Background and Purpose: Nuclear envelope proteins play important roles in the pathogenesis of hereditary cardiomyopathies. We recently discovered a new form of arrhythmic cardiomyopathy caused by a homozygous mutation (p.L13R) in the inner nuclear membrane (INM) protein LEMD2. We generated a knock-in (KI) mouse model carrying the Lemd2 p.L13R mutation to unravel the molecular mechanisms underlying the human mutation.

Methods and Results: KI mice were viable and phenotypically investigated at 6 months (6m) and 9 months (9m) old age. Aggravated susceptibility to cardiac arrhythmias was detected by surface electrocardiogram (ECG) in 6m KI mice, which developed into severe arrhythmias at 9m of age. ECG analysis demonstrated prolonged PR, QRS and corrected QT intervals indicating conduction and repolarization abnormalities. To determine cardiac function, we performed echocardiography, which indicated a severe form of cardiomyopathy with left ventricular (LV) systolic dysfunction, increased left ventricular end-systolic diameter and decreased LV wall thicknesses. Structural changes were observed by histology, which revealed mild cardiac fibrosis in 6m, while the 9m old KI hearts showed robust fibrosis. Despite a normal heart morphology and function at 3 months of age, we already observed cardiomyocyte hypertrophy measured as an enlarged cross-sectional area, which further increased until 9m of age. We hypothesized that cellular hypertrophy might be a result reduced proliferation capacity at the postnatal stage. Therefore, we performed a Ki-67 staining at postnatal day 5 (P5) showing reduced proliferation, which could be confirmed by lower cardiomyocyte cell counts at P5 using flow cytometry. To study the mechanisms towards premature cellular senescence, we scrutinized if activation of the p53/p21 pathway plays a role in the progression of disease and detected higher p53 protein levels in KI hearts. Senescence-associated secretory phenotype (SASP) including Gdf15, Tgfβ2 and Edn3 were up-regulated. Cellular senescence may occur as consequence of accelerated DNA damage response and we detected increased DNA damage (γH2AX) in 9m old KI hearts.

As LEMD2 plays a role in heterochromatin remodeling, we performed TEM, which showed nuclear membrane invaginations and detached heterochromatin from the inner nuclear membrane in 6m old KI cardiomyocytes. Furthermore, we detected an extended distance between the outer and inner nuclear membrane. Given that the p.L13R locates in the LEM domain which mediates the interaction between LEMD2 and Barrier-to-autointegration factor (BAF), we proposed that the interaction might be disrupted – an observation which we could demonstrate in vitro by co-immunoprecipitation. Further investigations will unravel if the process of repair following nuclear membrane ruptures might be disturbed requiring an interplay of BAF, LEMD2 and ESCRT-III. Potentially unrepaired ruptures compromise retention of DNA repair factors and favor sustained damage.

Conclusion: We showed that the Lemd2 p.L13R mutation in mice recapitulates the human phenotype leading to cardiomyopathy with cardiomyocyte hypertrophy, fibrosis and severe arrhythmias. The mutation leads to a decreased postnatal proliferation capacity, induces increased DNA damage and causes premature cellular senescence. We propose that a disturbed interaction between LEMD2 and BAF and subsequent compromised nuclear membrane repair may play a role in the pathogenesis of the disease.