Transcriptional regulation of endothelial cell cycle arrest at the transition from cardiac hypertrophy to heart failure

Introduction 

Chronic heart failure involves maladaptive left ventricular remodeling, including cardiomyocyte hypertrophy and fibrosis. Initially compensatory, hypertrophy later contributes to decompensation as capillary formation fails to meet oxygen demands of enlarged cardiomyocytes. Cardiomyocytes support endothelial proliferation via paracrine signals (e.g. VEGF), but impaired endothelial sprouting during remodeling disrupts this balance. This study investigates transcriptional regulators of endothelial proliferation in the transition from hypertrophy to heart failure to identify drivers of vascular adaptation in heart failure.

Methods and results 

C57BL/6 mice underwent transverse aortic constriction (TAC) or sham surgery (n= 4-5 per group) and were followed for 6 hours, one, three, seven, or 28 days post-surgery. Echocardiography showed a moderate decline of the ejection fraction already after 3 days, progressing to the decompensated stage between 7 and 28 days. While capillary density as assessed by immunohistochemistry initially matched hypertrophic growth, it declined significantly during the later stage, suggesting impaired angiogenesis is associated with the onset of heart failure.

To determine gene expression at the different time points, endothelial cells and cardiomyocyte nuclei were isolated for RNA sequencing or qPCR, respectively, using fluorescence assisted cell sorting. Vegfa expression in cardiomyocytes was upregulated after TAC, with a marked increase between 7 and 28 days post-intervention. Regulation of cell cycle marker genes such as Ki-67 (Mki67) in endothelial cells indicated high proliferation rates during the hypertrophic phase. However, despite high VEGF signaling from cardiomyocytes endothelial cells underwent cell cycle arrest between day 7 and 28.

We conducted a weighted coexpression network analysis across all time points, identifying 31 gene clusters in endothelial cells. One cluster of 180 genes showed a strong positive correlation with TAC in the compensated state (r = 0.8; p < 0.0001) and a negative correlation in the decompensated state (r = -0.27; p < 0.06). Functional annotation indicated enrichment in cell cycle processes, suggesting roles in angiogenesis.

Network analysis predicted ATAD2 and TCF19 to be key transcriptional regulators of endothelial proliferation and angiogenesis. siRNA knockdown of TCF19 or ATAD2 in HUVECs significantly impaired angiogenic capacity, with reduced tube formation (tube length, siTCF19 -72.51 %, q<0.001; siATAD2 -55.59%,  q=0.004 vs. siCTRL), decreased migration in scratch assay (wound closure, siTCF19 0.54, q=0.002; siATAD2 0.2, q<0.001 vs. siCTRL 0.84), and lower proliferation rates (cell count fold after 72h: siTCF19 1.25, q<0.001; siATAD2 1.44, q=0.002 vs. siCTRL 2.02). RNA-seq analysis after knockdown of either TCF19 or ATAD2 indicated that downregulated genes (log2FC<0; q<0.05) were primarily linked to mitotic cell cycle. Positive correlation of both gene expression profiles suggests overlapping roles of these genes in regulating endothelial cell proliferation (R²=0.4; p<0.001).

Conclusion

Gene network analysis identified key gene clusters and transcriptional regulators that control endothelial cell cycle during cardiac hypertrophic growth and are involved in the transition from compensated hypertrophy to heart failure. Interference with downstream signaling rather than increasing VEGF levels may be a promising strategy to improve angiogenesis in heart failure.