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

Abstract

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.

The Microenvironment of the Pathogenesis of Cardiac Hypertrophy

“…The alpha-isoform of PKC directly activates RAF1 [ 281 ], providing evidence for a complex link between the signaling pathway downstream of growth factor receptors in the context of cardiac hypertrophy. Others have reported that GPCR signaling can be linked directly to RAS GTPase ( Figure 2 ) [ 282 ], and GTP-bound RAS interacts with many downstream effectors which in turn transmit the signal for activating multiple signaling pathways [ 283 , 284 ], potentially promoting hypertrophic responses in cardiomyocytes. Additionally, others reported that the C-terminus of the AT1 receptor associates with JAK2 upon binding of the ligand, resulting in JAK2/STAT3 pathway activation [ 285 , 286 ], indicating another example of the crosstalk of signaling pathways in response to hypertrophy-associated stress signals.…”

SIRT4 as a novel interactor and candidate suppressor of C-RAF kinase in MAPK signaling

“…The MAPK signaling pathway plays a critical role in the regulation of various cellular processes such as differentiation, survival, and, in particular, proliferation ( Zhang & Liu, 2002 ; Guo et al, 2020 ; Ullah et al, 2022 ). Dysregulation of this pathway is frequently associated with the initiation and progression of human diseases, including cancer ( Degirmenci et al, 2020 ) and developmental disorders such as RASopathies ( Dar & Brady, 2022 ), the latter exemplified by the RAF1 S257L mutation causing cardiomyopathy ( Dhandapany et al, 2014 ; Jaffre et al, 2019 ; Nakhaei-Rad et al, 2023a ). As a key component of the MAPK pathway, C-RAF is activated by upstream receptor–RAS signaling and subsequently activates several downstream effectors, particularly MEK1/2 kinases and subsequently ERK1/2 signaling ( Wimmer & Baccarini, 2010 ; Matallanas et al, 2011 ; Ullah et al, 2022 ).…”

“…Jaffre et al, 2019;Nakhaei-Rad et al, 2023a). As a key component of the MAPK pathway, C-RAF is activated by upstream receptor-RAS signaling and subsequently activates several downstream effectors, particularly MEK1/2 kinases and subsequently ERK1/2 signaling(Wimmer & Baccarini, 2010;Matallanas et al, 2011;Ullah et al, 2022).…”

Identification of SIRT4 as a novel paralog-specific interactor and candidate suppressor of C-RAF kinase in MAPK signaling

“…The MAPK signaling pathway plays a crucial role in regulating various cellular processes such as differentiation, survival, and in particular proliferation (56)(57)(58). Dysregulation of this pathway is often associated with the development and progression of human diseases, including cancer (4) and developmental disorders like RASopathies (59), the latter exemplified by the RAF1 S257L mutation causing cardiomyopathy (60)(61)(62). As a key component of the MAPK pathway, C-RAF is activated by upstream receptor-RAS signaling and subsequently activates various downstream effectors, particularly MEK1/2 kinases and thus ERK1/2 signaling (21,22,57).…”