Lipodystrophy-associated LMNA mutations impair endothelial cell identity and promote senescence

Laminopathies such as Familial partial lipodystrophy type 2 (FPLD2) can be caused by mutations in the LMNA gene, coding for Lamin A/C. As part of the nuclear lamina, Lamin A/C plays an important role in nuclear integrity and chromatin organization. Besides the effects on adipose tissue, early clinical cardiovascular symptoms are more frequent in FPLD2 patients with LMNA mutations. Senescence or loss of endothelial cell (EC) identity, such as through endothelial-to-mesenchymal transition (EndMT), can lead to cardiovascular diseases.

We characterized 12 LMNA mutations identified in a FPLD2 patient cohort using DNA sequencing to analyze the effects on EC identity and integrity to identify potential pathological variants for the cardiovascular system.

All 12 identified FPLD2-associated LMNA mutations were introduced into the wild-type LMNA gene by site-directed mutagenesis and subsequently used to synthesize modified mRNAs for each variant. Transfer of the LMNA mRNA variants into human ECs resulted in a significant increase of LMNA levels. Interestingly, although overexpressed at the mRNA level, transfection of three mutations (c.1445G>A, c.1699-2A>G and c.1930C>T) resulted in reduced lamin A/C protein levels. Overexpression of the c.1444C>T LMNA variant induced EndMT and EC senescence as determined by increased mRNA levels of the mesenchymal marker CNN1 and the senescence-associated genes p16, p21, GDF15 and IL6. Immunofluorescence revealed increased nuclear abnormalities, such as increased nuclear invaginations, which are associated with cellular senescence, after overexpression of LMNA mutant variants (c.1304G>A, c.1444C>T and c.1445G>A). This was further enhanced when ECs were stressed by additional induction of EndMT, resulting in significantly increased nuclear abnormalities for LMNA mutations (c.1411C>G, c.1445G>A, c.1445G>C and c.1634G>A). Single-cell Multiome sequencing of EC overexpressing the LMNA c.1445G>A variant revealed lower RNA expression of EndMT-associated genes such as MGP1 or TIMP1, as well as the inflammatory marker CCL14, compared to LMNA wild-type transfected EC.

Taken together, these results indicate that LMNA mutations affect endothelial identity and survival potentially by interfering with nuclear integrity.