6132 Biochemistry, Vol. 49, No. 29, 2010 Vassilopoulos et al. FIGURE 1: Western blot and immunofluorescent analysis of the proteins of the calcium release complex in infected muscles. The experiments presented in panels A and B correspond to one representative experiment, which have been performed on three different infected muscles. The values have not been averaged among all the experiments as the infection level is not identical among all the infected muscles. (A) Expression of the components of the calcium release complex in skeletal muscle microsomes (20 μg) in Trisk 95-infected muscle (lane 1), Trisk 51-infected muscle (lane 2), or control DsRed-infected muscle (lane 3) as determined by Western blotting. (B) Level of proteins expressed as a fold of control DsRed- infected muscles, after loading correction using a quantity of myosin estimated by Coomassie blue staining. The quantification was performed on the Western blot presented in panel A. (C) Effect of Trisk 95 overexpression on proteins of the calcium release complex. Double immunofluorescent labeling was performed on transversal sections of muscle infected with AdV-T95 for 30 days, using antibodies directed against T95 and the R1s subunit of the DHPR (a-c), Trisk 95 and the RyR (d-f), T95 and CSQ (g-i), or T95 and T51 (j-l). The scale bar is 15 μm. (D) Effect of Trisk 51 overexpression on proteins of the calcium release complex. Double immunofluorescent labeling was performed on transversal sections of muscle infected with AdV-T51 for 30 days using antibodies directed against T51 and the R1s subunit of the DHPR (a-c), T51 and the RyR (d-f), T51 and CSQ (g-i), or T51 and T95 (j-l). The scale bar is 15 μm. we could observe a reduction in the level of Trisk 51 expres- sion in Trisk 95-infected fibers (Figure 1C), an effect that was not observed in a Western blot. Likewise, only the level of Trisk 95 expression was reduced in Trisk 51-infected fibers (Figure 1D). Effect of Trisk 95 and Trisk 51 Overexpression on the Muscle Histology. Histological analysis revealed no sign of muscle damage in control skeletal muscles injected with either saline (data not shown) or AdV-DsRed (Figure 2A, panel a). However, Trisk 95- and Trisk 51-infected skeletal muscles were characterized by the presence of fibrotic tissue, atrophic fibers, and regenerating fibers (Figure 2A, panels b and c). The extent of muscle damage was greater in T51-infected muscles than in T95- infected muscle, as assessed by the increased level of fibrosis and the presence of necrotic fibers. To specifically characterize Trisk 95- and Trisk 51-infected fibers, muscle sections were analyzed after immunostaining with caveolin 3 (Cav-3) as a plasma mem- brane marker (Figure 2B). In contrast to muscles infected with AdV-DsRed (Figure 2B, panels a-c and g-i), membrane stain- ing revealed that the size of fibers infected by AdV-Trisk 95 and AdV-Trisk 51 was abnormal. Trisk 95-infected fibers were smaller than noninfected fibers (Figure 2B, panels d-f), and Trisk 51-infected fibers had a round shape (Figure 2B, panels j-l) and lost the contact between them. In contrast with the control AdV-DsRed-infected fibers (Figure 2B, insets), the expression pattern of Cav-3 was altered in fibers overexpressing Trisk 95 and Trisk 51. In these latter fibers, Cav-3 was shown to accumulate intracellularly and to colocalize with the triadins. As Cav-3 is expressed during the differentiation of the satellite cells in myotubes (17), it could be hypothesized that the aberrant staining of Cav-3 specifically occurs in regenerating fibers. However, staining of nuclei indicated that none of the cells displaying an intracellular staining of Cav-3 were regenerating cells (data not shown). Immunoblot analysis showed a similar amount of Cav-3 in crude extracts of infected muscle as compared to control samples from DsRed-infected muscles (Figure 2C,D), suggesting most pro- bably a relocalization of Cav-3 upon triadin overexpression. Association of Cav-3 with the Component of the Skeletal Muscle CRC. In vivo modification of triadin expression results in modification of Cav-3, indicating a functional link between