S2 shows that after incubation of muscle mass pellet with 0.6 M KCl myofibrils almost fully disassemble, whereas desmin filaments are stable. presented as the percentage of fed control. = 6. *, P 0.05 vs. fed control; #, P 0.05 vs. shTrim32. Right: equivalent fractions of myofibrils were analyzed by Western blot using anti-actin and anti-MyHC. (E) Trim32 is not induced upon fasting. Top: soluble portion of muscle tissue, 1 or 2 2 d after food deprivation, were analyzed by SDS-PAGE and immunoblotting. Bottom: quantitative RT-PCR of mRNA preparations from atrophying and control muscle tissue using primers for MuRF1 and Trim32. Data are plotted as the mean fold change relative to control. = 6. Trim32 is necessary for the loss of thin filaments To learn if Trim32 catalyzes the loss of thin filament proteins from your myofibril, we analyzed the effect of Trim32 down-regulation on the total content of thin filament components upon fasting. Equivalent amounts of isolated myofibrils from muscle tissue transfected with shTrim32 or shLacz were analyzed by SDS-PAGE and Coomassie blue staining, and the intensity of VS-5584 specific protein bands was measured by densitometry. Previously, we recognized these different protein bands by mass spectrometry (Cohen et al., 2009; Fig. S1 C). To determine the absolute content of each myofibrillar protein in the muscle mass, the density of each band was multiplied by the total amount of myofibrillar proteins per muscle mass and then by the total muscle mass weight (observe Materials and methods). The total content of each myofibrillar component in the atrophying muscle mass was then expressed as the percentage of this proteins content in the corresponding muscle tissue in the fed mice (Fig. 1 D). The content of each major thin filament component, actin, tropomyosin (Tm), troponin I (TnI), and troponin T (TnT), and the Z-band protein VS-5584 -actinin decreased by more than 40% in the contralateral atrophying muscle tissue (expressing shLacz) below levels in control muscle tissue from fed animals (Fig. 1 D). This loss of myofibrillar proteins exceeded the relative loss of muscle mass; thus, these components decreased in fasting to a greater extent than the bulk of cell proteins and especially the soluble proteins. Trim32 down-regulation by transfection of shRNA blocked the loss of thin filament proteins and -actinin, whose content no longer differed significantly from that in muscle tissue of fed controls. By contrast, the shTrim32 only slightly reduced the loss of solid filament components, myosin heavy (MyHC) and light (MyLC2) chains and binding protein C (MyBP-C), which decreased by more than 40% (Fig. 1 D, Table S1). This selective sparing of thin filament components was further supported by Western blot analysis of actin and MyHC in equal fractions of myofibrils from transfected muscles (Fig. 1 D, right). Thus, upon fasting, Trim32 plays a critical role in the rapid atrophy, especially in the breakdown of thin filament components. In fact, the sparing of these proteins by shTrim32 in Fig. 1 D must underestimate the protective effects of Trim32 down-regulation because only about half the fibers were transfected. It is noteworthy that neither Trim32 protein nor mRNA increased upon fasting (Fig. 1 E), although this enzyme is clearly essential for the loss of muscle mass (Fig. 1, B and C; and see Fig. 5 B). Open in a separate window Figure 5. Depolymerization VS-5584 of desmin filaments promotes the loss of thin filaments during fasting. To test if disassembly of desmin filaments influences the stability of thin filaments, TA muscles were co-electroporated with Trim32-DN and either shLacz or a dominant-negative mutant of desmin (Desmin-DN) to induce filament disassembly. 4 d later, animals were deprived of food for 2 d. (A) Desmin-DN enhances disassembly of desmin filaments during Hsh155 fasting in muscles expressing Trim32-DN. Isolated desmin filaments and the soluble fraction from transfected.