INTRODUCTION: Pathological cardiac hypertrophy is a major cause of heart failure and is often induced by the stenosis of the aortic valve. The disease is multifactorial and accompanied by dysregulation of various signaling pathways. While molecular signatures of individual cell types have been extensively studied, less resources have been allocated to understand the intercellular cross-talk between the different cardiac cells during disease. Here, we applied high-resolution transcriptomic analysis on single-nuclei level, allowing us to identify disturbed intra- and intercellular communication in the hypertrophied heart. 

 

RESULTS: We analyzed single nuclei RNA sequencing data of cardiac tissue from five patients with cardiac hypertrophy caused by severe aortic stenosis and 14 location-matched healthy cardiac samples. In-silico analysis of inter- and intracellular communication pathways revealed striking downregulation of ligand-receptor interactions between cardiomyocytes (CMs) and other cell types in hypertrophied hearts compared to healthy controls (reduced by 74.41%). Several of the predominantly affected interacting receptors and ligands could be assigned to the FGF family and the ephrin-Eph family. In the first step we validated the expression regulation of the identified genes on mRNA level in a phenylephrine-induced CM hypertrophy model and on protein level in human and murine cardiac hypertrophic tissue. During validation FGF1, EphB1 and ephrin-A5 raised our attention since they were predominantly expressed and dysregulated in diseased CMs (on single-nuclei level FGF1: 0.16-fold, ephrin-A5: 0.07-fold, EphB1: 0.01-fold downregulated in disease, all p<0.0001). Furthermore, we characterized the function of the selected target genes. 

FGF1 is expressed in CMs and mural cells and has several interaction partners e.g. FGFR1, FGFR2 and TGFBR3, located on different cardiac cells. Interestingly, in vitro stimulation of neonatal rat cardiomyocytes with recombinant FGF1 protein provoked hypertrophy. Similar findings could be observed in a multicellular human organoid model, suggesting that FGF1 activates pro-hypertrophic pathways.

The ephrin ligand ephrin-A5 is predominantly expressed in CMs and is known to interact with EphA receptors, expressed on CMs, endothelial cells and fibroblasts. The in vitro stimulation of CMs with recombinant ephrin-A5 protein protected the cells from phenylephrine-induced hypertrophic response. 

The third target of interest, EphB1, is the Eph receptor with the highest expression level in CMs. We identified that its interaction with the endothelial-derived ephrin-B2 is necessary for the maintenance of cellular homeostasis of the CMs as disruption of the interaction in a co-culture experiment leads to CM hypertrophy and increased stress marker expression. Further, stimulation of the interaction by adding exogenous ligand ephrin-B2 to the cells or by overexpressing the receptor EphB1, protected the CMs from hypertrophy.

 

CONCLUSION: We identified impaired inter- and intra-cellular cardiac communication in the hypertrophic heart induced by aortic valve stenosis in humans. We identified FGFs, ephrins and Eph-receptors to be involved in the hypertrophic response of cardiomyocytes by interacting with other cardiac cell types. This study contributes to a better understanding of underlying mechanism contributing to pathological cardiac hypertrophy and identified potential therapeutic targets.