changes in all skeletal muscles analyzed (Fig. 3A–C). In transverse sections of fresh frozen specimens from the triceps brachii, tibialis anterior and vastus lateralis muscles, there was a marked variability in myofiber size in the treated double transgenic animals, with numerous small caliber fibers having a round shape, and multifocal clusters of mononuclear cells. As there was no elevation of atrogin-1 in the muscle from the double transgenic animals, the decrease in fiber size was not likely a result of muscle atrophy (data not shown). Many muscle fibers of both small and larger caliber contained internal nuclei. Measurement of the cross- sectional area confirmed the presence of many small fibers, most markedly within the vastus lateralis muscle (Fig. 3C). The small size of the muscle fibers was reflected by a decrease in the weight of each muscle (Fig. 3D). Loss of muscle mass may account for the global decrease in body weight observed in the double transgenic animals, as the weight of the other organs was not affected (Fig. 3E). Of the different muscles analyzed, the vastus lateralis was the most affected, demonstrated both by decreased cross-sectional area measurement and more pronounced weight loss. RNF5 overexpression is associated with increased muscle degeneration and extensive regeneration Although vacuolated fibers were not observed, rtTA RNF5 DTg animals exhibit myodegeneration, supported by various stages of myonecrosis and phagocytosis based on modified Gomori-trichrome stain (Fig. 4A). Levels of serum creatine kinase (CK) activity were consistently elevated after 6 wk of doxycyclin treatment in the rtTA RNF5 DTg animals, but not in their control littermates (Fig. 4B), supporting the presence of myofiber damage. To visualize the extent and distribution of myodegeneration, Evans Blue dye (EBD) was injected into the rtTA RNF5 DTg and their control littermates. Contrary to the skeletal muscle of their matching controls, the rtTA RNF5 DTg animals exhibited positive staining for EBD within a number of muscle fibers (Fig. 4C), consistent with degeneration (myonecrosis) and elevated serum CK activity. EBD-positive muscle fibers were negative for dystrophin staining (Fig. 4C) and surrounded by immune cells that showed positive staining for the pan-leukocyte marker CD45 and macrophage marker CD11b (data not shown), corresponding to macrophages clearing up necrotic debris. Ultra- structural analysis on DTg muscles further confirmed that the sarcomeric structure was conserved in non-degenerative fibers (Fig. 4D asterisk). In degenerating fibers, there was loss of the normal myofibrillar pattern, mitochondrial swelling and dilated tubular structures (Fig. 4D). Myodegeneration in RNF5 overexpressing animals was associ- ated with extensive fiber regeneration. Quantitative analysis revealed that 50% of the muscle fibers of rtTA RNF5 DTg, but not control animals, were centrally nucleated, and that 30% of the small fibers stained positively for embryonic myosin heavy chain (emb MHC), a known marker of early muscle regeneration (Fig. 4E,F). Consistent with this observation, positive staining for myogenin, a transcription factor expressed during differentiation of activated satellite cells[26], was also observed in small myofibers of rtTA RNF5 DTg animal muscle but not in their control littermates (Fig. 4E). Interestingly, clusters of mononuclear cells, staining positively with antibodies against CD45 (data nor shown) and CD11b (Fig. 4G), were also observed at sites of muscle regeneration, yet, neither lymphocytic infiltration nor fibrosis were observed (Fig. 4G, and data not shown). These data indicate that regeneration following overexpression of RNF5 is a result of myofiber degeneration and not an inflammatory process. Immunostaining using markers for DGC proteins including dystrophin and alpha-sarcoglycan, as well as the extracellular matrix protein laminin a2, did not show any major difference RNF5 in ER-Related Disorders between DTg and control muscles (Fig. S3), indicating that disruption of the DGC or extracellular matrix proteins was not a primary cause for myonecrosis. Consistently, in vitro differentiation of primary myoblasts cultured from the double transgenic animals treated with doxycyclin prior and during differentiation did not reveal changes in the sarcomeric structure as depicted by staining for alpha-sarcomeric actinin (Fig. S4). Taken together, these findings demonstrate that myofiber degeneration and necrosis account for the elevated serum CK activity associated with RNF5 overexpression, and that myodegeneration is not a primary inflammatory process nor is it a consequence of the alteration of common sarcolemmal structural proteins. Myofiber degeneration-coupled regeneration in rtTA RNF5 DTg mice is associated with altered expression of ER chaperones without the formation of protein aggregates Given the link between ER stress and sIBM [16,17] we next examined the status of ER stress markers in the muscles of RNF5 overexpressing animals. Consistent with the increase in ER stress markers observed in s-IBM muscles, a clear and consistent, albeit moderate, increase in expression of PDI, Grp78, Grp94 and calnexin was seen in rtTA RNF5 DTg but not control mice (Fig. 5A), suggesting that ER stress occurs in the muscles of RNF5 overexpressing animals. Grp94 upregulation was confirmed by immunohistochemistry of the DTg muscle sections (Fig. 5B). However, contrary to what has been observed in s-IBM patients, this ER chaperone did not localize to aggregates (Fig. 5B) and congophilic material was not detected in the muscle fibers of DTg animals (data not shown). These data suggest that the myodegen- eration observed following RNF5 overexpression is associated with ER stress yet occurs at earlier stage in the development of this disease, prior to aggregate formation. ER stress is commonly considered a protective response. However, high levels of ER stress or ER dysfunction may also impair cell survival by triggering apoptotic pathways (review by [27]. Therefore, the onset of ER stress could cause fiber death with concomitant induction of muscle regeneration without directly affecting the muscle structural components [28,29,30]. Analysis of TUNEL and cleaved caspase 3 as markers of apoptosis did not reveal any changes in programmed cell death (data not shown). Similarly, no increase was observed in the levels of CHOP or in cleavage of caspase 12 (data not shown). Rather CHOP levels were decreased in DTg animals (Fig. 5A), implying that overexpression of RNF5 may induce ER stress by limiting the activation of some UPR components. These data also indicate that the degeneration process observed in rtTA RNF5 DTg animals is not linked with ER- associated programmed cell death. Grp94 was previously reported to be important for myoblast fusion [31], a critical step in regenerating muscle. Change in Grp94 levels and localization is expected to impact its contribution to myoblast fusion and therefore affect the regeneration process [30]. Analysis of GRP94 expression revealed increased expression and possible post translational modification in rtTA RNF5 DTg but not in their matching controls (Fig. 5A). Further, RNF5 staining exhibited dense perinuclear staining in the endoplasmic reticulum of mature and regenerative fibers of the transgenic mice (Fig. 5C). Notably, RNF5 staining was higher in regenerating fibers and localized along the nascent sarcoplasmic reticulum network and sarcolemma (Fig. 5C), suggesting that its function may prevail during the regeneration process. These findings suggest that RNF5 may exert its effect on muscle physiology by modulating the function/localization of specific components of the ER, including Grp94. PLoS ONE | www.plosone.org 5 February 2008 | Volume 3 | Issue 2 | e1609