Functional Characterization of a Novel Truncating Mutation in Lamin A/C Gene in a Family with a Severe Cardiomyopathy with Conduction Defects

Background/Aims: Truncating LMNA gene mutations occur in many inherited cardiomyopathy cases, but the molecular mechanisms involved in the disease they cause have not yet been systematically investigated. Here, we studied a novel frameshift LMNA variant (p.D243Gfsâ 4) identified in three members of an Italian family co-segregating with a severe form of cardiomyopathy with conduction defects. Methods: HEK293 cells and HL-1 cardiomyocytes were transiently transfected with either Lamin A or D243Gfsâ 4 tagged with GFP (or mCherry). D243Gfsâ 4 expression, cellular localization and its effects on diverse cellular mechanisms were evaluated with western blotting, laser-scanning confocal microscopy and video-imaging analysis in single cells. Results: When expressed in HEK293 cells, GFP- (or mCherry)-tagged LMNA D243Gfsâ 4 colocalized with calnexin within the ER. ER mislocalization of LMNA D243Gfsâ 4 did not significantly induce ER stress response, abnormal Ca2+ handling and apoptosis when compared with HEK293 cells expressing another truncated mutant of LMNA (R321X) which similarly accumulates within the ER. Of note, HEK293-LMNA D243Gfsâ 4 cells showed a significant reduction of connexin 43 (CX43) expression level, which was completely rescued by activation of the WNT/β-catenin signaling pathway. When expressed in HL-1 cardiomyocytes, D243Gfsâ 4 significantly impaired the spontaneous Ca2+ oscillations recorded in these cells as result of propagation of the depolarizing waves through the gap junctions between non-transfected cells surrounding a cell harboring the mutation. Furthermore, mCh-D243Gfsâ 4 HL-1 cardiomyocytes showed reduced CX43-dependent Lucifer Yellow (LY) loading and propagation. Of note, activation of β-catenin rescued both LY loading and LMNA D243Gfsâ 4 -HL-1 cells spontaneous activity propagation. Conclusion: Overall, the present results clearly indicate the involvement of the aberrant CX43 expression/activity as a pathogenic mechanism for the conduction defects associated to this LMNA truncating alteration.