Skeletal muscle adapts to physiological and pathological signals by undergoing remodeling. To sustain muscle performance, myofibers hypertrophy (for example, due to exercise training) and increase muscle bulk through elevated protein synthesis and addition of nuclei by satellite cell-mediated fusion. Muscle protein loss or atrophy in response to cancer cachexia, food deprivation, denervation, or inactivity is accomplished primarily through the ubiquitin proteasome pathway. Two muscle-specific E3 ligases, TRIM63 and FBXO32, have been shown to be major contributors to skeletal muscle atrophy through the ubiquitin proteasome pathway (17
). Since TRIM63 is a TRIM family member, it was logical to presume that TRIM32 might perform a similar biological role as TRIM63, especially since TRIM32 was shown to bind to myosin and ubiquitinate actin (2
). In this investigation, we examined the role of TRIM32 in muscle atrophy and growth. Our data demonstrate that, unexpectedly, TRIM32 does not play a role in protein degradation processes that take place during muscle atrophy caused by either food deprivation or inactivity. Rather, TRIM32 appears to play an important role in muscle growth after disuse atrophy. The process of muscle growth requires activation, proliferation, and differentiation of muscle satellite cells. Our data demonstrate that TRIM32 deficiency leads to premature senescence of myogenic cells, which become unable to provide adequate muscle growth in response to growth-demanding stimuli. Premature senescence contributes to satellite cell proliferation and differentiation defects in Trim32–/–
myogenic cultures, leading to impaired myotube formation. These findings are in agreement with the observations that senescent myoblasts are able to fuse less well and more slowly, forming thinner myotubes with fewer nuclei (55
). Whether aberrant differentiation in Trim32–/–
myoblasts is caused solely by senescence or involves additional mechanisms is yet to be explored; however, these results provide what we believe to be the first suggestion that senescence in myoblasts could underlie pathogenic mechanisms in LGMD2H.
Our data link the E3 ubiquitin ligase TRIM32 and myoblast senescence via the E3 SUMO ligase PIAS4, which accumulates in myoblasts and skeletal muscle tissue in the absence of TRIM32. Similar to fibroblasts overexpressing PIAS4 (21
), accumulation of PIAS4 in Trim32–/–
myoblasts also leads to premature senescence. TRIM32 and PIAS4 have been shown to occupy different cellular compartments under basal conditions: TRIM32 is mainly a cytoplasmic protein, while PIAS4 is primarily nuclear (19
). However, both proteins can shuttle between the cytoplasm and nucleus: while PIAS4 translocation from nucleus to cytoplasm seems to occur only upon cellular stress conditions (19
), recently published data ascertain that TRIM32 can be relocated from the cytoplasm to the nucleus upon interaction with defined types of E2 ubiquitin-conjugating enzymes, such as UBE2N and UBE2V2 (57
). Strikingly, these E2 enzymes have been implicated in the cell cycle progression and cellular responses to DNA damage upon genotoxic stress (58
). These data not only open up the possibility for an in vivo interaction between TRIM32 and PIAS4, but also suggest a fine adjustment for such an interaction in the cell.
Interestingly, our data demonstrated that PIAS4 concentration increases during unloading and increases even further after reloading of Trim32+/+
murine skeletal muscles. This observation is in agreement with a recent report of global gene expression patterns after 48 hours of unloading and 24 hours of reloading in humans (60
). It was observed that human PIAS
gene expression was upregulated in both unloaded and reloaded muscles; however, this finding is yet to be addressed with regard to its role in skeletal muscle remodeling.
