Abstract 1926: Mutant Lamin A/C Expression Mediates Mutation-Specific Alteration of Sumoylation in Murine Myoblasts and in Myocytes From the LMNAH222P/H222P Mouse
Lamins A and C are major components of the nuclear lamina and are important for nuclear shape, strength, protein scaffolding, chromatin binding and have roles in transcription, cell cycle and anchoring nuclear pores. LMNA encodes the alternatively spliced transcript that gives rise to lamin A/C and mutations are implicated in over ten human disease phenotypes that include dilated cardiomyopathy (DCM), Emery-Dreifuss muscular dystrophy, and premature aging. Previous studies have shown that in vitro expression of mutant lamin A/C can result in its aggregation and one mutant was found to sequester the post-translational modification protein, sumo1, inside lamin C aggregates. Conjugation of sumo1 to target proteins (sumoylation) regulates substrate localization, activity, and stability. We hypothesized that lamin A/C is sumoylated and that mutant lamin A/C may affect the regulation of sumoylation. We transfected mouse myoblasts with fluorescently tagged wild-type and myopathy-related lamin A/C mutants and wild-type sumo1. We did not detect sumoylation of lamin A/C however we observed a mutation-dependent aggregation of lamins and the mislocalization of sumo1 with or inside the aggregates. Western blotting showed an increase in both sumoylated proteins and non-conjugated sumo1 in aggregating mutants. This suggests defective de-sumoylation of modified proteins and deficient turnover of the sumo1 protein mediated by the sequestration in mutant lamin A/C aggregates. The LMNAH222P/H222P knock-in mouse model of laminopathy develops late-onset muscular dystrophy and DCM comparable to the human phenotype. Primary myoblasts transfected with sumo1 showed an increase in non-conjugated sumo1 as well as the sumoylation of one unknown protein. Cross-sections of affected skeletal muscle showed a striking endogenous sumo1 staining at the nuclear envelope in a sub-set of nuclei from mutant mice that was not observed in the wild-type. As many sumoylation enzymes are targeted there, this suggests a possible conjugation deficiency mediated by defective lamin A/C protein scaffolds. Our results support that mutation-dependent effects on sumoylation may contribute to the variability in tissue specific phenotypes associated with LMNA mutations.