The bulk of muscle proteins (50–70%) exists in actomyosin complexes and myofibrils, and in different catabolic conditions, contractile proteins in these complexes are degraded by the Ub-P’some system (1
). The response is complicated, however, as shown by Solomon and Goldberg, who constructed a reconstituted system containing the components of the Ub-P’some system and found that it degrades monomeric actin or myosin but does not degrade actomyosin complexes (14
). They concluded that disassociation of actomyosin complexes is the rate-limiting step in muscle protein degradation.
Our results indicate that caspases play a critical, initial role in muscle protein degradation. We found that caspase-3 cleaves actomyosin in vitro and in muscle cells; products of caspase-3 action are rapidly removed by the ATP-dependent, Ub-P’some system. Moreover, the characteristic 14-kDa actin fragments are present in atrophying muscles of animals with diabetes or chronic uremia. In response to diabetes, caspase-3 activity in muscle increases. In addition, Ac-DEVD-CHO, the inhibitor of caspase-3, suppresses both the accelerated muscle proteolysis and the accumulation of actin fragments that is induced by acute diabetes. The finding that Ac-DEVD-CHO suppresses the accelerated rate of protein degradation in muscle of rats with acute diabetes is enlightening for two reasons. First, exposure of muscle to Ac-DEVD-CHO for only a relatively brief period did suppress total protein degradation. Inhibiting caspase-3 activity during the incubation could only block the breakdown of additional actomyosin complexes and would not suppress the degradation of proteins already released from previously cleaved actomyosin complexes. Thus, over a short period, the caspase-3 inhibitor could only exert limited suppression of total protein breakdown compared with an inhibitor of the proteasome (11
), and the results in Table are consistent with caspase-3 acting as an initial step triggering the breakdown of muscle protein complexes. We also found that the caspase-3 inhibitor did not significantly suppress protein degradation in muscles of paired, control rats. This result suggests that the stimulation of caspase-3 activity is a response to a catabolic condition rather than regulating basal protein turnover in muscle.
How much does actomyosin breakdown contribute to total protein degradation in muscle? To examine this question, we prepared a cell-free homogenate of rabbit skeletal muscle containing the elements of the Ub-P’some system (14
). When we activated the Ub-P’some system in the muscle homogenate by adding ATP, we confirmed that there was an increase in the degradation of endogenous proteins (Table ). In contrast, when we added the breakdown products of actomyosin resulting from its digestion by recombinant caspase-3, there was an additional 125% increase in ATP-stimulated protein degradation over that measured when undigested actomyosin was present. The large increase in total protein degradation indicates that actomyosin cleavage converts actomyosin to individual myofibrillar proteins that are degraded by the Ub-P’some system. This response occurred in conditions in which we limited the cleavage of actin in actomyosin to about 1%, based on its detection by the anti-actin antibody. Notably, acute diabetes or chronic uremia is associated with accumulation of the actin fragment in muscle to approximately the same degree (Figure ). Since a somewhat small accumulation of the actin fragment is associated with a substantial increase in total protein degradation, we conclude that the cleavage of actomyosin in the muscle of diabetic or uremic rats is a major contributor to the increase in total protein breakdown.
What other systems could be involved in accelerating muscle proteolysis? Calpains are a possibility, in light of the reports of others that calpains clip myofibrillar proteins into fragments in a septic rat model (32
). In addition, Huang and Forsberg reported that expression of the endogenous inhibitor of calpains, calpastatin, or a dominant inhibitor of calpains in L8 muscle cells suppressed the cleavage of fodrin (31
). They also found a lower rate of total protein degradation, suggesting that the calpain inhibitors had suppressed actomyosin degradation. Tidball and Spencer reported that muscle atrophy due to hindlimb unloading was attenuated in transgenic mice that overexpress calpastatin in a muscle-specific manner (43
). In contrast, we and others find that inhibiting calcium-dependent proteases does not suppress either total or myofibrillar protein degradation in muscles isolated from rats with acute diabetes or chronic uremia (1
). In the present study as well, we found no evidence that calpains are critical mediators of the accelerated muscle protein degradation found in the conditions we studied, although we cannot exclude the possibility that activation of calpains in some other pathologic conditions could lead to muscle atrophy.
