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

Cell Physiol Biochem. 2017;44(4):1559-1577. doi: 10.1159/000485651. Epub 2017 Dec 4.


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.D243Gfs*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 D243Gfs*4 tagged with GFP (or mCherry). D243Gfs*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 D243Gfs*4 colocalized with calnexin within the ER. ER mislocalization of LMNA D243Gfs*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 D243Gfs*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, D243Gfs*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-D243Gfs*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 D243Gfs*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.

Keywords: Ca2+ signaling; Cardiomyocytes; Connexin; Endoplasmic reticulum; Lamin A/C gene; Laminopathies; Nucleus.

MeSH terms

  • Apoptosis
  • Base Sequence
  • Calcium / metabolism
  • Calnexin / metabolism
  • Cardiac Conduction System Disease / complications
  • Cardiac Conduction System Disease / genetics*
  • Cardiac Conduction System Disease / pathology
  • Cardiomyopathies / complications
  • Cardiomyopathies / genetics*
  • Cardiomyopathies / pathology
  • Cell Line
  • Connexin 43
  • Endoplasmic Reticulum / metabolism
  • Female
  • Gap Junctions / metabolism
  • HEK293 Cells
  • Humans
  • Lamin Type A / genetics*
  • Lamin Type A / metabolism
  • Microsatellite Repeats / genetics
  • Microscopy, Confocal
  • Middle Aged
  • Mutagenesis, Site-Directed
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / metabolism
  • Pedigree
  • Polymorphism, Single Nucleotide
  • Time-Lapse Imaging
  • Wnt Signaling Pathway


  • Connexin 43
  • LMNA protein, human
  • Lamin Type A
  • Calnexin
  • Calcium