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. 2012 Aug 20;198(4):575-89.
doi: 10.1083/jcb.201110067.

Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy

Shenhav Cohen  1 , Bo Zhai, Steven P Gygi, Alfred L Goldberg
Affiliations

Ubiquitylation by Trim32 causes coupled loss of desmin, Z-bands, and thin filaments in muscle atrophy

Shenhav Cohen et al. J Cell Biol. .

Abstract

During muscle atrophy, myofibrillar proteins are degraded in an ordered process in which MuRF1 catalyzes ubiquitylation of thick filament components (Cohen et al. 2009. J. Cell Biol. http://dx.doi.org/10.1083/jcb.200901052). Here, we show that another ubiquitin ligase, Trim32, ubiquitylates thin filament (actin, tropomyosin, troponins) and Z-band (α-actinin) components and promotes their degradation. Down-regulation of Trim32 during fasting reduced fiber atrophy and the rapid loss of thin filaments. Desmin filaments were proposed to maintain the integrity of thin filaments. Accordingly, we find that the rapid destruction of thin filament proteins upon fasting was accompanied by increased phosphorylation of desmin filaments, which promoted desmin ubiquitylation by Trim32 and degradation. Reducing Trim32 levels prevented the loss of both desmin and thin filament proteins. Furthermore, overexpression of an inhibitor of desmin polymerization induced disassembly of desmin filaments and destruction of thin filament components. Thus, during fasting, desmin phosphorylation increases and enhances Trim32-mediated degradation of the desmin cytoskeleton, which appears to facilitate the breakdown of Z-bands and thin filaments.

