Defects in DNA Repair and Formation of Senescence-Associated Heterochromatin Foci (SAHF) in Lemd2-Associated Cardiomyopathy

Background and Purpose: Nuclear envelope proteins are crucial in the pathogenesis of hereditary cardiomyopathies. LEMD2, an inner nuclear membrane protein interacting with LMNA, maintains nuclear membrane integrity and chromatin organization. We generated a knock-in (KI) mouse model with the Lemd2 p.EL8_9del mutation, an in-frame deletion at amino acids 8 and 9 within the LEM domain, to investigate its role in cardiomyopathy pathogenesis and underlying mechanisms.

Methods and Results: KI mice survive up to five weeks of age and were phenotypically investigated at postnatal day 1.5 (P1.5) and postnatal day 21 (P21) of age. At P21, KI mice exhibited reduced body weight and an increased heart weight-to-body weight ratio compared to wild-type (WT) mice. To determine cardiac function, echocardiography was conducted, which indicated a severe form of dilated cardiomyopathy with a pronounced reduction in ejection fraction and fraction shortening at P21. Histology showed cardiomyocyte hypertrophy at P21, accompanied by a robust increase in fibrosis, which starts at P7.5 and is significantly present at P21.

To evaluate the impact of the mutation on protein expression, western blotting was performed, which revealed that Lemd2 protein levels in KI hearts were reduced by 97% compared to WT hearts at both P1.5 and P21. Given that Lemd2 plays a role in maintaining nuclear structural integrity maintenance, transmission electron microscopy was performed to observe the presence of dense heterochromatin aggregation throughout the nucleus in KI cardiomyocytes. H3K9me2, a marker for lamin-associated domains (LADs) interacting with the nuclear lamina, was observed in both the nuclear membrane and the nucleoplasm in KI cardiomyocytes. This suggests LAD detachment from the nuclear lamina, indicating nuclear instability and potential senescence-associated heterochromatin foci (SAHF) formation due to the Lemd2 deletion mutation.

Subsequently, a bulk RNA-seq analysis of P1.5 hearts revealed a downregulation of DNA repair and replication pathways and an upregulation of the immune response. DNA damage was detected using the γ-H2AX marker in western blots comparing KI with WT hearts at P1.5. A proliferation defect in cardiomyocytes was shown through Ki-67 staining. Furthermore, RNA-seq in P21 hearts revealed the upregulation of cellular senescence and P53 signaling pathways, indicating that DNA repair defects and increased DNA damage contribute to Lemd2-associated cardiomyopathy by inducing cellular senescence.

Conclusion: The Lemd2 p.EL8_9del mutation leads to Lemd2 deficiency, resulting in nuclear structure damage, LAD detachment, and SAHF formation. Lemd2 deficiency downregulates DNA replication, induces proliferation defects that promote cardiomyocyte hypertrophy, and downregulates DNA repair pathways, leading to increased DNA damage and cellular senescence, which the senescence-associated secretory phenotype further drives an immune response and fibrotic remodeling.