Lamin A mutation impairs interaction with nucleoporin NUP155 and disrupts nucleocytoplasmic transport in atrial fibrillation

Abstract

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Here, we show the identification and functional characterization of one AF-associated mutation p.Arg399Cys in lamin A/C. Co-immunoprecipitation and GST pull-down assays demonstrate that lamin A/C interacts with NUP155, which is a nucleoporin and causes AF when mutated. Lamin A/C mutation p.Arg399Cys impairs the interaction between lamin A/C and NUP155, and increases extractability of NUP155 from the nuclear envelope (NE). Mutation p.Arg399Cys leads to aggregation of lamin A/C in the nucleus, although it does not impair the integrity of NE upon cellular stress. Mutation p.Arg399Cys inhibits the export of HSP70 mRNA and the nuclear import of HSP70 protein. Electrophysiological studies show that mutation p.Arg399Cys decreases the peak cardiac sodium current by decreasing the cell surface expression level of cardiac sodium channel Na_v 1.5, but does not affect I_Kr potassium current. In conclusion, our results indicate that lamin A/C mutation p.Arg399Cys weakens the interaction between nuclear lamina (lamin A/C) and the nuclear pore complex (NUP155), leading to the development of AF. The findings provide a novel molecular mechanism for the pathogenesis of AF.

Keywords: LMNA; NUP155; SCN5A/Nav1.5; atrial fibrillation (AF); mutation.

FIGURE 1

Identification of the p.Arg399Cys mutation in LMNA associated with AF. (a) DNA sequences of LMNA from an affected patient and a normal individual. A heterozygous missense mutation (c.1195C>T, p.Arg399Cys) was identified in the affected patient. (b) Multiple amino acid sequence alignment of lamin A proteins. The p.Arg399Cys mutation occurs at an arginine (R) residue that is evolutionarily conserved. (c) Schematic structure of the LMNA gene and lamin A protein. The p.Arg399Cys mutation was found in exon 7. NLS, nuclear localization signal

FIGURE 2

Mutation p.Arg399Cys affects the localization of lamin A. (a) Immunofluorescence analysis of EGFP-tagged WT or p.Arg399Cys lamin A expressed in HL-1 cells. WT lamin A was correctly localized to the nuclear periphery in the majority of the cells, while p.Arg399Cys showed a higher frequency of a “foci” phenotype. Data are presented as mean ± SEM, *P < 0.05. (b) Analysis of lamin B1 localization in cells expressing WT or mutant lamin A with p.Arg399Cys. EGFP-tagged WT or p.Arg399Cys mutant LMNA plasmid was transfected into HL-1 cells. The p.Arg399Cys mutation did not disturb the distribution of endogenous lamin B1 on the nuclear rim. Scale bar: 10 μm

FIGURE 3

Lamin A interacts with NUP155. (a) Co-immunoprecipitation (Co-IP) assays. HeLa cells were transiently transfected with a FLAG-LMNA-WT plasmid, and cell lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting analysis (IB) with an anti-NUP155 antibody. Emerin was used as a positive control, and normal IgG were used as negative control. (b) Reciprocal co-immunoprecipitation assays. HeLa cells were transiently transfected with a FLAG-LMNA-WT plasmid. Cell lysates were immunoprecipitated with anti-NUP155 antibody and analyzed by immunoblotting analysis (IB) with an anti-FLAG antibody. NUP205 was used as a positive control, and normal IgG were used as negative control. (c) Bacterial expression of recombinant GST-lamin A. Protein expression was induced by 0 or 0.5 mM IPTG, the recombinant protein was analyzed by SDS-PAGE and Coomassie brilliant blue staining. The result indicated that the N-terminal or the C-terminal truncated lamin A protein was expressed. The arrow indicated the estimated size of the truncated fusion proteins. Asterisk indicated the location of the full length fusion proteins. (d) GST pull-down assays. The cell lysates extracted from HeLa cells were incubated with a purified GST fusion N-terminal or C-terminal truncated lamin A protein or negative control GST. GST-LMNA-C-WT, but not GST-LMNA-N-WT or control GST, pulled down the endogenous NUP155. Coomassie brilliant blue staining showed the amount of protein. The arrow indicated the estimated size of fusion proteins