Regarding the cellular mediator that could activate caspase-3 in the conditions associated with accelerated muscle protein degradation, insulin resistance is common to many of these disorders (e.g., sepsis or chronic uremia), while other conditions are characterized by a low level of insulin (e.g., acute diabetes or starvation) (12
). Not only is PI3K signaling integral for insulin responses, but it also is critical for antiapoptotic responses (41
). Consequently, dysfunction of the PI3K signaling process could be a common initiator of muscle protein degradation. In support of this possibility, we found evidence that serum withdrawal (which reduces PI3K activity; Figure ) stimulates actomyosin cleavage in muscle cells and insulin blocks this response. But when PI3K activity is blocked, the ability of insulin to suppress actomyosin cleavage is abolished (Figure ). These in vitro results indicate that an essential role of PI3K signaling is to suppress actomyosin cleavage. Regarding the relevance of PI3K activity in vivo, certain catabolic conditions including those we studied are associated with decreased PI3K activity in muscle. For example, we found that the PI3K activity associated with insulin receptor substrate-1 (IRS-1) is low in the muscle of rats with chronic uremia (J.L. Bailey et al., unpublished observations). Rats with chronic uremia exhibit a marked increase in the rate of muscle protein degradation (11
) as well as evidence of actin cleavage (Figure ). We also find that acute diabetes stimulates muscle proteolysis (12
) and is associated with decreased IRS-1–associated PI3K activity and evidence of caspase-3 activation (S.W. Lee et al., unpublished observations). Since PI3K is an essential regulator of apoptotic pathways (41
), it would provide a link between a deficiency of insulin or impaired insulin-signaling processes and activation of caspase-3 resulting in loss of muscle protein. We extended these results by examining whether the proapoptotic protein, Bax, is activated. In serum-starved muscle cells that exhibit suppressed PI3K activity (Figure ), the level of Bax in the conformational state that activates caspase-3 is increased (Figure ). Serum starvation increases the appearance of the 14-kDa actin fragment (Figure ). These results are consistent with the reported sequence of events that link decreased PI3K activity to caspase-3 (28
We have emphasized that caspase-3 is involved in the catabolic response because we found that recombinant caspase-3 will degrade actomyosin complexes and because Ac-DEVD-CHO not only prevents the generation of the actin fragment but also suppresses protein breakdown in muscle of rats with acute diabetes. Since the specificity of an inhibitor may not be absolute, we do not exclude the possibility that other proteases could be involved in initiating muscle proteolysis. Others have reported that caspases can cleave actin in vitro (20
), but Song et al. (45
) did not find actin fragments in leukemia or lymphoma cells that were undergoing apoptosis. They concluded that actin is not degraded by apoptosis-associated proteases. A potential reason for the difference from our results is that actin fragments in their experiments were rapidly degraded by the Ub-P’some system. Alternatively, the cells they were examining may have different pathways of protein degradation than those in muscle cells.
In summary, muscle atrophy is a frequent complication of many catabolic conditions. Our findings indicate that muscle protein loss occurs in two steps: (i) catabolic stimuli cause the breakdown of actomyosin to release monomeric actin and myosin; (ii) the resulting fragments and monomeric proteins are then degraded by the Ub-P’some system. Blocking caspase activity in intact muscle of rats with accelerated muscle proteolysis reduces both the accumulation of actin and the acceleration of protein degradation. Armstrong and colleagues published preliminary results suggesting a beneficial, cardioprotective effect of infused caspase inhibitors following a myocardial infarction (46
). Their results, obtained in an intact rat, suggest that the responses we identified could provide new targets for preventing muscle atrophy in catabolic conditions.