefore, if calpain-3 was activated in these experiments, it seems likely that calpeptin prevented this stimulation, since calpeptin prevented talin cleavage.

In summary, this study demonstrates that the proteasome is necessary for calpain-activated proteolysis, and that calpain activation is sufficient to increase proteasome-dependent proteolysis, evidence that the calpain proteases act upstream of the proteasome. In addition, we report that calpain activation adversely affects the Akt–mTOR–GSK-3β signalling pathway, which is indicative of a role for calpain in cell signalling and reduced protein synthesis. Taken together, these data suggest that the calpain proteases play a dual role in protein metabolism, altering pathways of protein degradation and protein synthesis. These findings provide new insight concerning the role of calpains in skeletal muscle and may have important implications for developing interventions to prevent muscle atrophy.

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  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix
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Appendix

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Acknowledgements

This work was supported by National Aeronautics and Space Administration (NASA) Graduate Student Researchers Program (NGT5-50443 I.J.S.). Partial support for Dr Smith was provided by National Institutes of Health (NIH) grant 5T32 AG000029-30 (I.J.S.).