Cellular interplay via cytokine hierarchy causes pathological cardiac hypertrophy in RAF1-mutant Noonan syndrome

Noonan syndrome (NS) is caused by mutations in RAS/ERK pathway genes, and is characterized by craniofacial, growth, cognitive and cardiac defects. NS patients with kinase-activating RAF1 alleles typically develop pathological left ventricular hypertrophy (LVH), which is reproduced in Raf1(L613V/+) knock-in mice. Here, using inducible Raf1(L613V) expression, we show that LVH results from the interplay of cardiac cell types. Cardiomyocyte Raf1(L613V) enhances Ca2+ sensitivity and cardiac contractility without causing hypertrophy. Raf1(L613V) expression in cardiomyocytes or activated fibroblasts exacerbates pressure overload-evoked fibrosis. Endothelial/endocardial (EC) Raf1(L613V) causes cardiac hypertrophy without affecting contractility. Co-culture and neutralizing antibody experiments reveal a cytokine (TNF/IL6) hierarchy in Raf1(L613V)-expressing ECs that drives cardiomyocyte hypertrophy in vitro. Furthermore, postnatal TNF inhibition normalizes the increased wall thickness and cardiomyocyte hypertrophy in vivo. We conclude that NS-cardiomyopathy involves cardiomyocytes, ECs and fibroblasts, TNF/IL6 signalling components represent potential therapeutic targets, and abnormal EC signalling might contribute to other forms of LVH.