https://doi.org/10.1007/s00392-024-02526-y
1Universitätsklinikum Würzburg Deutsches Zentrum für Herzinsuffizienz Würzburg, Deutschland; 2Institut für Humangenetik Würzburg, Deutschland
Background:
LEMD2-associated dilated cardiomyopathy (DCM) manifests as mild impairment of systolic left ventricular function accompanied by extensive fibrotic tissue deposition, 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 dermal fibroblasts exhibited abnormal nuclear morphology, chromatin disorganization, compromised proliferative capacity, and premature senescence. This pathological phenotype of the heart has been successfully recapitulated in a Lemd2 p.L13R mouse model and is now being investigated using CRISPR/Cas9 editing and the generation of human induced pluripotent stem cell (hiPSC) derived microtissues (MTs).
LEMD2-associated dilated cardiomyopathy (DCM) manifests as mild impairment of systolic left ventricular function accompanied by extensive fibrotic tissue deposition, 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 dermal fibroblasts exhibited abnormal nuclear morphology, chromatin disorganization, compromised proliferative capacity, and premature senescence. This pathological phenotype of the heart has been successfully recapitulated in a Lemd2 p.L13R mouse model and is now being investigated using CRISPR/Cas9 editing and the generation of human induced pluripotent stem cell (hiPSC) derived microtissues (MTs).
Methods and Results:
The CRISPR/Cas9 system was used to introduce the homozygous LEMD2 p.L13R mutation into hiPSCs derived from a healthy donor. Characterization of these cells was done to establish a robust cell line, including assessment of genotype, pluripotency, differentiation potential, and other relevant parameters. Accordingly, CRISPR/Cas9 was utilized to correct the mutation of hiPSCs reprogrammed from LEMD2 p.L13R patient fibroblasts generating an isogenic control hiPSC line within the patient background. Both mutant hiPSCs and the respective controls were differentiated into the main cell types found in the heart: cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs). Following differentiation, each cell type displayed cell-specific marker expression. First analyses of CMs revealed extensive nuclear and cellular hypertrophy in mutant CMs only, which indicated an accelerated maturation process in the diseased cells.
Moreover, analysis of senescence revealed a trend towards increased senescence rates accompanied by reduced proliferation capacity mainly in mutant CFs compared to control indicating a contribution of accelerated CF transition to the disease phenotype of fibrosis such as seen in our mouse and human heart tissue. To better recapitulate the human phenotype, 3D MTs were generated by co-assembling the three cell types in a ratio of 70 % of CMs and 15 % each of CFs and ECs. After a 21-day maturation period, the resultant MTs demonstrated spontaneous contraction. Immunofluorescence staining using cell type-specific markers revealed a proper composition and organization of the MTs, providing valuable insights into their cellular architecture.
Conclusion:
A comprehensive model consisting of hiPSCs with the LEMD2 p.L13R mutation and healthy controls was established using the CRISPR/Cas9 system. After differentiation into the three cardiac cell types, analyses of mutant hiPSC-CFs suggested that they might also be a driver of fibrosis in the disease. However, beyond analyzing individual cardiac cell types, MTs provide a valuable 3D model for investigating the intricate interactions underlying the LEMD2 mutation.
Outlook:
To gain a deeper insight into LEMD2-associated DCM, a more comprehensive approach involves MTs not only from cell types derived from one hiPSC line, but rather employing a mixed model. For instance, combining healthy CMs and ECs and LEMD2 p.L13R CFs could reveal the different contributions of the individual cell types to the development of the disease phenotype.