The Functional Expression of the Lamin A/C Mutant Q517X in HL1 Cardiomyocytes Induces Electrophysiological Impairments Through Impaired Ca2+‐Homeostasis at the Endoplasmic Reticulum.

Abstract

Mutations in the LMNA gene, which encodes A‐type nuclear Lamins, are among the most frequent genetic cause of dilated cardiomyopathy (DCM). We identified a novel LMNA nonsense mutation (Q517X) co‐segregating with DCM and permanent atrial fibrillation. Aim of this study is to gain insights into the unknown pathogenic mechanism induced by the expression of Q517X at cellular level.HEK293 cells and HL‐1 cardiomyocytes were transiently transfected with either Lamin A or Q517X both tagged with mCherry. Q517X expression, localization and its effects on diverse cellular mechanisms were evaluated by western blotting, laser‐scanning confocal microscopy and real time FRET/imaging analysis in single cell. Also, patch clamp experiments were used to evaluate the effect of Q517X expression on HL‐1 spontaneous action potentials (APs).When expressed in both cell types, Q517X mislocalized within the nucleoplasm where it clustered in aggregates of different sizes. Emerin was also irregularly distributed along the nuclear envelope probably indicating a general impairment of the inner nuclear membrane organization upon Q517X expression. HL‐1 cardiomyocytes expressing the mutant showed a significant reduction in APs frequency induced by an increased duration of both single APDs (APD100) and cycle length (i.e. the distance between two sequential APDs’ thresholds). In addition, after depolarizations events were constantly recorded in cells expressing the mutant. When we measured intracellular Ca2+ level in cardiomyocytes expressing either LMNA wild type or Q517X we found similar patterns of oscillation. However, in the absence of electrical activity at the plasma membrane, cardiomyocytes expressing the mutant showed spontaneous Ca2+ spikes, in free‐extracellular Ca2+, likely induced by abnormal release by the endoplasmic reticulum (ER) as directly confirmed by steady state FRET experiment with the ER Ca2+ probe ERD1.So far, these results depict a pathogenic mechanism in which the exogenous expression of Q517X changed the geometry of emerin within the nucleus and altered the electrical activity at the plasma membrane by probably depleting ER Ca2+ content.Support or Funding InformationThis research was supported by funding from the ‘CLUSTER TECNOLOGICO REGIONALE DICLIMAX’ (project # MTJU9H8)