Function alteration of engineered heart tissues carrying a shorter filamin C protein induced by CRISPR/Cas9
Dilated cardiomyopathy (DCM) is characterized by ventricular dilation and systolic dysfunction beginning in young adults and leading to heart failure with few treatment options. Loss-of-function mutations in the FLNC gene, encoding Filamin C, including splicing mutations, have been strongly associat...
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| Published in: | Archives of Cardiovascular Diseases Supplements 2022-06, Vol.14 (2), p.182-182 |
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| Main Authors: | , , , , , , |
| Format: | Article |
| Language: | eng |
| Online Access: | Get full text |
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| Summary: | Dilated cardiomyopathy (DCM) is characterized by ventricular dilation and systolic dysfunction beginning in young adults and leading to heart failure with few treatment options. Loss-of-function mutations in the FLNC gene, encoding Filamin C, including splicing mutations, have been strongly associated with an arrhythmogenic DMC phenotype.
FLNC-related cardiomyopathies are autosomal dominant disease and the mechanisms leading to the phenotype are not fully understood. In vitro models are needed to improve knowledge of pathogenic mechanisms.
A control iPSc line (ICAN-403.3) was established from healthy donor fibroblasts and a mutated FLNC subclone (ICAN-FLNC-42.1) was derived using CRISPR/Cas9 technology. The iPSc cells were then differentiated into cardiomyocytes and beating pseudo-tissues in order to study the functional effects of the mutation.
We generated two iPS clones, one derived from the other after mutation induced by non-homologous CRISPR/Cas9-dependent DNA repair. The clones were characterized for their pluripotency (immunoflurescence and qPCR of genes expressed in the pluripotent state, alkaline phosphatase test), their genomic integrity (exome, karyotype) and for their capacity for differentiation in the 3 layers (ScoreCard). More specifically, these cells can be differentiated into beating cardiomyocytes (>85% of cells express troponin T) and exhibit sarcomeric striation by immunostaining of actinin-2. Sequencing shows that the mutation induced by CRISPR/Cas9 is a deletion of the 3′ terminal of exon 42 of the FLNC gene found in the homozygous state [Chr7(GRCh38):g.128854898_128854915del] and lead to the deletion of the consensus donor splice site of exon 42. Additionally, no off-target in cardiac expressed genes has been detected by exome sequencing. Analysis by RT-PCR and immunoblot shows that the mutation induces a exon 42 skipping and reduced expression of a protein of lower molecular weight. Cardiac tissues records for 3weeks [engineered heart tissue (EHT) generated from the two iPS clones differentiated into cardiomyocytes] show, for the mutant, a lower beat frequency and a higher arrhythmia index.
This model will make it possible to understand the pathophysiological mechanism of DCM due to FLNC mutations. These models are also useful for testing potential therapeutic pathways. |
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| ISSN: | 1878-6480 1878-6502 |