Background: Activated cardiac fibroblasts play an essential role in the pathological remodeling during heart failure via the production and degradation of the extracellular matrix. Fibroblast activation protein (FAP) is a serine protease involved in matrix remodeling which is upregulated in heart failure. Here we investigated the effects of a stromal cell-specific FAP depletion on Engineered Human Myocardium (EHM) function.

Methods and Results: A homozygous and heterozygous FAP knockout was created by CRISPR/Cas9 editing in wildtype (WT) human iPSC. The iPSC lines were then subjected to a cardiac stromal cell differentiation protocol. The FAP knockout was confirmed via Western Blot and flow cytometry. No differences during differentiation were observed. Expression of POSTN, PDGFRα and VIM confirmed stromal cell identity for WT- and FAP-KO stromal cells. Notably, FAP-KO stromal cells demonstrated an increased abundance of characteristic myofibroblast proteins (3-fold increase of ACTA2 and POSTN, 5-fold increase of CTGF vs. WT). High purity iPSC-derived cardiomyocytes were combined with either FAP-KO or WT stromal cells (70:30 ratio) to generate EHM. No significant differences in tissue functionality were observed during the maturation of the EHM. WT and FAP-KO EHM were then submitted to a 7-day treatment with 5 ng/ml TGFβ-1 and 10 µM L-Noradrenaline to simulate heart failure. This treatment regimen was associated with increased expression of myofibroblast genes (POSTN, COL1A1, FAP) and contractile dysfunction with reduced EHM shortening (-60±6% and -61±3%, WT [n=10] vs FAP-KO [n=26]) and increase in beating frequency (+43±7% and +48±15%, WT [n=10] vs FAP-KO [n=25]); no differences between WT and FAP-KO were observed.

Summary and Conclusion: The knockout of FAP leads to an increased basal expression of myofibroblast protein in iPSC-derived human stromal cells, but does not alter the functional phenotype of EHM in a model of neurohumoral stress.