Alteration of myocardial structure and function in RAF1-associated Noonan syndrome: Insights from cardiac disease modeling based on patient-derived iPSCs

Abstract

Noonan syndrome (NS), the most common among the 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.770C-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 disease phenotype was partly reverted by using both MEK inhibition, and a gene-corrected isogenic RAF1L257S cell line. 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. These insights represent a basis to develop future targeted therapeutic approaches.

LZTR1 polymerization provokes cardiac pathology in recessive Noonan syndrome

“…Interestingly, no myofibrillar disarray was observed in our cell model. However, the presence of myofibril disarray in NS remains controversial: whereas structural defects were described in RAF1 -associated iPSC models, 15,41 we and others did not observe any impact on sarcomere structures or myofibril organization in LZTR1 -related, PTPN11 -related, and BRAF -related iPSC-CMs, 10,14,16 implying potential genotype-dependent differences in the manifestation of myofibril disassembly in NS. Missense variants in LZTR1 located within the Kelch domain are predicted to affect substrate recognition, whereas missense variants in the BTB-BACK domain are assumed to impair either binding of cullin 3, proper homo-dimerization, or correct subcellular localization.…”

“…Interestingly, no myofibrillar disarray was observed in our cell model. However, the presence of myofibril disarray in NS remains controversial: whereas structural defects were described in RAF1-associated iPSC models, 15,41 we and others did not observe any impact on sarcomere structures or myofibril organization in LZTR1-related, PTPN11-related, and BRAF-related iPSC-CMs, 10,14,16 implying potential genotype-dependent differences in the manifestation of myofibril disassembly in NS.…”

The Microenvironment of the Pathogenesis of Cardiac Hypertrophy

“…This ultimately triggers stress signals released by different cell types of the microenvironment to compensate for the wall tension increase, resulting in a hypertrophic growth response [ 4 , 5 ]. Individual cardiomyocytes can increase in length and/or width in response to hypertrophic stimuli depending on the intracellular signaling cascades involved [ 6 , 7 , 8 ].…”

Modeling (not so) rare developmental disorders associated with mutations in the protein-tyrosine phosphatase SHP2

“…In contrast, both cardiac tissue from NS patients with CRAF and SHP2 variants, and iPSCs with SHP2-N308S variant, display cellular hyperplasia, rather than hypertrophy, which is presumably due to the increased cell cycle progression (Meier et al, 2021). However, hypertrophic phenotypes are observed in cardiomyocytes differentiated from NS-derived iPSCs with CRAF mutations (Jaffré et al, 2019;Nakhaei-Rad et al, 2022). The observed discrepancy may be related to the genetic background of the patients and/or the difference in the mutated gene itself: whereas NS-patients with a mutation in the CR2 domain of CRAF develop HCM in more than 90% of cases, this is the case for only 10% of NS-patients with PTPN11 mutations.…”