Background: Cardiomyocyte hypertrophy and impaired cardiac angiogenesis are major drivers of maladaptive myocardial remodelling in the diseased human heart, which can be partially attributed to an impaired intercellular communication between cardiomyocytes and endothelial cells. Therefore, identifying mediators that regulate myocardial repair and cardiac angiogenesis is of high translational relevance.

Methods: Single-nuclear RNA sequencing of human heart tissue was performed to discover the transcriptional heterogeneity and molecular changes of cardiomyocytes from two healthy heart samples, five patients with cardiac hypertrophy and two samples with advanced heart failure on single-cell resolution. Unsupervised clustering of 25,084 nuclei led to the identification of 9 distinct cardiomyocyte clusters.

Results: Based on canonical markers (NPPA, NPPB, ACTA1) a cardiomyocyte disease score was applied to dissect diseased cardiomyocytes at the molecular level. Interestingly, analysis of differentially expressed genes revealed downregulation of “Endothelial growth factor like 7” (EGFL7) in diseased cardiomyocytes. Consistently, in silico analysis of bulk RNA sequencing data confirmed decreased Egfl7 expression in isolated cardiomyocytes upon cardiac injury in mice. While EGFL7 has been primarily studied for its role in endothelial cells, we investigated its intrinsic function in cardiomyocyte biology. Since endogenous EGFL7 expression was downregulated in human and murine cardiomyocytes upon cardiac injury, we assessed the functional consequences of siRNA mediated silencing on the intrinsic regenerative capacity in vitro. Surprisingly, silencing of Egfl7 in cardiomyocytes revealed a shift towards an increased proportion of cardiomyocytes during cell cycle S-phase, which was accompanied by an 1.58±0.069-fold elevated number of phosphorylated histone H3 (pH3) positive nuclei in immunofluorescence stainings (p<0.05). Consistently, we detected reactivation of the embryonic gene program on mRNA level (Nppa, Nppb, Acta1) in cardiomyocytes following siRNA mediated knockdown of Egfl7compared to scrambled control. In addition to its cell-intrinsic impact on cardiomyocyte biology, we assessed whether disease-induced downregulation of EGFL7 in cardiomyocytes might also affect cardiac angiogenesis by paracrine crosstalk. Hence, we exposed human endothelial cells to conditioned media of Egfl7-deficient cardiomyocytes compared to control media. While the migratory capacity was not affected, endothelial cells treated with medium of Egfl7-deficient cardiomyocytes exhibited an impaired cell sprouting and tube formation. 

Conclusions: Together, our study identifies cardiomyocyte-derived EGFL7 as an autocrine and paracrine regulator of cardiomyocyte proliferation and vascular growth.