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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.
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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.