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Abstract

Dilated Cardiomyopathy (DCM) is frequently leading to heart failure. Familial analysis revealed 30% - 60 % of cases to be of genetic cause, predominantly occurring in genes encoding for structural proteins. The underlying mechanisms and signalling events that translate DCM causing mutations into the clinical phenotype are poorly understood. Previously, our group identified the Core- Binding Factor beta (CBFb) to be an essential factor for maintaining the sarcomeric Z-disc structure of the heart muscle. CBFb is mainly located in the sarcomere and is retained in the cytoplasm via the interaction with Filamin A (FLNA). However it is hypothesized that under stress conditions, CBFb is translocated to the nucleus, where it regulates the expression of several genes. To analyze the contribution of FLNA to the development of heart failure we created a transient and a stable loss-of-function model of FLNA in zebrafish. FLNA transient knockdown (MO-flna) resulted in systolic dysfunction, defects in re-absorption of cardiac matrix, a dilated atrium, blood regurgitation and maturation defects shown by uncompleted heart looping. We observed a significantly reduced heart rate (MO-control vs MO-flna; mean±SD: 148±6.9 vs 108±25.1 beats/min, respectively; p<0.05) and fractional shortening (MO-control vs MO-flna; mean±SD: 64.7±4.0 vs 19.3±10.8 beats/min, respectively; p<0.0001). We further established the CRISPR-CAS9 mediated FLNA stable knockout (FLNA KO). Indeed, in F0 embryos we observed a significantly reduced heart rate (wildtype vs FLNA KO; mean±SD: 144±17.6 vs 106±12.3 beats/min, respectively; p<0.0001) and fractional shortening (wildtype vs FLNA KO; mean±SD: 64±8.9 % vs 43±8.8 %, respectively; p<0.0001), as observed for the FLNA knockdown. F0 mosaic embryos were grown to adulthood and outcrossed with wildtype fish. Two FLNA KO lines (with a predicted stop codon on flna exon 4) were selected for propagation. Heterozygous adults from both lines showed a significantly reduced ejection fraction (wildtype vs FLNA KO; mean±SD: 62±6.9 % vs 41±12.2 %, respectively; p<0.01) and increased ventilation frequency (wildtype vs FLNA KO; mean±SD: 7±4.2 vs 15±7.0 buccal movements, respectively; p<0.001). Compound heterozygous, with both lines mutations, developed a pericardial edema with a dilated heart, stressing the potential role of FLNA in maintaining heart function. Homozygous embryos from one of the lines, did not show a heart failure phenotype. In accordance, flnb was shown to be up-regulated in FLNA KO adults, illustrating possible compensatory mechanisms activation. In regard to CBFb translocation mechanism, for the first time, beta-adrenergic stress was identified as a trigger for CBFb translocation to the nucleus. In conclusion, FLNA knockdown and knockout in zebrafish leads to a heart failure phenotype. By deeply investigating FLNA regulation (such as cleavage, phosphorylation, response to external cellular stress, etc) and its interacting partners (such as CBFb), we will further our understanding of the genetic pathogenic pathways involved in heart failure.