A Comprehensive hiPSC-Based Model to Investigate the Cellular and Metabolic Impact of the LEMD2 p.L13R Mutation

Background:
LEMD2-associated dilated cardiomyopathy (DCM) is characterized by mild left ventricular dysfunction, extensive fibrosis, and severe arrhythmias leading to sudden cardiac death. This is caused by a ubiquitously expressed homozygous missense mutation in the inner nuclear membrane protein LEMD2 (LEMD2 p.L13R). Patient fibroblasts displayed nuclear abnormalities, chromatin disorganization, reduced proliferation, and premature senescence. The cardiac phenotype has been successfully recapitulated in a Lemd2 p.L13R mouse model and is now being investigated in human induced pluripotent stem cell (hiPSC) derived cardiac cell types and 3D microtissues (MTs).

Methods:
To recapitulate the human phenotype, a system of different hiPSC lines was established using CRISPR/Cas9. In addition to a healthy control, a LEMD2-KI (p.L13R) and LEMD2-KO generated in this line, a hiPSCs line reprogrammed from patient fibroblasts and a repaired patient line (LEMD2 rescue) were used. These hiPSC lines were differentiated into the main cell types found in the heart: cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs), which were analyzed with regard to cell size, senescence, proliferative capacity and metabolism. To better recapitulate the human phenotype, 3D MTs were generated by co-assembling the three cell types and after 21 days maturation, the resultant MTs were used for experiments. In addition to the analysis of whole MTs based on structure, size and metabolism, the MTs were dissociated into single cells to analyze their composition.

Results:
Patient CMs revealed extensive nuclear and cellular hypertrophy, which indicated an accelerated maturation process in the diseased cells, accompanied by increased DNA damage and cellular senescence. CFs and ECs also demonstrated changes in cellular senescence and additionally in the cell cycle in the LEMD2-KI, LEMD2-KO and the patient line when compared with the healthy control. Data of CFs indicate a contribution of accelerated CF transition to the disease phenotype of fibrosis such as seen in our mouse and human heart tissue. Initial metabolic analyses revealed reduced spare respiratory capacity in ECs and CFs with a LEMD2 mutation (KI, KO, patient). However, first data from the MTs suggested that the respiratory capacity of the crispered lines (KI, KO) was also reduced, but that of the patient MTs was significantly increased. Moreover, the MTs displayed spontaneous contraction with proper composition and organization.

Conclusion:
A comprehensive hiPSC-based model was established to analyze the LEMD2 p.L13R mutation. These cell lines could be successfully differentiated into the three main cell types in the heart, whereby the cells with a LEMD2 mutation displayed changes, both in the cell cycle and in the metabolism. Metabolic changes were also detected in the 3D model of MTs, which serve as a valuable 3D model for investigating the intricate interactions underlying the LEMD2 mutation.

Outlook: To gain a deeper insight into LEMD2-associated DCM and to reveal the different contributions of individual cell types to the development of the disease phenotype, a more comprehensive approach is required, in which MTs are not only derived from cell types from one hiPSC line, but a mixed model will be used.