The sumoylation pathway has been implicated in the process of cellular senescence (61
). There are several SUMO isoforms in mammals that differ in structure and functional activity. While SUMO-2 and SUMO-3 are 95% identical to each other (and therefore are referred to as SUMO-2/3), SUMO-1 shares only 50% sequence identity with SUMO-2/3. Moreover, in contrast to SUMO-1, SUMO-2/3 can form poly-SUMO chains on a target protein (62
). Under normal conditions SUMO-1 is mainly conjugated to target proteins, while free unconjugated pools of SUMO-2/3 are abundant and ready to be used under stress conditions. The E3 SUMO ligase PIAS4 is able to use all SUMO isoforms in sumoylation reactions (63
). Interestingly, global levels of both SUMO-1 and SUMO-2/3 are increased in Trim32–/–
myoblasts, though only SUMO-2/3 has been implicated in cellular senescence (23
). Despite the structure/functional dissimilarities between the isoforms, sumoylation is widely regarded as a protective mechanism against cellular stresses such as heat shock, hypoxia, and osmotic and high oxidative stress, upon which global levels of SUMO-1 (68
) and SUMO-2/3 (69
) are elevated. Thus, elevated levels of both SUMO-1 and SUMO-2/3 conjugation suggest that Trim32–/–
cells may experience additional cellular stresses besides senescence.
It is noteworthy that, in contrast to sumoylation, total ubiquitination levels are not perturbed in the absence of TRIM32, neither under normal nor under pathological conditions (Figure C and Figure A), suggesting a highly selective role for TRIM32’s E3 ubiquitin ligase activity, which has been implicated in a number of diverse biological processes and most likely functions in a cell type–dependent manner. A controversial role for TRIM32 in apoptosis has recently emerged in studies using different cell models. TRIM32 has been shown to be elevated in epidermal and epithelial tumorigenic cells, where its antiapoptotic and carcinogenic roles have been discovered (19
). However, TRIM32 overexpression/knockdown experiments in established cell culture lines have suggested a proapoptotic, tumor suppressive role for TRIM32 (40
). Our data demonstrate that apoptosis is not perturbed in TRIM32-deficient skeletal muscle and that it is not an essential pathogenic feature of Trim32–/–
myopathy. In support of our finding, no signs of apoptosis were found in the muscle biopsies from patients with LGMD2H/STM (8
Skeletal muscle is a dynamic tissue capable of regeneration and remodeling. Its plasticity depends on satellite cell function. Loss of skeletal muscle mass (sarcopenia) progresses with age at a rate of 0.5%–1% a year and is associated with failure of the regeneration machinery to replace damaged muscle fibers (70
). Factors other than aging that lower the ability of satellite cells to replicate and/or differentiate will contribute to premature sarcopenia. The importance of normal replicative capacity of satellite cells for effective skeletal muscle maintenance has recently emerged. The abrogation of age-related atrophy by satellite cell transplantation was demonstrated to arise from an increased regenerative capacity of donor stem cells (73
). Moreover, the introduction of a null mutation in the telomerase gene dramatically exacerbates muscular dystrophy in the mdx mouse, a model for Duchenne muscular dystrophy (74
). Lack of telomerase activity in this model results in decreased proliferative ability of satellite cells with shortened telomeres, leading to their reduced regenerative capacity. These data further emphasize the importance of replicative senescence of satellite cells in the pathogenesis of muscular dystrophy. In Duchenne muscular dystrophy, however, sarcopenia represents a secondary mechanism of satellite cell exhaustion, as it occurs due to the large number of cycles of degeneration/regeneration. In contrast, sarcopenia seems to play a primary role in the pathogenesis of LGMD2H due to TRIM32 deficiency, through accumulation of PIAS4, leading to premature myoblast senescence and resulting in inadequate adaptive muscle growth. Even though the majority of the LGMD2H pathogenic mutations in TRIM32 are point mutations, at least some of these mutations result in TRIM32 loss in vivo (7
). Concordantly, our recently published data analyzing a knock-in mouse carrying the common D489N TRIM32 mutation revealed a dramatic reduction of mutated TRIM32 at the protein level, which leads to a muscle pathology similar to the myopathy described for the Trim32
-null mouse (75
). Therefore, at least in some cases, LGMD2H can arise from insufficiency of TRIM32 due to protein instability caused by pathogenic mutations. Thus, our data not only reveal a role for TRIM32 in implementing myoblast senescence in skeletal muscles but also suggest a pathogenic mechanism of muscular dystrophy that involves accumulation of PIAS4 and satellite cell senescence in vivo, leading to premature sarcopenia.