Background: 
Nuclear membrane (NM) proteins play important roles in the nuclear structural integrity maintenance and pathogenesis of laminopathies. As one of the major contributors to cardiac laminopathy, nuclear envelope ruptures (NER) could be caused by two main factors: compromised nuclear integrity and extensive mechanical stress. 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. Previous work could show a disrupted interaction between LEMD2 p.L13R and the barrier to autointegration factor (BAF) which is involved in NER and repair mechanisms.

Purpose: 
To investigate the involvement of the mutant LEMD2 in NER occurrence and DNA damage in LEMD2 p.L13R knock-in (KI) and LEMD2 knock-out (KO) HeLa cell lines which were generated by CRISPR/Cas9 technology.

Methods and Results: 
To determine the effect of mutant LEMD2 compared to controls on nuclear morphology at baseline, nuclear area size and circularity of the NM were measured by Image J whereby the KO and KI cells indicated larger nuclear areas and decreased circularity.

To investigate the impact of LEMD2 KI and KO on the NM integrity maintenance, NER were analyzed by performing immunofluorescence (IF) co-staining against DNA repair factor Ku80 and Lamin A/C under basal and stress conditions. NERs were determined by cytoplasmic Ku80 distribution whereas invaginations and micronuclei were visualized by Lamin/Dapi co- staining. At baseline KI cells displayed an increased cell ratio with abnormal nuclei while the KO cells showed a significant increase of ruptured nuclei, micronuclei and invaginations of the NM compared to controls. 

Next, we performed a stress protocol to determine the dynamics of NER and its repair by 5Hz electric stimulation for 10mins and analyzed at different time points post-stimulation (p-s). The results demonstrated apronounced increase of ruptured nuclei for KI and KO cells versus controls. Importantly, the rupture peak was reached after 30 min p-s for KO and KI cells and after 10 min p-s for controls. Thus, the repair of NER was delayed in both mutant cell lines. Additionally, KI and KO cells showed a remarkable increase of micronucleated cells and invaginations of the NM. 

Loss of DNA repair factors results in DNA damage which could be accumulated. Therefore, we examined DNA damage using IF staining of γH2AX as a marker for DNA damage and adopted the same stress conditions as for the nuclear rupture assay. γH2AX intensity peaked at 2h p-s in all cell lines, whereas a significant higher intensity was detected in KI and KO cells compared to controls at this timepoint. Interestingly, DNA damage levels of controls recovered to untreated levels after 24h while prominent DNA damage maintained unrepaired in KI and KO cells indicating sustained DNA damage. As DNA damage can induce cell cycle arrest, we additionally performed flow cytometry analyses and detected mitotic arrest in KI and KO cells. 

Conclusion: 
We showed that loss of LEMD2 and the p.L13R mutation alter nuclear morphology which results in increased NER and a disturbed NER repair process. Consequently, obvious DNA damage with DNA damage repair defects and cell cycle arrest occur. Taken together, we propose that the disturbed interaction between mutant LEMD2 and BAF and subsequent impaired NER repair capacity and DNA damage may play roles in the pathogenesis of cardiomyopathy caused by LEMD2 mutation.