Lemd2 haploinsufficiency results in defects in cardiomyocyte proliferation

Background and Purpose: Nuclear envelope proteins play a crucial role in hereditary cardiomyopathies. We have identified a novel form of arrhythmic cardiomyopathy caused by a homozygous mutation (p.L13R) in the inner nuclear membrane (INM) protein LEMD2. Utilizing a Lemd2 p.L13R knock-in (KI) mouse model, we effectively recapitulated the cardiac phenotype observed in patients. Comprehensive cardiac assessments, including echocardiography and electrocardiogram, revealed severe dilated cardiomyopathy and pronounced arrhythmias at 9 months. Histological analysis illustrated the progression of cardiac fibrosis as age advanced. Given LEMD2’s involvement in cell cycle process and cell proliferation, our objective is to investigate its role in the early stages of postnatal heart development and growth before the manifestation of the phenotype.

Methods and Results: At 3 months of age, KI mouse hearts did not exhibit evident collagen deposition despite a significant reduction in Lemd2 protein levels, as determined by western blotting, indicating protein haploinsufficiency due to the Lemd2 p.L13R mutation. However, cardiomyocyte hypertrophy was observed at this stage. To investigate earlier effects, we examined hearts harvested on postnatal day 5 (P5) and identified increased DNA damage in KI hearts, evidenced by γ-H2AX staining. Additionally, p53, a downstream component of the DNA damage response, displayed a substantial increase. Cardiomyocyte counts at P5 were analyzed using flow cytometry with Troponin T (TnT) staining, revealing a decreased cardiomyocyte cell count in KI hearts. To gain insights into cell fates, an apoptosis assay using a TUNEL kit was conducted, but no obvious changes were observed. Furthermore, a proliferation assay was performed via co-immunofluorescence (IF) staining for postnatal mice injected with EdU, indicating a decreased ratio of EdU-positive proliferating cardiomyocytes in KI hearts. This suggested that reduced Lemd2 protein content restricted cellular proliferation capacity, potentially leading to compensatory cardiomyocyte hypertrophy. In parallel, another Lemd2 knock-out (KO) mouse model exhibited embryonic lethality and a robust decrease in proliferating cardiomyocytes, as determined by co-IF staining using TnT and Ki-67.

Conclusion: Our investigation revealed that Lemd2 p.L13R resulted in protein haploinsufficiency, initiating DNA damage as early as the postnatal stage. The increased DNA damage and activated p53 pathway, potentially impacting cyclin-dependent kinase inhibitors, inhibited cardiomyocyte proliferation and resulted in a reduced cardiomyocyte cell count and compensatory hypertrophy at a young age. This may potentially trigger fibroblast recruitment, advancing the progression of cardiac fibrosis. This sequence of events may further exacerbate the phenotype with aging.