The present findings clarify the critical role that MuRF1 plays in muscle atrophy. Although other ubiquitin ligases are also induced during atrophy (i.e., atrogin-1 [Gomes et al., 2001
], which is also essential for rapid atrophy [Bodine et al., 2001
], as well as E3α-II [Kwak et al., 2004
]), MuRF1 is necessary for most of the increase in ubiquitin conjugates during denervation atrophy (). Thus, MuRF1 either ubiquitylates most proteins being degraded 10 d after denervation, or its actions allow other ligases to act. In either case, MuRF1 clearly is a very general ligase affecting many contractile (), soluble, and nuclear proteins (unpublished data), as was also suggested by two-hybrid studies (Witt et al., 2005
; Hirner et al., 2008
). Moreover, our studies implicate ubiquitylation by MuRF1 in the selective loss of MyBP-C, MyLC1, and MyLC2, as well as the slower degradation of MyHC, and thereby in the disassembly and degradation of myofibrillar proteins, which is the defining feature of the atrophy process.
Inactivation of MuRF1's capacity to bind E2s by deletion of its Ring-finger motif attenuated atrophy and prevented the differential loss of MyBP-C, MyLC1, and MyLC2 from the myofibril (). Their selective loss is a new feature of atrophying muscles and seems to be of regulatory importance. Because these proteins appear to be critical in stabilizing the myofibril (see below), the reduced loss of MyHC in the ΔR-MuRF1myc/myc mice might be a direct consequence of the sparing of MyLC1, MyLC2, and MyBP-C. Also surprising were the findings that these components of the thick filaments are degraded sooner and by a distinct mechanism from the thin filament components, actin, tropomyosin, and α-actinin, whose degradation is independent of MuRF1.
Because MuRF1 is required for rapid atrophy, but not for normal muscle growth or function, identification of its substrates is essential to understand the mechanisms of protein loss. We have identified a number of myofibrillar components as novel substrates for MuRF1 in vivo, all of which are important for muscle integrity and contractile activity. Mutations in each of them cause severe disorders characterized by disarrayed myofibrils and compromised contractile function (Clark et al., 2002
). Although cardiac Troponin-I was previously reported to be a MuRF1 substrate (Kedar et al., 2004
), Troponin-I could not be immunoprecipitated with MuRF1 from these skeletal muscles and was not lost by a MuRF1-dependent mechanism after denervation. MyHC had been shown to accumulate in mice lacking MuRF1 and MuRF3 (Fielitz et al., 2007
) and to be degraded in a MuRF1-dependent manner upon dexamethasone treatment of embryonic myotubes (Clarke et al., 2007
). Accordingly, decreased content of MyHC by a MuRFl-dependent mechanism was clearly evident at 14 d denervation (). However, within the myofibril from innervated muscles, MyHC is not ubiquitylated by MuRF1 and is not a preferred substrate of MuRF1 even 10 d after denervation ().
It is noteworthy that these various myofibrillar proteins could be precipitated with GST-MuRF1 from the cytosolic fraction, and were ubiquitylated by pure MuRF1. Soluble pools of these myofibrillar components have been reported in chicken (Horvath and Gaetjens, 1972
) and were found in the mouse muscle extracts (unpublished data). Possibly, these soluble components function either as precursors to the mature contractile apparatus or as components released during myofibrillar turnover, or may represent a fraction of myofibrillar proteins that are easily released upon homogenization (Etlinger et al., 1975
). However, these proteins did not accumulate in the cytosol during atrophy, when they were lost from the myofibril (unpublished data).
Although it has long been recognized that the major myofibrillar proteins are degraded by the ubiquitin–proteasome pathway during atrophy (Solomon and Goldberg, 1996
), the identification of MuRF1 as the critical ligase that acts on the myofibrillar compartment (perhaps together with other E3s) suggests a role for ubiquitylation in disassembly of the myofibril. It is noteworthy that purified monomeric actin and MyHC were efficiently ubiquitylated by MuRF1, but not when associated with each other in the actomyosin complex or in the intact myofibril (, unpublished data). Previously, Solomon and Goldberg (1996)
demonstrated in crude homogenates that the ubiquitin–proteasome system rapidly degrades monomeric actin and myosin, but this process was not demonstrable in the extracts, when these proteins were associated with each other in the actomyosin complex or a myofibril. These early findings suggested that during atrophy a mechanism may exist to release these components from the myofibril or to enhance the susceptibility of actin, myosin, and other myofibrillar proteins to ubiquitylation. However, based upon the present findings, it no longer seems necessary to postulate such a release system (see below).
MuRF1 could ubiquitylate these substrates together with UbcH1, which forms K-48–linked polyubiquitin chains, and UbcH13/Uev1, which forms processively K-63–linked chains, and UbcH5, which forms mixed chains (Kim et al., 2007
) (). However, it is unclear which E2s function in proteolysis with MuRF1 or which types of ubiquitin chains it forms in normal or atrophying muscles. Although K-63–linked ubiquitin chains were believed to serve functions other than proteasomal degradation in vivo, recent findings indicate that K63 chains can target substrates to the proteasome in vitro and in vivo (Saeki et al., 2009
). Also, the nature and content of the E2s can influence the rates of protein degradation and thus the rate of atrophy.
