Pathological cardiac hypertrophy is a leading cause of heart failure, but knowledge of the full repertoire of cardiac cells and their gene expression profiles in the human hypertrophic heart is missing. Here, by using large-scale single-nucleus transcriptomics, we present the transcriptional response of human cardiomyocytes to pressure overload caused by aortic valve stenosis and describe major alterations in cardiac cellular crosstalk. Hypertrophied cardiomyocytes had reduced input from endothelial cells and fibroblasts. Genes encoding Eph receptor tyrosine kinases, particularly EPHB1, were significantly downregulated in cardiomyocytes of the hypertrophied heart. Consequently, EPHB1 activation by its ligand ephrin (EFN)B2, which is mainly expressed by endothelial cells, was reduced. EFNB2 inhibited cardiomyocyte hypertrophy in vitro, while silencing its expression in endothelial cells induced hypertrophy in co-cultured cardiomyocytes. Our human cell atlas of the hypertrophied heart highlights the importance of intercellular crosstalk in disease pathogenesis and provides a valuable resource.
Introduction The pathophysiology of cardiac hypertrophy is multifactorial and is accompanied by the dysregulation of various signaling pathways contributing to cardiac dysfunction and heart failure. While the hypertrophic response of cardiomyocytes (CM) has been extensively studied, the interplay of CMs with the non-parenchymal cells in the heart is less explored. Here, we apply high-resolution transcriptomic analysis on single cell level allowing the identification of cellular responses and communication in the hypertrophic human heart. Results We analyzed single nuclei RNA sequencing data of cardiac tissues from five patients with aortic stenosis and cardiac hypertrophy and 13 matched healthy subjects. Bioinformatic data analysis of 88,536 nuclei followed by clustering led to the identification of specific heterogenic cell type signatures. Analyzing cell type specific gene expression signatures, we found the expected up-regulation of the cardiac stress MYH7 (4.15-fold), CMYA5 (4.89-fold) and XIRP2 (6.13-fold) in cardiomyocytes (CM) (all p<0.0001). Fibroblasts showed increased expression of genes associated with fibrosis and activation markers such as periostin (POSTN; 6.84-fold, p<0.0001). In-silico analysis of intercellular communication pathways revealed a striking downregulation of ligand-receptor interactions between CMs and other cells in hypertrophic compared to healthy controls indicating that CMs are less responsive to signals from fibroblasts and endothelial cells (ECs) in the hypertrophied heart. Particularly, CM showed reduced expression of receptor tyrosine kinases of the Ephrin family and FGF-family members. Specifically, Ephrin-B1 was significantly downregulated in CMs of the hypertrophic hearts (0.01-fold, p<0.0001). The down-regulation of Ephrin-B1 was additionally validated on protein level using histological sections of hypertrophic cardiomyopathy patients (n=6) versus healthy controls (n=5) (0.66-fold, p=0.02). In-vitro studies in neonatal cardiomyocytes further demonstrated that activation of the Ephrin-B1 receptor by the agonist Ephrin-B2 induced cardioprotective effects. Thus, Ephrin-B2 inhibited phenylephrine (PE) induced Nppb expression by 0.775-fold (vs. PE) and hypertrophic growth (0.774-fold reduction of cell size vs. PE). Similar findings were observed in PE-stimulated human cardiac organoids, which showed a 0.58-fold reduction of size in response to Ephrin-B2 treatments compared to PE alone. Conclusion Investigating the cross-talk in cardiac hypertrophy reveals novel disturbed communication signatures, with a striking reduction in the intercellular communication pathways of CMs. Reduced expression of receptors of the Ephrin family, particularly Ephrin-B1, in CM may prevent cardioprotective signaling by the agonist Ephrin-B2, which is highly expressed in ECs, leading to inhibition of cardioprotective cross-talk between ECs and CMs in the hypertrophic heart. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Dr. Rolf M. Schwiete StiftungDie Deutsche ForschungsgemeinschaftGerman Center for Cardiovascular Research
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