Molecular and cellular evidence for the impact of a hypertrophic cardiomyopathy-associated RAF1 variant on the structure and function of contractile machinery in bioartificial cardiac tissues.

Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1-associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1S257L-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease.

Generation of a genetically-modified induced pluripotent stem cell line harboring an oncogenic gene variant KRAS p.G12V.

Activating KRAS codon 12 gene variants are known to cause severe RAS-MAPK and PI3K-AKT signaling pathway hyperactivity and are frequently involved in the development of various carcinomas. Here, we describe the generation of a human iPSC line harboring the common oncogenic KRAS p.G12V variant by using CRISPR/Cas9 technology. The established KRASG12V iPSC line allows the study of oncogenic KRAS-induced signaling dysregulation and its impact on cell physiology in various iPSC-derived cell types and tissues. Furthermore, it might serve as a powerful platform for drug and toxicity screenings to identify new chemotherapeutic drugs.