Emery-Dreifuss Muscular Dystrophy-Associated Mutant Forms of Lamin A Recruit the Stress Responsive Protein Ankrd2 into the Nucleus, Affecting the Cellular Response to Oxidative Stress

Abstract

Background: Ankrd2 is a stress responsive protein mainly expressed in muscle cells. Upon the application of oxidative stress, Ankrd2 translocates into the nucleus where it regulates the activity of genes involved in cellular response to stress. Emery-Dreifuss Muscular Dystrophy 2 (EDMD2) is a muscular disorder caused by mutations of the gene encoding lamin A, LMNA. As well as many phenotypic abnormalities, EDMD2 muscle cells also feature a permanent basal stress state, the underlying molecular mechanisms of which are currently unclear. Methods: Experiments were performed in EDMD2-lamin A overexpressing cell lines and EDMD2-affected human myotubes. Oxidative stress was produced by H₂O₂ treatment. Co-immunoprecipitation, cellular subfractionation and immunofluorescence analysis were used to validate the relation between Ankrd2 and forms of lamin A; cellular sensibility to stress was monitored by the analysis of Reactive Oxygen Species (ROS) release and cell viability. Results: Our data demonstrate that oxidative stress induces the formation of a complex between Ankrd2 and lamin A. However, EDMD2-lamin A mutants were able to bind and mislocalize Ankrd2 in the nucleus even under basal conditions. Nonetheless, cells co-expressing Ankrd2 and EDMD2-lamin A mutants were more sensitive to oxidative stress than the Ankrd2-wild type lamin A counterpart. Conclusions: For the first time, we present evidence that in muscle fibers from patients affected by EDMD2, Ankrd2 has an unusual nuclear localization. By introducing a plausible mechanism ruling this accumulation, our data hint at a novel function of Ankrd2 in the pathogenesis of EDMD2-affected cells.

Samp1 Mislocalization in Emery-Dreifuss Muscular Dystrophy

“…In fact, double immunolabeling of Samp1 and laminin alpha 2, which lines the basal lamina of myofibers, demonstrated that nuclei inside control muscle fibers were Samp1 positive and the protein was located at the nuclear periphery, which was previously reported in Reference [ 30 ], while interstitial nuclei presented a faint staining ( Figure 4 d). In EDMD2 myonuclei bearing the H506P LMNA mutation [ 32 ], Samp1 was mis-localized in the vast majority (80%) of examined myofibers ( Figure 4 d) possibly due to a loss of interaction with farnesylated prelamin A, which is dramatically reduced in EDMD2 muscle fibers [ 15 ].…”

“…In the EDMD2 myoblasts used in this study, the R190Q/R249Q LMNA mutations had been determined in a single allele [ 31 ]. Moreover, we used myoblasts from an EDMD2 patient harboring the H506P LMNA mutation [ 32 ] and a muscular dystrophy linked to the compound heterozygous P68D/G338S SUN1 mutation [ 17 ]. Accumulation of non-farnesylated prelamin A was obtained after 18 h of treatment of differentiating myoblasts with 20 µM Mevinolin (Sigma).…”

Elevated TGF β2 serum levels in Emery-Dreifuss Muscular Dystrophy: Implications for myocyte and tenocyte differentiation and fibrogenic processes

“…Human myoblast and fibroblast cultures were obtained from healthy donors, LGMD1B patients carrying the Y259D LMNA mutation [ 6 ] or EDMD2 patients bearing the H506P LMNA mutation [ 35 ]. Tenocytes were established from biopsies of healthy donors undergoing orthopaedic surgery.…”

PCAF Involvement in Lamin A/C-HDAC2 Interplay during the Early Phase of Muscle Differentiation

“…However, prelamin A has also been shown to regulate early events of myoblast differentiation, as caveolin 3 expression [ 16 ] and nuclear positioning in human muscle cells [ 17 ]. We also demonstrated that lamin A and prelamin A are complexed to Ankrd2, a protein involved in the transcriptional response to stress during myoblast differentiation [ 18 , 19 ]. LMNA gene mutations are responsible for the onset of autosomal dominant Emery–Dreifuss muscular dystrophy (EDMD2), which is characterized by skeletal muscle weakness and wasting, joint contractures and cardiomyopathy [ 18 , 19 ].…”