Background and Purpose: LEMD2 is a key inner nuclear membrane protein that maintains nuclear envelope integrity and chromatin organization via interactions with lamins and the chromatin-binding factor BAF. Mutations within its conserved LEM-domain disrupt nuclear envelope repair after rupture, leading to DNA damage accumulation, premature senescence, and fibrotic remodeling.
Methods and Results: To investigate the impact of the LEM-domain, we generated a Lemd2 knock-in (KI) mouse model carrying a deletion mutation within the LEM-domain (ΔE8_L9/ΔE8_L9). Homozygous mice exhibited severe growth retardation and survived up to five weeks of age. Echocardiographic examinations at postnatal day 21 (P21) revealed a pronounced dilated cardiomyopathy (DCM) in KI mice. Western blotting analysis demonstrated a complete loss of Lemd2 in homozygous KI hearts. Computational modeling revealed that the ΔE8_L9/ΔE8_L9 mutation causes increased conformational destabilization of the LEM domain, resulting in protein instability, whereas the previously investigated p.L13R variant induces only moderate structural perturbations.
Transmission electron microscopy (TEM) showed dense heterochromatin aggregation throughout the KI cardiomyocyte nuclei. Immunofluorescence (IF) of H3K9me2 staining at postnatal day 1.5 (P1.5) demonstrated detachment of lamina-associated domains (LADs) from the nuclear periphery. Further punctate DAPI foci indicated the formation of senescence-associated heterochromatin foci (SAHF). RNA-sequencing identified downregulation of DNA replication and DNA repair pathways in KI hearts at P1.5, consistent with proliferation defect visualized by IF staining, and supported by increased DNA damage levels detected by western blotting. Heterochromatin reorganization disrupts genome stability and gene expression regulations essential for cardiomyocyte proliferation and survival.
Conclusions: These findings provide compelling evidence for the pivotal role of the LEM-domain in the protein homeostasis of Lemd2, and the indispensable role of Lemd2 in nuclear envelope integrity and chromatin organization. The loss of Lemd2 leads to heterochromatin reorganization, suppressing DNA replication and DNA repair. The subsequent proliferation defect and DNA damage accumulation trigger a senescence-associated secretory phenotype (SASP), promoting immune activation and fibrotic cardiac remodeling.
