Phosphorylation of Lamin A/C at serine 22 modulates Nav 1.5 function

Abstract

Variants in the LMNA gene, which encodes for Lamin A/C, are associated with cardiac conduction disease (CCD). We previously reported that Lamin A/C variants p.R545H and p.A287Lfs*193, which were identified in CCD patients, decreased peak I_Na in HEK-293 cells expressing Na_v 1.5. Decreased peak I_Na in the cardiac conduction system could account for patients' atrioventricular block. We found that serine 22 (Ser 22) phosphorylation of Lamin A/C was decreased in the p.R545H variant and hypothesized that lamin phosphorylation modulated Na_v 1.5 activity. To test this hypothesis, we assessed Na_v 1.5 function in HEK-293 cells co-transfected with LMNA variants or treated with the small molecule LBL1 (lamin-binding ligand 1). LBL1 decreased Ser 22 phosphorylation by 65% but did not affect Na_v 1.5 function. To test the complete loss of phosphorylation, we generated a version of LMNA with serine 22 converted to alanine 22 (S22A-LMNA); and a version of mutant R545H-LMNA that mimics phosphorylation via serine 22 to aspartic acid 22 substitution (S22D-R545H-LMNA). We found that S22A-LMNA inhibited Lamin-mediated activation of peak I_Na by 63% and shifted voltage-dependency of steady-state inactivation of Na_v 1.5. Conversely, S22D-R545H-LMNA abolished the effects of mutant R545H-LMNA on voltage-dependency but not peak I_Na . We conclude that Lamin A/C Ser 22 phosphorylation can modulate Na_v 1.5 function and contributes to the mechanism by which R545H-LMNA alters Na_v 1.5 function. The differential impact of complete versus partial loss of Ser 22 phosphorylation suggests a threshold of phosphorylation that is required for full Na_v 1.5 modulation. This is the first study to link Lamin A/C phosphorylation to Na_v 1.5 function.

Keywords: Lamin phosphorylation; Nav1.5; cardiac conduction disease.

FIGURE 1

Confirmation of LMNA phosphorylation plasmids. (a) Electropherographs of sequencing results confirming mutagenesis of targeted codon (blue highlight) for each respective LMNA phosphorylation plasmid. (b) Western blot on protein lysates extracted from HEK‐293 cells transfected with each respective LMNA plasmid

FIGURE 2

Serine 22 phosphorylation in LMNA‐transfected cells. (a, b) Representative western blots of HEK‐293 cells transfected with the combination of plasmids noted. (c, d) Densitometry showing fold change of Ser 22 phosphorylation in: (c) WT‐LMNA (N = 6) and R545H‐LMNA (N = 6); *p < 0.05. (d) WT‐LMNA (N = 6) and A287Lfs‐LMNA (N = 4); p = N.S. Data are mean ± SE

FIGURE 3

S22A‐LMNA phosphorylation mutant alters peak I _Na. (a) Representative superimposed current traces drawn to scale obtained from cells co‐transfected with SCN5A and LMNA variants; step‐pulse protocol (inset). (b) I–V relationship comparing cells co‐transfected with SCN5A + WT‐LMNA (black), N = 10 versus SCN5A + S22A‐LMNA (red), N = 10; p < 0.0005. Peak current for SCN5A + WT‐LMNA = 1. (c) I–V relationship comparing cells co‐transfected with SCN5A + S22A‐LMNA (red), N = 10 versus SCN5A + S22D‐R545H‐LMNA (black), N = 7; p = N.S. Peak current for SCN5A + WT‐LMNA is set at 1

FIGURE 4

4 S22A‐LMNA phosphorylation mutant alters steady‐state inactivation. Voltage‐dependency of the steady‐state inactivation and activation of cells co‐transfected with SCN5A + WT‐LMNA (black) versus SCN5A + S22A‐LMNA (red, a) or S22D‐R545H‐LMNA (red, b). Inactivation: SCN5A + WT‐LMNA versus SCN5A + S22A‐LMNA p < 0.05. No significant differences in other inactivation or activation curves. Insets show pre‐pulse inactivation (left) and step‐pulse activation (right) protocols. Data are mean ± SE

FIGURE 5

LBL1 decreases Ser 22 Phosphorylation but does not alter I _Na . (a) Representative western blot of HEK‐293 cells transfected with WT‐LMNA and treated with 10 µM LBL1 (top) or 5 µM LBL1 (bottom). (b, c) Quantification of Ser 22 phosphorylation in cells treated with vehicle, 10 µM (b) or 5 µM (c) LBL1 (red bars). N = 6; *p < 0.05. Ser 22 phosphorylation in vehicle‐treated cells was set at 1. (d) Representative superimposed current traces obtained from respective cells. (e) I–V relationship of cells co‐transfected with SCN5A + WT‐LMNA; vehicle (black), N = 10 versus 10 µM LBL1 (red), N = 7. Peak current for vehicle is set at 1. (f) Voltage‐dependency of the steady‐state inactivation and activation of cells co‐transfected with SCN5A + WT‐LMNA. Inactivation: vehicle (black), N = 10 versus 10 µM LBL1 (red), N = 8; p = N.S. Activation: vehicle (black), N = 10 versus 10 µM LBL1 (red), N = 7; p = N.S. Insets show pre‐pulse inactivation (left) and step‐pulse activation (right) protocols. Data are mean ± SE