Background and Purpose: We have recently discovered a homozygous mutation (p.L13R) in the nuclear envelope protein LEMD2, which results in a severe form of arrhythmic cardiomyopathy with sudden cardiac death in affected patients of the Canadian Hutterite population. LEMD2 has been implicated in chromatin tethering, nuclear reassembly, cell cycle and DNA damage response, gene regulation, signaling and mechano-transduction. However, the role of LEMD2 in disease and the sequence of molecular events leading to cardiomyopathy remains unknown.

We aimed to generate and initially characterize an induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model to elucidate the role of mutant LEMD2 in human cardiomyopathy.

Methods and Results: We generated an in vitro model by reprogramming patient derived (L13R-P) dermal fibroblasts into iPSCs using non-integrative Sendai virus. Next, we wanted to generate an isogenic control by correcting the nucleotide substitution c.G38>T back to the normal sequence in the patient background (rescue) using CRISPR/Cas9 technology. In addition, we introduced the c.T38G knock-in (KI) mutation into a healthy (Ctr) genetic background. We used the Cas9 system and Cas9 nickase (Cas9n) system in parallel, as the latter can avoid unspecific double-strand breaks.

For the KI, the Cas9n system was finally able to generate two clones, one of which carried a heterozygous Kl whereas the classical Cas9 system failed. For the rescue, the Cas9n system created three iPSC clones with the homozygous repair. During the gene editing process, we additionally generated by chance a LEMD2 knock-out (KO) in the Ctr background.

For initial characterization of our model system we differentiated the KO, the L13R-P and the Ctr to iPSC-CMs and cultured them from 30-125 days depending on the read-out. To confirm

the expression of LEMD2 we performed immunoblotting that showed almost absent LEMD2 in KO cells whereas the L13R-P showed remarkably reduced LEMD2 compared to Ctr CMs. Next, we studied the morphology of iPSCs and CMs via immunofluorescence (IF) and observed significantly increased nuclear areas in L13R-P cells compared to KO and Ctr cells. The L13R-P CMs demonstrated extensive hypertrophy that indicated an accelerated maturation process in contrast to KO and Ctr CMs.

Furthermore, we characterized some phenotypic features of iPSC-CMs. Therefore, we performed the β galactosidase (ß-gal) staining in combination with cTnT to measure the senescent CM ratio in young and old iPSC-CMs. The L13R-P CMs showed increased senescent CM ratios compared to Ctr CMs. Senescent cells displayed persistent DNA damage response (DDR), so we stained for DNA damage markers γ-H2AX and 53BP1 to evaluate DDR in iPSC-CMs at 30 and 60 days of age. We observed an increased DNA damage in particular in L13R-P CMs compared to Ctr. Finally, we investigated the MAPK pathways in mutant and Ctr CMs for p38 phosphorylation (pi-p38) levels by IF staining and interestingly, we detected a hyperactivation of pi-p38 in KO CMs but the opposite, a decreased pi-p38 level in L13R-P CMs compared with Ctr CMs.

Conclusion: We generated an iPSC model for LEMD2 associated cardiomyopathy. Phenotypic characterization revealed that mutant LEMD2 induces CM senescence and increased DNA damage, which may be regulated by altered MAPK signaling pathway. Further validation of these phenotypes will be continued to unravel the mechanisms of LEMD2 associated cardiomyopathy.