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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 RAF1 S257L 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 RAF1 S257L 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 RAF1 S257L -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., (© 2023. The Author(s).)

Background: More than 90% of individuals with Noonan syndrome (NS) with mutations clustered in the CR2 domain of RAF1 present with severe and often lethal hypertrophic cardiomyopathy (HCM). The signaling pathways by which NS RAF1 mutations promote HCM remain elusive, and so far, there is no known treatment for NS-associated HCM., Methods: We used patient-derived RAF1 S257L/+ and CRISPR-Cas9-generated isogenic control inducible pluripotent stem cell (iPSC)-derived cardiomyocytes to model NS RAF1-associated HCM and to further delineate the molecular mechanisms underlying the disease., Results: We show that mutant iPSC-derived cardiomyocytes phenocopy the pathology seen in hearts of patients with NS by exhibiting hypertrophy and structural defects. Through pharmacological and genetic targeting, we identify 2 perturbed concomitant pathways that, together, mediate HCM in RAF1 mutant iPSC-derived cardiomyocytes. Hyperactivation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), but not extracellular regulated kinase 1/2, causes myofibrillar disarray, whereas the enlarged cardiomyocyte phenotype is a direct consequence of increased extracellular regulated kinase 5 (ERK5) signaling, a pathway not previously known to be involved in NS. RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iPSC-derived cardiomyocytes and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways that could contribute to the NS-associated HCM., Conclusions: Taken together, the results of our study identify the molecular mechanisms by which NS RAF1 mutations cause HCM and reveal downstream effectors that could serve as therapeutic targets for treatment of NS and perhaps other, more common, congenital HCM disorders.

RASopathies are caused by mutations in genes that alter the MAP kinase pathway and are marked by several malformations with cardiovascular disorders as the predominant cause of mortality. Mechanistic insights in the underlying pathogenesis in affected cardiac tissue are rare. The aim of the study was to assess the impact of RASopathy causing mutations on the human heart. Using single cell approaches and histopathology we analyzed cardiac tissue from children with different RASopathy-associated mutations compared to age-matched dilated cardiomyopathy (DCM) and control hearts. The volume of cardiomyocytes was reduced in RASopathy conditions compared to controls and DCM patients, and the estimated number of cardiomyocytes per heart was ∼4–10 times higher. Single nuclei RNA sequencing of a 13-year-old RASopathy patient (carrying a PTPN11 c.1528C > G mutation) revealed that myocardial cell composition and transcriptional patterns were similar to <1 year old DCM hearts. Additionally, immaturity of cardiomyocytes is shown by an increased MYH6/MYH7 expression ratio and reduced expression of genes associated with fatty acid metabolism. In the patient with the PTPN11 mutation activation of the MAP kinase pathway was not evident in cardiomyocytes, whereas increased phosphorylation of PDK1 and its downstream kinase Akt was detected. In conclusion, an immature cardiomyocyte differentiation status appears to be preserved in juvenile RASopathy patients. The increased mass of the heart in such patients is due to an increase in cardiomyocyte number (hyperplasia) but not an enlargement of individual cardiomyocytes (hypertrophy). [Display omitted] • Pediatric patients with RASopathies exhibit an increased heart mass. • Cardiomyocyte size and volume are reduced in pediatric patients with RASopathies. • Cardiomyocyte numbers per heart are increased in pediatric RASopathy patients. • Cardiomyocytes in juvenile RASopathy have an immature state. • Elevated LV mass in RASopathy patients is mainly due to cardiomyocyte hyperplasia. [ABSTRACT FROM AUTHOR]

Introduction: Noonan Syndrome (NS) is caused by mutations that affect the RAS/ERK1/2-MAPK pathway. More than 90% of NS individuals with mutations clustered in the CR2 domain of RAF1 present with severe and often lethal hypertrophic cardiomyopathy (HCM). The signaling pathways by which NS RAF1 mutations promote HCM remain elusive, and so far, there is no known treatment for NS-associated-HCM. Methods: Here, we used patient-derived RAF1S257L/+ and CRISPR-Cas9-generated isogenic control iPSC-derived cardiomyocytes (iCMs) to delineate the molecular mechanisms underlying HCM in NS-associated RAF1 mutations. Results: We found that mutant iCMs phenocopy the pathology seen in NS by exhibiting hypertrophy and structural defects (n=5). At the molecular level, while RAF1S257L/+ iCMs had elevated RAF1 and MEK1/2 activities, ERK1/2 itself was modestly enhanced. To test whether ERK1/2 activation was responsible for increased size of RAF1S257L/+ iCMs, we inhibited MEK1/2 activity with three different inhibitors (U0126, PD or Tram) and found that, while neither PD nor Tram reduced mutant iCMs area, both rescued myofibrillar disarray (n=3), suggesting that MEK1/2 and/or ERK1/2 regulate myofilament organization but not cell size. Since, PD and Tram block MEK1/2 activation by RAF, whereas U0126 prevents activation of ERK1/2 by active MEK1/2, we hypothesized that MEK1/2 rather than ERK1/2 regulates myofilament organization. Accordingly, we found that specific inhibition or ERK1/2 activity by ectopic expression of MKP3 did not rescue myofibrillar disarray in mutant RAF1 iCMs. Strikingly, U0126 was able to reduce iCMs surface area, suggesting that an ERK1/2-independent pathway was responsible for hypertrophy. Since U0126 also targets MEK5, we treated RAF1S257L/+ iCMs with a specific MEK5/ERK5 inhibitor and found that hypertrophy was drastically reduced (n=3), indicating that the ERK5 pathway triggered hypertrophy in RAF1S257L/+ iCMs. Finally, RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iCMs and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways and known as critical regulators of cardiac development, as well as extracellular matrix, cytoskeletal and actin assembly homeostasis that could contribute to the NS-associated HCM. Conclusion: Taken together, our study identifies the molecular mechanisms by which NS RAF1 mutations cause HCM and reveals downstream effectors that could serve as therapeutic targets for treatment of HCM in NS patients. [ABSTRACT FROM AUTHOR]