Role of TRIM32, the ubiquitin ligase mutated in Limb Girdle Muscular Dystrophy R8

Mutations in TRIM32 result in the inherited disease Limb-girdle muscular dystrophy Recessive 8 (LGMDR8), a neuromuscular disorder manifesting in the proximal muscles around the hips and shoulders. TRIM32 is a RING E3 ligase belonging to the tripartite motif family (TRIM), which functions in the last step of ubiquitination, transferring the ubiquitin from E2 conjugating enzyme to specific substrates based on its ability to recognize different proteins. In literature, some studies report the critical role of TRIM32 in muscle physiology, muscle atrophy, muscle regeneration, as well as neuronal differentiation, however the exact LGMDR8 pathogenic mechanism is still unclear. Moreover, several possible substrates and interactors of TRIM32 have been found in these studies. On the other hand, evidence support that the pathogenic mutations on TRIM32 result in the loss function of TRIM32. Not only the full-length deletion of TRIM32 has been discovered in LGMDR8 patients, but also the Trim32 knock-out and Trim32 pathogenic mutation knock-in mice share similar neuromuscular phenotypes. Therefore, the impaired myogenic process is very likely caused by the loss function of TRIM32 as the mechanism underlying LGMDR8.  In this project, we used an immortalized mouse myoblast cell line C2C12 to generate the Trim32 knock-out and wild-type clones. Using the gene-edited C2C12 cells as the research tool, we determined the impairment of the myogenic program caused by the loss function of TRIM32, including low activity of myogenic-relative signaling pathways, the delayed and abnormal myotubes formation, and the down regulation of myogenesis marker myosin heavy chain (MHC). This deficient myogenesis is not caused by the malfunction of MyoD, a master myogenic regulatory factor (MRF), but likely due to the downregulation of myogenin, which is a downstream target of MyoD predominately expressed at the late stage of differentiation. At last, we identify c-Myc, a proto-oncogene and antagonist of MyoD activity on myogenin expression, as likely substrate of TRIM32 at the beginning of differentiation. Taken together, these results strongly indicate that TRIM32 functions is crucial in the myogenic differentiation program at its onset and provide new insight into LGMDR8 pathogenic mechanisms.