Article Biochemistry, Vol. 49, No. 29, 2010 6133 FIGURE 2: Alteration of muscle morphology in infected muscles. (A) Histological analysis by Hematoxylin-eosin staining of gastrocne- mius infected for 30 days with AdV-DsRed (CTRL, panel a), AdV- Trisk 95 (inf T95, panel b), or AdV-Trisk 51 (inf T51, panel c). Only in muscle infected with T95 or T51 is the presence of fibrotic tissue observed, as well as a few atrophic fibers (asterisks), necrotic fibers stained in pink (inf T51 only), and a few regenerating fibers, cha- racterized by their centrally located nuclei (arrows). The scale bar is 50 μm. (B) Double immunofluorescent labeling with anti-Cav-3 and anti-T95 antibodies (a-f) or anti-Cav-3 and anti-T51 (g-l) antibo- dies was performed on muscle sections from CTRL DsRed-infected muscle (a-c and g-i), T95-infected muscle (d-f), or T51-infected muscle (j-l). Cav-3 and T95 (insets of panels a-c and d-f) or Cav-3 and T51 (insets of panels g-i and j-l) intracellular staining was com- pared by confocal microscopy. (C) The expression level of Cav-3 was analyzed by Western blotting on T95-infected muscle (lane 1), T51- infected muscle (lane 2), or DsRed-infected muscle (lane 3). Five micrograms of mouse skeletal microsomes was loaded in each lane. (D) The quantification of the amount of caveolin 3 in each lane of panel C was performed, and the results were compared to the amount in DsRed-infected muscle (CTRL), after correction of loading by myosin quantification. the two proteins. We studied their possible association by immunoprecipitation in rat skeletal muscle (Figure 3A). Using isoform specific antibodies, both Trisk 95 and Trisk 51 co- immunoprecipitated Cav-3 (Figure 3A, lanes 2 and 3). Trisk 95 and Trisk 51 also co-immunoprecipitated each other and the RyR, which indicates that Cav-3 is associated with the CRC. An association between the DHPR and Cav-3 was proposed in skeletal muscle (18) and shown in cardiac muscle (19). Thus, it is possible that the molecular complex between the triadins and Cav-3 requires a direct interaction of Cav-3 with the DHPR. To test the direct interaction between the DHPR and Cav-3, we ana- lyzed the co-immunoprecipitation in rat skeletal muscle of Cav-3 with DHPR or the triadins (Figure 3A). The DHPR efficiently co-immunoprecipitated Cav-3 and the RyR (Figure 3A, lane 5), FIGURE 3: Association of Cav-3 with the CRC and the triadins. (A) Co-immunoprecipitation on rat skeletal muscle. Immunoprecipita- tion was performed on 500 μg of rat skeletal muscle with antibodies against Trisk 95 (lane 2), with antibodies against Trisk 51 (lane 3), with preimmune serum (lane 4), or with antibodies against the DHPR (lane 5). The immunoprecipitated proteins were then analyzed by Western blotting with antibodies against the RyR, against the DHPR, against the N-terminal end of triadin, or against Cav-3. Lane 1 contained 5 μg of rat skeletal muscle microsomes as a control. (B) Co-immunoprecipitation in L6 cells. Immunoprecipitation was performed on 300 μg of Trisk 95-infected cells (lane 2), Trisk 51- infected cells (lane 3), or control cells (lane 4), with antibodies specific for T95 or T51. The immunoprecipitated proteins were then analyzed by Western blotting (lanes 5-8) with the anti-RyR, anti-Nter triadin, or anti-Cav 3. Lane 1 contained 5 μg of rat skeletal muscle micro- somes as a control. The RyR is absent from L6 cells (infected and control, lanes 2-4) and is not co-immunoprecipited with triadin isoforms (lanes 5-8). but not the triadins. Conversely, both Trisk 95 and Trisk 51 co- immunoprecipitated Cav-3 but not the DHPR (Figure 3A, lanes 2 and 3), suggesting that the association of triadin and Cav-3 is not DHPR-dependent. To test whether Cav-3 and the triadins are able to associate directly, we studied their association in the L6 rat muscle cell line that does not express the known CRC proteins but expresses Cav-3. L6 cells were infected with AdV-Trisk 95 or AdV-Trisk 51 to induce the expression of each triadin isoform (Figure 3B, lanes 2-4). The absence of the RyR and the presence of Cav-3 were confirmed in these cells (Figure 3B, lanes 2-4). Direct association of Cav-3 with either triadin was studied by co- immunoprecipitation with the anti-T95 and anti-T51 antibodies. Although Trisk 95 and Trisk 51 were efficiently immunoprecipi- tated, Cav-3 was co-immunoprecipitated with none (Figure 3B, lanes 5-7), indicating that Cav-3 is not engaged in a direct interaction with any of the triadins. As both triadins are linked in skeletal muscle, as demonstrated by their co-immunoprecipita- tion (Figure 3A), it is possible that the simultaneous presence of Trisk 95 and Trisk 51 is required for association with Cav-3. To test this hypothesis, L6 cells were co-infected with Adv-Trisk 95 and AdV-Trisk 51. However, Cav-3 was not co-immunoprecipi- tated with either anti-triadin isoform antibody (data not shown), suggesting that the presence of the two isoforms is not sufficient for the association with Cav-3 RyR1 Is Associated with Cav-3. An association between RyR2 and Cav-3 was shown in cardiac muscle (20). To test whether Cav-3 directly interacts with RyR1 in skeletal muscle, we took advantage of the triadin mouse model (7) to analyze the co-immunoprecipitation of Cav-3 with RyR1 in the absence of triadin. As shown in Figure 4A, Cav-3 was efficiently co-immun- oprecipitated with the RyR in both WT (Figure 4A, lane 1) and triadin skeletal muscle microsomes (Figure 4A, lane 3), de- monstrating that triadin was not necessary for this association and that the RyR-Cav-3 interaction could be direct. To confirm the direct interaction of the RyR and Cav-3, immunoprecipitation