Inhibition of dysregulated il-8-cxcr1 paracrine cellular crosstalk reverses cardiac hypertrophy and fibrosis in noonan syndrome

Abstract Background Pathogenic variants in the RAS/MAPK signalling pathway cause severe, early-onset hypertrophic cardiomyopathy (HCM) in Noonan syndrome (NS). In particular, hyperactivating mutations in RIT1 and RAF1, as well as loss-of-function mutations in LZTR1 are associated with HCM development, showing strong genotype–phenotype correlations. Despite the high prevalence of NS-associated HCM, there are currently no curative therapies available. Although the cell-autonomous effects of these mutations are well characterized, the role of paracrine signalling in cardiac hypertrophy and fibrosis remains poorly defined. Purpose This study investigates how cardiomyocytes (CMs) and non-CMs contribute to HCM via molecular and paracrine mechanisms, with the aim of identifying novel therapeutic targets. Methods We established 2D and multicellular 3D human iPSC models of LZTR1 deficiency, including NS patient-derived iPSCs harbouring pathogenic RAS-MAPK variants. These models were analysed at cellular, molecular, and functional levels using advanced imaging, proteomics, transcriptomics, and engineered tissue platforms. Results NS cardiac fibroblasts (CFs) exhibited increased RIT1-dependent cell cycle activity, which was fully reversed by RIT1 knockdown. 3D engineered connective tissues (ECTs) from NS CFs displayed a profibrotic signature, including persistent myofibroblast activation, excessive extracellular matrix deposition, and increased tissue stiffness. Notably, NS CFs secreted elevated levels of interleukin-8 (IL-8), resulting in impaired paracrine signalling. Both conditioned medium from NS CFs and recombinant IL-8 induced hypertrophic remodeling in neighbouring CMs. Mechanistic studies utilizing a fluorescent collagen reporter iPSC line and receptor-specific blockade revealed that IL-8 signalling predominantly occurs via the CXCR1 receptor, with minimal involvement of the canonical CXCR2. Pharmacological inhibition of IL-8–CXCR1 signalling using a CXCR1 receptor antagonist reversed disease phenotypes in NS CFs, CMs, and ECTs. Furthermore, CXCR1 receptor inhibition restored normal contractile function in NS engineered 3D cardiac tissues, highlighting its potential as a therapeutic target for NS-associated HCM. Conclusions We identified aberrant IL-8–CXCR1 signalling as a convergent pathogenic mechanism across genetically distinct NS genotypes. Therefore, targeting the IL-8–CXCR1 axis with a CXCR1 receptor inhibitor represents a promising and translatable strategy to mitigate cardiac fibrosis and hypertrophy in NS-associated HCM, providing a novel therapeutic avenue for NS-driven cardiac disease.