The expression of Lamin A mutant R321X leads to endoplasmic reticulum stress with aberrant Ca²⁺ handling

Abstract

Mutations in the Lamin A/C gene (LMNA), which encodes A‐type nuclear Lamins, represent the most frequent genetic cause of dilated cardiomyopathy (DCM). This study is focused on a LMNA nonsense mutation (R321X) identified in several members of an Italian family that produces a truncated protein isoform, which co‐segregates with a severe form of cardiomyopathy with poor prognosis. However, no molecular mechanisms other than nonsense mediated decay of the messenger and possible haploinsufficiency were proposed to explain DCM. Aim of this study was to gain more insights into the disease‐causing mechanisms induced by the expression of R321X at cellular level. We detected the expression of R321X by Western blotting from whole lysate of a mutation carrier heart biopsy. When expressed in HEK293 cells, GFP‐ (or mCherry)‐tagged R321X mislocalized in the endoplasmic reticulum (ER) inducing the PERK‐CHOP axis of the ER stress response. Of note, confocal microscopy showed phosphorylation of PERK in sections of the mutation carrier heart biopsy. ER mislocalization of mCherry‐R321X also induced impaired ER Ca2+ handling, reduced capacitative Ca2+ entry at the plasma membrane and abnormal nuclear Ca2+ dynamics. In addition, expression of R321X by itself increased the apoptosis rate. In conclusion, R321X is the first LMNA mutant identified to date, which mislocalizes into the ER affecting cellular homeostasis mechanisms not strictly related to nuclear functions.

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

“…2+ levels with either Fura-2 or FRET-based probe D1ER Steady state FRET experiments were performed as previously described [8]. Briefly, HEK293 cells seeded on poly-l-lysine-coated glass coverslips were transiently cotransfected with plasmids encoding either mCh-Lamin A or mCh-D243Gfs*4 and the FRET-based probe D1ER (gift from Prof. Roger Tsien) [15].…”

“…The synthetic gene mCherry (mCh) was designed with XbaI and PspXI flanking restriction sites, inserted into pMA-RQ (ampR) and synthesized by Life technologies™ (Thermo Fisher Scientific, Waltham, MA, USA). The generation of Lamin A mCherry-tagged constructs was previously described [8].…”

“…The frameshift nucleotide deletion LMNA p.D243Gfs*4 was performed with Stratagene's Quik Change II XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA), using the PCR product of LMNA cDNA cloned in the pcDNA™6.2/N-EmGFP-DEST (Thermo Fisher Scientific, Waltham, MA, USA) vector system as a template [5,8]. Mutagenic primers were designed using Quik Change Primer Design Program available online at www.agilent.com/genomics/qcpd/.…”

“…At cellular level, the R321X mutant was found to accumulate within the endoplasmic reticulum (ER), likely unable to continue its biosynthetic pathway. This alteration also deeply impacts with the ability of this organelle to properly handle key physiological processes such as Ca 2+ homeostasis, ER stress, and apoptosis [8]. Here, we studied another novel LMNA truncating alteration (c.728_747del,p.D243Gfs*4), identified in an Italian family showing a severe form of cardiomyopathy with poor prognosis.…”

“…In order to better exploring the functional effects of LMNA truncating alterations associated to a cardiac phenotype, we recently studied a PTC mutation (R321X) upstream the LMNA Nuclear Localization Sequence (NLS) associated to severe cardiomyopathy [8]. At cellular level, the R321X mutant was found to accumulate within the endoplasmic reticulum (ER), likely unable to continue its biosynthetic pathway.…”