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Figures

Figure 1.
Figure 1.
Trim32 is required for muscle atrophy induced by fasting. TA muscles were electroporated with shRNA vectors against Trim32 (shTrim32) or Lacz (shLacz), and 4 d later mice were deprived of food for 2 d. In fed mice, muscles were dissected 6 d after electroporation. Electroporation of shLacz into fibers of fasted or fed control mice did not affect fiber size (Sandri et al., 2004). (A) shRNA-mediated knockdown of Trim32 in HEK293 cells (top) and muscles from fasted animals (bottom). Soluble extracts were analyzed by immunoblotting. (B) Down-regulation of Trim32 in muscles from fasted mice reduces fiber atrophy. Measurement of cross-sectional area of 500 fibers transfected with shTrim32 (and expressing GFP; black bars) vs. 500 nontransfected fibers (open bars) in the same muscle. Data acquired from six mice. Green fibers express shTrim32 and laminin staining in red. Bar, 75 µM. (C) Down-regulation of Trim32 attenuates the loss of muscle mass during fasting. shTrim32 was delivered to more than 70% of muscle fibers. Mean weights of electroporated muscles are plotted as the percent weight loss. n = 6. *, P < 0.001 vs. fed control; #, P < 0.05 vs. shLacz (see also data in Fig. 5 B). (D) Down-regulation of Trim32 prevents the loss of thin and slightly reduced the loss of thick filament components during fasting. Left: isolated myofibrils from shLacz- (shaded) or shTrim32-expressing muscles (open) were analyzed by SDS-PAGE and Coomassie blue staining. To obtain the absolute content of each myofibrillar protein, densitometric measurement of specific bands was performed, and the values were multiplied by the total amount of myofibrils per muscle and then by muscle weight. The content of each myofibrillar protein is presented as the percentage of fed control. n = 6. *, P < 0.05 vs. fed control; #, P < 0.05 vs. shTrim32. Right: equal fractions of myofibrils were analyzed by Western blot using anti-actin and anti-MyHC. (E) Trim32 is not induced upon fasting. Top: soluble fraction of muscles, 1 or 2 d after food deprivation, were analyzed by SDS-PAGE and immunoblotting. Bottom: quantitative RT-PCR of mRNA preparations from atrophying and control muscles using primers for MuRF1 and Trim32. Data are plotted as the mean fold change relative to control. n = 6.
Figure 2.
Figure 2.
Actin and other myofibrillar proteins are substrates for Trim32. (A) In vitro ubiquitylation of actin, tropomyosin (Tm), and MyLC by Trim32 was analyzed by immunoblotting with specific antibodies. Asterisk represents nonspecific bands. (B) Muscle extracts contain soluble forms of actin, α-actinin, Tm, MyHC, MyLC2, and MyBP-C, whose loss requires Trim32 during fasting. Soluble fractions of normal and atrophying muscles expressing shLacz or shTrim32 were analyzed by SDS-PAGE, and immunoblot analysis using antibodies against the indicated proteins. Each lane is a separate muscle, and representative bands were chosen based on similar atrophy rates in transfected muscles.
Figure 3.
Figure 3.
Desmin is lost during atrophy induced by fasting. (A) Paraffin-embedded longitudinal sections of TA muscle from fed mice and ones deprived of food (2 d) were stained with an antibody against desmin. Bar, 25 µM. (B) During fasting, depolymerization of desmin filaments is prevented in muscle fibers expressing Trim32-DN. Paraffin-embedded longitudinal sections of TA muscles expressing Trim32-DN (green) from fasted mice were stained with an antibody against desmin (red). Desmin filaments are degraded in two fibers, which do not express Trim32-DN, at the two opposite edges of the image. Expression of Trim32-DN in the three fibers in the center of the image markedly attenuated the loss of desmin. Bar, 25 µM.
Figure 4.
Figure 4.
Trim32 promotes disassembly and destruction of phosphorylated desmin filaments. (A) Desmin filaments are phosphorylated during fasting. Top: the pellet from atrophying muscle expressing shTrim32 was analyzed by isoelectric focusing 2D gel electrophoresis and Coomassie blue staining. Four spots were observed at ∼53 kD and were identified by mass spectrometry as vimentin and desmin phosphorylated at serines 28, 32, and 68. Bottom: the three phosphorylated serine residues are located in desmin’s head domain. (B) Trim32 promotes disassembly and destruction of phosphorylated desmin filaments during fasting. Left: desmin filaments were isolated from normal and atrophying muscles expressing shLacz or shTrim32, and analyzed in parallel to the soluble fraction by SDS-PAGE and immunoblotting. Phosphorylated desmin was detected with phospho-serine antibody. The AKT blot serves as a loading control. Right: densitometric measurement of blots which were stained for phospho-serine. *, P < 0.05 shTrim32 vs. fed control; #, P < 0.005 shTrim32 vs. shLacz in fasting; n = 13. (C) During fasting, desmin accumulates as a phosphorylated species in the cytosol of muscles deficient in active Trim32. Desmin was immunoprecipitated from the soluble fraction of muscles expressing shLacz or dominant-negative to Trim32 (Trim32-DN) from fed or fasted mice. Precipitates were analyzed by immunoblotting using anti-desmin and phospho-serine. (D) Trim32 ubiquitylates desmin during fasting in vivo. Desmin filaments, which were purified from normal or atrophying muscles expressing shLacz or Trim32-DN, were analyzed in parallel to the soluble fraction by Western blot analysis using a desmin antibody. (E) Phosphorylation of desmin filaments facilitates their ubiquitylation by Trim32. Isolated desmin filaments from normal (lanes 2 and 3) and atrophying muscles expressing shLacz (lanes 4 and 5) or Trim32-DN (lanes 6–11) were treated with protein phosphatase 1 (PP1; lanes 9–11) or left untreated (lanes 6–8) and then subjected to ubiquitylation by pure Trim32 and UbcH5 using 6His-tagged ubiquitin. The 6His-tagged ubiquitin conjugates were purified with a Ni column and analyzed by SDS-PAGE and immunoblotting using anti-desmin. The band above the dashed line, which is also marked with an asterisk, is nonspecific.
Figure 5.
Figure 5.
Depolymerization 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 fasting in muscles expressing Trim32-DN. Isolated desmin filaments and the soluble fraction from transfected muscles were analyzed by SDS-PAGE and immunoblotting. Representative bands were chosen based on similar atrophy rates in transfected muscles. Ponceau S staining is shown as a loading control for the pSER membrane. (B) The ability of Trim32-DN to protect against loss of muscle mass is not affected by the coexpression of desmin-DN. Mean weights of electroporated muscles are plotted as percent weight loss. n = 6. *, P < 0.005. (C) Depolymerization of desmin filaments by Desmin-DN during fasting reduces the sparing of thin filament proteins and MyHC by Trim32-DN. Left: myofibrils purified from atrophying muscles expressing shLacz alone (black bars), Trim32-DN together with shLacz (shaded bars), or Trim32-DN together with Desmin-DN (open bars) were analyzed by SDS-PAGE and Coomassie blue staining. The absolute content of each myofibrillar protein was obtained as described in Fig. 1 D. Data are expressed as percentages of fed control. n = 10, *, P < 0.05 vs. fed control; #, P < 0.05 vs. Trim32-DN/Lacz; §, P < 0.05 vs. Trim32-DN/Lacz. Right: equal fractions of myofibrils from different muscle samples were analyzed by immunoblotting using anti-actin and anti-MyHC. Ponceau S staining is shown as a loading control for each membrane. (D) Electroporation of desmin-DN into normal muscle to promote desmin disassembly does not trigger destruction of thin filaments. Myofibrils from normal muscles expressing desmin-DN were analyzed by SDS-PAGE and Coomassie blue staining, and the absolute content of each protein was calculated as in Fig. 1 D. Data are presented as percentage of control (expressing shLacz). n = 6. (E) During fasting, loss of desmin precedes degradation of myofibrillar proteins and Z-bands. Time course of the loss of desmin and myofibrillar proteins was analyzed by immunoblotting of equal amounts of myofibrils from TA muscles of fed mice and ones deprived of food for 1 or 2 d. Representative bands were chosen based on similar atrophy rates. Ponceau S staining is shown as a loading control.
Figure 6.
Figure 6.
Proposed mechanism for thin filament loss during atrophy. Upon fasting, when insulin and IGF-1 levels are low and glucocorticoid levels rise, there is an enhanced phosphorylation of desmin filaments leading to ubiquitylation by Trim32 (Fig. 4), disassembly, and degradation (Fig. 3). Because desmin filaments are important for the stability of the Z-band (Fuseler and Shay, 1982) and the attached thin filaments (Conover et al., 2009), this loss of desmin filaments probably loosens the structure of the Z-band and reduces the stability of thin filaments (Fig. 6). As a result, Z-band components (e.g., α-actinin) as well as actin and associated thin filament proteins should be more susceptible to ubiquitylation by Trim32.
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