It is particularly intriguing that MyLC1, MyLC2, and MyBP-C are selectively degraded in a MuRF1-dependent manner after denervation (), and that these proteins, unlike MyHC and actin, are efficiently ubiquitylated by MuRF1 within the myofibril (, unpublished data). Thus, these critical stabilizing proteins can be substrates within the myofibril without dissociation or proteolytic processing. Furthermore, the susceptibility of MyHC to ubiquitylation and degradation is enhanced 14 d after denervation (), presumably because the levels of MyBP-C and MyLCs in the myofibril are reduced. Accordingly, myosin can be extracted from the cardiac thick filament only after MyBP-C content has been reduced by 20% (Kulikovskaya et al., 2007
). By contrast, the thin filament components, actin, tropomyosin, and α-actinin, were lost from the myofibril 14 d after denervation by a mechanism that does not require MuRF1 or the MuRF1-dependent loss of MyBP-C and MyLCs. However, it is also possible that MuRF1 may target for degradation proteins associated within the Z-disk lattice because Filamin C was precipitated together with MuRF1 (). Alternatively, another E3 induced in atrophy may promote disassembly of the Z-disk or thin filaments, and function in the ΔR-MuRF1 animals.
Although no reduction in the absolute levels of contractile proteins was demonstrable by these techniques until 10 d after denervation, their degradation must have been accelerated much sooner because the loss of proteins results from the integrated effects of enhanced proteolysis over time. Any small changes in myofibrillar content (e.g., 10–20% decreases) that occur sooner after denervation probably cannot be detected by the methods used here. Accordingly, our finding that MuRF1 is induced by 3 d after denervation () implies that MuRF1 functions maximally early in atrophy (and before any loss of myofibrillar proteins is detectable). MuRF1 may promote the degradation of soluble cytosolic components, many of which associate with MuRF1 (unpublished data).
It is noteworthy that decreases in muscle mass can be demonstrated during the first week after denervation (Furuno et al., 1990
) and do not seem attributable to MuRF1-dependent loss of myofibrillar components (). However, other E3s are induced rapidly during atrophy (e.g., atrogin-1) and may help accelerate degradation. Also, recent investigations have shown that autophagy is activated rapidly during denervation-atrophy and seems to promote destruction of mitochondria and cytosolic proteins, perhaps during the first week after denervation (Mammucari et al., 2007
; Zhao et al., 2007
; O'Leary and Hood, 2009
), before any loss of myofibrillar myosin and actin can be detected.
Thus, during atrophy, MuRF1 seems to function at the initial steps of thick filament disassembly, apparently by selectively ubiquitylating certain key regulatory components, whose degradation should facilitate the breakdown of the remaining thick filament components. The extraction of these ubiquitylated regulatory proteins from the myofibril might be directly linked to their degradation by the 26S proteasome, whose 19S component catalyzes ATP-dependent unfolding and translocation of substrates into the core 20S proteasome (Smith et al., 2005
). Alternatively, ATP-dependent extraction of these proteins may be catalyzed by the p97/VCP complex, which extracts ubiquitin-conjugated proteins from ER membranes in the ER-associated degradation pathway (Ye et al., 2001
) and plays an important role in myofibril assembly (Janiesch et al., 2007
). Additional evidence for a critical regulatory role of myofibrillar MyBP-C is that mRNAs for this protein also decrease selectively during denervation atrophy (unpublished data).
MyBP-C and MyLC2 are critical modulators of muscle contractility, and their loss may account for some of the alterations in contractile properties seen after denervation (Zorzato et al., 1989
; Trachez et al., 1990
). Extraction of MyBP-C from skinned fibers leads to an increase in the Ca2+
sensitivity of force development (Hofmann et al., 1991
). Removal of myofibrillar MyLC2 has a similar effect on the Ca2+
-tension curve as removal of MyBP-C. Both proteins thus appear to reduce inappropriate contractions by decreasing the probability of the myosin heads binding to actin (Moss et al., 1983
). In addition, in heart, increasing MyBP-C content enhances the ATPase activity of actomyosin but not if MyLC2 was removed, and this effect reappeared when MyLC2 was restored (Margossian, 1985
Several days after denervation, the muscle's contractile properties change (i.e., there is a prolongation of the contraction and relaxation periods), apparently due to alterations in intracellular concentrations of Ca2+
(Zorzato et al., 1989
) as well as an increased myofibrillar Ca2+
sensitivity (Trachez et al., 1990
). As noted above, extraction of MyBP-C or MyLC2 also leads to increased myofibrillar sensitivity to Ca2+
. Because Ca2+
is less efficiently pumped out of the cytosol of denervated muscle (Finol et al., 1981
), the selective loss of these regulatory components might contribute to their altered contractile properties. Another characteristic of atrophied muscle is a progressive reduction in the twitch force. Because MyLC1 is required for full force production by muscle myosin (VanBuren et al., 1994
), its selective loss (as well as subsequent loss of MyHC) should contribute to the loss of tension after denervation.
Although the ubiquitylation on the myofibril and selective degradation of the critical stabilizing proteins MyBP-C, MyLC1, and MyLC2 by MuRF1 is an attractive new mechanism for loss of the thick filaments, it remains to be proven that these sequential steps are essential for the loss of MyHC and rapid atrophy. It will also be important to establish the nature of the subsequent steps, which lead to a loss of thin filaments during atrophy and also during the slower turnover of myofibrillar components in normal muscle. This mechanism (ubiquitylation of key regulatory components in the myofibril) is very different from the frequent suggestion that the initial attack on the contractile apparatus is by endoproteases, such as caspases (Du et al., 2004
) or calpains (Tidball and Spencer, 2002
). Although such mechanisms may function in certain apoptotic conditions (Du et al., 2004
), direct evidence that these proteases play an essential or primary role is lacking, and the present findings demonstrate a very different MuRF1-dependent mechanism that appears to function in vivo at the level of the myofibril.