panels c,d) and the formation of blue-rimmed vacuoles containing inclusions (Fig. 6D panels e–h). These findings establish that muscle- specific overexpression of RNF5 is sufficient to cause muscle degeneration with extensive muscle regeneration. Moreover, prolonged RNF5 overexpression leads to the formation of vacuole containing inclusions and accumulation of congophilic material, as seen in sIBM patients, further substantiating a role for RNF5 in the development of the disease. Similar to what we have observed in rtTA RNF5 DTg mice, ER stress markers such as GRP94 was up regulated (possibly a post- translationally modified) in MCK-tTA RNF5 DTg animals (Fig. 6E) and elevated expression was observed in small regenerating fibers by immunohistochemistry (Fig. 6F). The two mouse models used provide important confirmation for the role of RNF5 in ER stress- associated muscle degeneration phenotype. RNF5 KO mice exhibit a delay in regeneration from cardiotoxin-induced muscle damage To further assess the potential role of RNF5 in normal muscle physiology we used an RNF5 KO model established in our laboratory. In these mice, exons 1-3 of the RNF5 gene were replaced by a Neo cassette (Fig. 7A). The chimeric mice gave a germline transmission of the disrupted RNF5 gene (see Materials and Methods for further details) and heterozygous mice were interbred to produce litters that included homozygous offspring. Southern blot analysis of mouse DNAs isolated from the tails of these mice revealed the expected three genotypes (+/+, +/2, 2/ 2), as judged by diagnostic probes for the correct 59 and 39 homologous recombination events (Fig. 7A). RNF5 KO and WT animals were subjected to cardiotoxin treatment, which induces muscle damage followed by its regener- ation [33], thereby allowing us to monitor possible changes in the ability of each genotype to promote muscle regeneration. The toxin, or PBS control, was injected directly into the tibialis muscle and the muscles were collected after 3, 4, 6, 12 and 28 days. Significantly, the expression of RNF5 in WT muscle was elevated after cardiotoxin treatment (Fig. 7B), thereby supporting a role for RNF5 in muscle physiology/regeneration. To assess whether increase of RNF5 levels in physiological conditions would affect ER stress markers, we monitored the levels of GRP94 in RNF5 KO and WT during the course of muscle regeneration. Importantly, both induction of Grp94 expression and its post translational modification, as defined by the disappearance of the slower migrating form, were delayed in RNF5 KO mice, compared with WT animals (Fig. 7B and Fig. S5). Of interest, induction of calnexin was slightly delayed whereas expression of PDI was elevated in RNF5 KO. Consistent with the changes observed in induction of ER stress markers, muscle regeneration in RNF5 KO mice was attenuated, compared to control animals, as evident from (i) delayed production of myosin heavy chain (Fig. 7B) and (ii) a high number of centrally nucleated fibers observed 28 days after cardiotoxin treatment in the KO but not WT animals (Fig. 7C). These data substantiate the observations made with RNF5 Tg mice and suggest that RNF5 is playing an important role in muscle regeneration process associated with ER stress response. Discussion The present study establishes a link between RNF5 and specific muscular disorders associated with ER stress. Prompted by our observation that RNF5 is overexpressed and mislocalized to aggregates in muscle biopsies from sIBM patients and deregulated in an animal model for hereditary IBM, we have investigated the RNF5 in ER-Related Disorders role of RNF5 in muscle using 3 genetic mouse models: an inducible transgenic mouse in which the E3 ligase RNF5 is conditionally overexpressed (globally and selectively in skeletal muscle) and an RNF5 KO mouse. RNF5 overexpression led to myodegeneration and regeneration in skeletal muscle independent of the classical dystrophic mechanism of sarcomeric disruption. In addition, prolonged RNF5 overexpression leads to the formation of blue-rimmed vacuoles containing inclusions and congo red positive fibers, as seen in sIBM. Further, RNF5 KO mice subjected to muscle damage exhibited a milder muscle phenotype which is consistent with a slower regenerative process. Both RNF5 overexpression and KO models suggest that the muscle phenotypes are associated with altered ER stress response, as shown by the increase in the ER stress markers PDI, GRP78, GRP94 and calnexin in the muscle of the RNF5-overexpressing animals and the delay in GRP94 and calnexin induction in RNF5 KO. These observations establish a link between RNF5, ER stress and muscle physiology. The mouse lines established and characterized in this study offer new models to study ER-related myodegenerative process that occur in myopathies such as sporadic or hereditary IBM. Impairment of the ERAD and UPR pathways has been linked with the onset of degenerative diseases such as sIBM, and is thought to have a contributing role by allowing improper accumulation of malfolded proteins. Consistent with this hypothesis, we found that RNF5, an E3 ligase involved in ERAD, exhibit an altered pattern of expression in biopsies from sIBM patients. Interestingly, RNF5 has also been shown to be upregulated in brain (Substantia Nigra) from Parkinson Disease patients [34], therefore pointing to a possible broader role of this E3 ligase in degenerative disorders. The increase in RNF5 expression seen in these degenerative diseases could be a consequence of ER overload, ERAD dysfunction, or part of the mechanism engaged in the development of these disorders. Our data supports the latter possibility since RNF5 overexpression is sufficient to induce the myodegenerative process associated with both the formation of blue-rimmed vacuole over time and an elevation of ER stress markers, characteristic of sIBM. Our observation that RNF5 over-expression was not sufficient to promote the formation of vacuoles containing inclusions at an early time point when ER stress markers were already upregulated suggests that ER stress and RNF5 overexpression are among early events in the pathology of these muscle disorders. That RNF5 is deregulated in the DMRV mouse model, where a sialation defect has previously been shown to promote the IBM- like phenotype, implies that RNF5 deregulation may not be the primary cause for the disease but rather be one of the early components contributing to the pathological process. Among mechanisms that may account for the ability of RNF5 to cause myodegeneration is the effect of RNF5 on ER stress response and ERAD. RNF5 contributes to ubiquitination of misfolded proteins, with a concomitant effect on their clearance of the proteins by the proteasomes [23]. RNF5 also controls ERAD through its effect on JAMP, an ER anchored 7 transmembrane protein which is important for proteasome recruitment to the ER (our unpublished observation). Thus, RNF5 overexpression is expected to impair the ER stress response, consistent with the modification of ER stress associated chaperones observed in our mouse model. Of importance is that accumulating pathological evidence now links mutations or dysregulation of ER-related proteins with muscular disorders. For example, mutations in the ER-associated ATPase p97/VCP have been associated with a specific type of inclusion body myopathy associated with Paget disease [35]. In addition, the p97/VCP and ERAD pathways were recently implicated in the degradation of sarcomeric myosin and its chaperone Unc-45 [36,37], thereby offering a link between PLoS ONE | www.plosone.org 10 February 2008 | Volume 3 | Issue 2 | e1609