Background and Purpose: Nuclear envelope proteins play important roles in the pathogenesis of hereditary cardiomyopathies. Recently, our group has discovered a new form of arrhythmic cardiomyopathy caused by a homozygous mutation (p.L13R) in the inner nuclear membrane 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) of 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 severe ventricular arrhythmias and cardiac conduction and repolarization abnormalities. To unravel the underlying mechanisms we investigated calcium homeostasis and transmembrane action potential (AP) in isolated cardiomyoytes (CMs). Calcium handling was measured with Indo-1 which showed slightly increase in diastolic and systolic calcium concentrations in KI CMs. Transmembrane APs were recorded in isolated CMs exposed to increasing pacing rates. Interestingly, significantly elevated membrane voltage was determined in 9m KI which could be a potential mechanism of arrhythmias. Consistently, mRNA expression data suggest the involvement of several voltage-gated potassium channels in particular a down-regulation of KCNJ2 and KCNJ5 which are responsible for resting membrane potential maintenance.

To determine cardiac function and structure, we performed echocardiography and histology, which indicated a severe form of dilated cardiomyopathy with left ventricular (LV) systolic dysfunction at 9m of age. Histology showed cardiomyocyte hypertrophy and an increase in fibrosis which starts at 6m of age and is significantly present at 9m of age.

As LEMD2 plays a role in nuclear structural integrity maintenance, we performed transmission electron microscopy and observed a robust increase of abnormal nuclei in KI CMs with a 4-fold increase in nuclear membrane invaginations and nuclear membrane rupture (NER). NER, NER repair capacity and DNA damage was further investigated in mice and a cell-culture model upon stress. The LEMD2 p.L13R mutation leads to compromised NER repair capacity and subsequent DNA damage.

Finally, we performed the RNA sequencing for 9m old mouse hearts and it showed notable upregulations of interferons and CM specific senescence-associated secretory phenotype. NER occurrence could be responsible for the interferon up-regulations due to NER with exposed DNA being recognized by cyclic GMP–AMP synthase (cGAS) which activates the cGAS-STING-interferon pathway.

Conclusion: We showed that the Lemd2 p.L13R mutation in mice recapitulates the human phenotype leading to cardiomyopathy with CM hypertrophy, fibrosis, severe arrhythmias and conduction defects. Arrhythmias occur before significant fibrosis occurs suggesting an independent mechanisms due to dysregulations of cardiac voltage gated potassium channels. As the p.L13R mutation is located in the LEM-domain, we propose a disrupted interaction between mutant LEMD2 and barrier-to-autointegration factor (BAF) which is required for NER repair initiation process. Impaired NER repair capacity associated subsequent DNA damage, chromatin remodeling and transcriptional alterations play pivotal roles in the pathogenesis of cardiomyopathy caused by LEMD2 mutations.