BACKGROUND: More than 90% of Noonan syndrome (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: We used patient-derived RAF1 and CRISPR-Cas9-generated isogenic control iPSC-derived cardiomyocytes (iCMs) to model NS RAF1-associated HCM and to further delineate the molecular mechanisms underlying the disease.

RESULTS: We show that mutant iCMs phenocopy the pathology seen in patient NS hearts by exhibiting hypertrophy and structural defects. Through pharmacological and genetic targeting, we identify two perturbed concomitant pathways that, together, mediate HCM in RAF1 mutant iCMs; hyper-activation of MEK1/2, but not ERK1/2, causes myofibrillar disarray, whereas the enlarged cardiomyocyte phenotype is a direct consequence of increased ERK5 signaling, a pathway not previously known to be involved in NS. RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iCMs and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways that could contribute to the NS-associated HCM.

CONCLUSIONS: 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 NS, and perhaps other, more common, congenital HCM disorders as well.