Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

Generation and initial characterization of a human in vitro model system for LEMD2-associated cardiomyopathy

A. Cirnu1, A. Janz1, A. Kroth1, M. Regensburger1, R. Chen1, B. Gerull1, für die Studiengruppe: DZHI

1Deutsches Zentrum für Herzinsuffizienz, Universitätsklinikum Würzburg, Würzburg;

Introduction: Dilated cardiomyopathy (DCM) with arrhythmias is caused by an autosomal recessive missense mutation c.T38G (p.L13R) in the inner nuclear membrane protein LEMD2. The mutation affects a highly conserved amino acid in the LEM domain, which is crucial for tethering chromatin to the nuclear periphery. However, the role of mutant LEMD2 for nuclear integrity and its contribution to arrhythmic DCM in a human setting remains unknown. Purpose: To generate and initially characterize an induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model system to elucidate the role of mutant LEMD2 in human cardiomyopathy. Methods and Results: We generated an in vitro system by reprogramming patient (L13R-P)-derived dermal fibroblasts into iPSCs using non-integrative Sendai virus. Next, we aimed for additional iPSC lines by first introducing the c.T38G knock-in mutation (KI) into the healthy genetic background of control iPSCs and by second correcting the nucleotide substitution back to the healthy sequence in the patient background (rescue) using CRIPSR/Cas9 technology. We compared the mutation efficiencies of catalytically intact Cas9 enzyme for creating double-strand breaks with the mutated p.D10A Cas9n that effects single-strand nicks only. By designing two different single-guide RNAs, Cas9n was adapted to a double-nicking system to create a more extended DNA breakage. A mutation-carrying single-strand oligo of 144 bp served as a template for homology-directed repair (HDR template) in both systems to exclusively introduce a point mutation without any other alterations. The gene editing compounds were delivered to the individual iPSC lines by nucleofection, followed by puromycin selection and single colony cultivation. Potentially positive clones were sequenced to confirm the presence of the desired mutations. For the KI, the Cas9n system was finally able to generate two clones, one of which carried a heterozygous KI whereas the classical Cas9 system was not successful. Regarding the rescue, the double-nicking system produced 25 surviving iPSC clones of which three displayed the homozygous repair. Interestingly, a shorter HDR template (80 bp) yielded higher survival rates, but did not result in positive clones, highlighting the importance of a proper design. Hence, the usage of the Cas9n system with the 144 bp HDR template significantly increased gene editing efficiency in iPSCs. For initial characterization of the in vitro model, we additionally generated a LEMD2 knock-out (KO) in the healthy isogenic background of a control iPSC line (Ctr) via classical CRISPR/Cas9. The KO and patient L13R-P iPSCs as well as the Ctr have been differentiated to iPSC-CMs and cultured for 30 days before evaluation. To confirm the expression of LEMD2 in our model system we performed immunoblotting that showed almost absent LEMD2 expression in KO cells whereas L13R-P had reduced LEMD2 compared to Ctr CMs. Next, we studied the morphology of iPSCs and CMs via immunofluorescence and observed significantly increased areas of nuclei in L13R-P cells compared to KO and Ctr cells. Additionally, the L13R-P CMs demonstrated extensive hypertrophy that indicated an accelerated maturation process in contrast to KO and Ctr CMs. Conclusion: The application of the Cas9n double-nicking system is efficient in generating a human iPSC-CM in vitro model system for the investigation of DCM and has the power to enable valuable insight into the pathogenesis of LEMD2.

https://dgk.org/kongress_programme/jt2022/aP1878.html