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

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

Author information

1
Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, Bari, Italy.
2
Medical Genetics, Department of Molecular Medicine, Sapienza University, San Camillo-Forlanini Hospital, Rome, Italy.
3
Cardiology Division, Cardiac Arrhythmia Center and Cardiomyopathies Unit, San Camillo-Forlanini Hospital, Rome, Italy.
4
Department of Sciences, University of Basilicata, Via dell'Ateneo Lucano, Potenza, Italy.

Abstract

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

PMID:
29197877
DOI:
10.1159/000485651
[Indexed for MEDLINE]
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