LOSS OF SPARC IN MOUSE SKELETAL MUSCLE CAUSES MYOFIBER ATROPHY KATSUYUKI NAKAMURA, DVM, SHIN-ICHI NAKANO, PhD, TAKAHIRO MIYOSHI, DVM, KEITARO YAMANOUCHI, PhD, and MASUGI NISHIHARA, PhD Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113–8657, Japan Accepted 11 February 2013 ABSTRACT: Introduction: The expression of secreted protein acidic and rich in cysteine (SPARC) in skeletal muscle decreases with age. Here, we examined the role of SPARC in skeletal muscle by reducing its expression. Methods: SPARC expression was suppressed by introducing short interfering RNA (siRNA) into mouse tibialis anterior muscle. Myofiber di- ameter, atrogin1, and muscle RING-finger protein 1 (MuRF1) expression, and tumor necrosis factor-a (TNFa) and transform- ing growth factor-b (TGFb) signaling were then analyzed. Results: Reduced SPARC expression caused decreases in the diameter of myofibers, especially fast-type ones, accompanied by upregulation of atrogin1, but not MuRF1, at 10 days after siRNA transfection. The expression of TNFa and TGFb and the phosphorylation status of p38 were not affected by SPARC knockdown, whereas Smad3 phosphorylation was increased at 2 days after siRNA transfection. Conclusions: The loss of SPARC not only upregulates atrogin1 expression but also enhances TGFb signaling, which may in turn cause muscle atrophy. Muscle Nerve 48:791–799, 2013 Skeletal muscle mass is maintained by the balance between anabolism and catabolism. When the bal- ance is biased toward excessive catabolism, myofiber atrophy occurs. Two muscle-specific ubiquitin ligases, atrogin1 (muscle atrophy F box) and muscle RING-finger protein 1 (MuRF1), have been found to be induced in conditions associated with muscle atrophy, such as denervation, hindlimb suspension, and aging, 1–6 and their expression was shown to be regulated by some cytokines. For example, tumor necrosis factor-a (TNFa) induces the expression of atrogin1 and MuRF1, 7 whereas transforming growth factor-b (TGFb) and phosphorylation of its down- stream molecule, Smad2/3, induce the expression of atrogin1, but not MuRF1. 8,9 Skeletal muscle mass also decreases with age, 10 a phenomenon called sarcopenia. 11 Specifically, both the number and diameter of myofibers in the elderly are decreased, 12 and it is common that the decrease in diameter is more severe in fast-type myofibers than in slow-type myofibers and that the proportion of slow-type myofibers increases with age. 12 In addition, the expression levels of atrogin1 and MuRF1 are upregulated with age in rats. 6,13 Secreted protein acidic and rich in cysteine (SPARC; also known as osteonectin or BM-40) is a secretory nonstructural, matricellular glycoprotein that plays multiple roles in cell adhesion, angiogen- esis, interactions with growth factors, and cell differ- entiation. 14,15 For instance, SPARC was shown to promote myogenic differentiation of C2C12 and MM14 myoblasts by inhibiting fibroblast growth fac- tor (FGF)-2/FGF receptor-1 signaling, which in turn promotes myoblast proliferation and prevents termi- nal differentiation. 16,17 Moreover, SPARC-null mice exhibit early-onset cataracts, 18 osteopenia-like decline of bone formation, 19 increased obesity, and decreased skeletal muscle mass. 20 These phenotypes overall are typically seen in the elderly. Further- more, we reported previously that the responsive- ness to SPARC in rat muscle-derived cells declines with age. 21 SPARC expression was also found to be decreased in skeletal muscle of older mice 22 and rats (our unpublished data) compared with that in younger animals. In addition, SPARC is known to modify TGFb signaling. 14 Specifically, a recent study showed that SPARC inhibited TGFb by means of interaction with endoglin, an accessory receptor of TGFb. 23 Therefore, dysregulation of TGFb signal- ing, which serves as a regulator of atrogin1 expres- sion, may cause muscle atrophy; however, the precise role of SPARC in skeletal muscle remains to be elucidated. Short interfering RNA (siRNA) is a 21 to 23 base pair duplex of RNA complementary with the target gene, by which the expression of target gene can be specifically suppressed. In this study, we per- formed an in vivo siRNA-targeting method to in- hibit SPARC expression in skeletal muscle and examined whether reduction of SPARC in skeletal muscle causes a decline in myofiber diameter. MATERIALS AND METHODS Animals. C57BL/6J male mice (8 weeks old) pur- chased from Charles River Japan, Inc. (Hino, Shiga, Japan) were maintained in our laboratory under Abbreviations: HE, hematoxylin–eosin stain; MHC, myosin heavy chain; MuRF1, muscle RING-finger protein 1; PFA/PBS, paraformaldehyde in phosphate buffered saline; qPCR, quantitative polymerase chain reaction; siRNA, short interfering RNA; SPARC, secreted protein acidic and rich in cysteine; TA, tibialis anterior muscle; TGFb, transforming growth factor-b; TNFa, tumor necrosis factor-a Key words: atrogin1; myofiber atrophy; Smad3; SPARC; TGFb Correspondence to: K. Yamanouchi; e-mail: akeita@mail.ecc.u- tokyo.ac.jp This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant (24-7714, 23228004, 21221008). VC 2013 Wiley Periodicals, Inc. Published online 20 February 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mus.23822 SPARC Knockdown in Muscle MUSCLE & NERVE November 2013 791