FIGURE 4

LMNA mutation p.Arg399Cys weakens binding of lamin A to NUP155, and increased extractability of NUP155 from NE. (a) GST pull-down assays. Nuclear extracts from HeLa cells were incubated with GST-LMNA-C-WT or GST-LMNA-C- p.Arg399Cys. Coomassie brilliant blue staining showed the approximately equal amount of WT and mutant fusion proteins. The interaction between endogenous NUP155 and mutant lamin A with p.Arg399Cys was significantly reduced by 42% compared with WT lamin A. The arrow indicated the estimated size of fusion proteins. Data are presented as mean ± SEM, *P < 0.05. (b) Mutant lamin A with p.Arg399Cys did not affect the expression level of endogenous NUP155 protein. EGFP-LMNA-WT or EGFP-LMNA-R399C was transferred into HeLa cells, and cell lysates were analyzed by Western blotting with an anti-NUP155 antibody or a control anti-α-tubulin antibody. (c) Mutant lamin A with p.Arg399Cys did not affect the NUP155 localization at NE (nuclear envelope). EGFP-LMNA-WT or EGFP-LMNA-R399C was transferred into HeLa cells, and the cytoplasmic and NE extracts were analyzed by Western blotting with antibodies for NUP155 and lamin B1. Cyto: cytoplasm; NE: nuclear envelope. (d) Mutant lamin A with p.Arg399Cys increased the extractability of NUP155 from NE. EGFP-LMNA-WT or EGFP-LMNA-R399C was transferred to HeLa cells, and the insoluble NE fraction was prepared by treatment with TrionX-100. The extracted NE (eNE) was further re-extracted with increasing concentrations of NaCl, and analyzed by Western blotting with an anti- NUP155 antibody or an anti-lamin B1 as a positive control. S, supernatant; P, precipitate. The relative NUP155 protein level at the Y-axis was expressed as either S/S+P or P/S+P. Data are presented as mean ± SEM, *P < 0.05

FIGURE 5

LMNA mutation p.Arg399Cys reduces the export of HSP70 mRNA, and nuclear import of HSP70 protein. (a) Analysis of nuclear import of HSP70 protein in HL-1 cells transiently transfected using a EGFP-LMNA-WT or EGFP-LMNA-R399C LMNA expression plasmid for 48 hr. Data are presented as mean ± SEM, *P < 0.05, **P < 0.01; n, number of images analyzed as indicated. (b) Analysis of the nuclear export of HSP70 mRNA in HL-1 cells. After heat shock, total RNA from nuclear or cytoplasmic fractions was isolated and reverse-transcribed with random primers. Quantitative real time RT-PCR analysis was used to estimate the levels of HSP70 mRNA in the cytoplasm and nucleus. LMNA mutation p.Arg399Cys inhibits HSP70 mRNA export into the cytoplasm. Data are presented as mean ± SEM, *P < 0.05

FIGURE 6

LMNA mutation p.Arg399Cys decreases cardiac sodium current density in HEK293/Na_v1.5 cells. (a) Representative whole-cell sodium currents recorded from HEK/Na_v1.5 cells transfected with a EGFP-LMNA-WT or p.Arg399Cys mutant expression plasmid. (b) The relationship of average current densities (current normalized to cell capacitance) and voltages. (c) Peak sodium current densities at –25 mV. Data are presented as mean ± SEM, *P < 0.05. (d) Steady-state activation curve. (e) Steady-state inactivation curve

FIGURE 7

LMNA mutation p.Arg399Cys decreases the cell surface expression level of Na_v1.5 without affecting the expression of total Na_v1.5 in HEK293/Na_v1.5 cells. (a) Western blotting analysis of total Na_v1.5 protein in HEK293/Na_v1.5 cells transiently transfected with a EGFP-LMNA-WT or EGFP-LMNA-R399C LMNA expression plasmid for 48 hr. Data are presented as mean ± SEM. ns, not significant. (b) Western blotting analysis of cell surface expression levels of Na_v1.5 in HEK293/Na_v1.5 cells. Data are presented as mean ± SEM, *P < 0.05