Endothelial NADPH oxidases protect against sympathetic nervous system hyperactivity-induced cardiac dysfunction and remodeling in mice: contribution of SGLT2

Abstract

Abstract Background NADPH oxidase-derived reactive oxygen species (ROS) contribute to cardiac dysfunction, often characterized by coronary microvascular dysfunction, an inflammatory response and cardiomyocyte hypertrophy. Hyperactivity of the sympathetic nervous system (SNS) induces oxidative stress, promoting cardiac dysfunction and the development of heart failure. Selective inhibitors of sodium-glucose co-transporter 2 (SGLT2), have shown remarkable cardioprotective effects in clinical studies. Recently, SGLT2 inhibitors have been reported to prevent endothelial dysfunction and pro-inflammatory responses in endothelial cells in response to angiotensin II involving NADPH oxidases. Purpose Therefore, the aim of the study was to determine whether endothelial NADPH oxidases promote SNS-induced cardiac dysfunction and to clarify the role of SGLT2. Methods Male wild-type mice and mice lacking the NADPH oxidase subunit p22phox in the endothelium (p22phox ecKO, 11-week-old) were treated with isoproterenol (100 mg/kg) for five consecutive days and sacrificed at day 14. Hemodynamic measurements of left (LV) and right (RV) ventricles were performed by a transthoracic approach. Heart tissue sections were stained with Sirius red to evaluate fibrosis and wheat germ agglutinin to assess cardiomyocyte size. Cultured human microvascular endothelial cells (HMEC-1) were stimulated with 100 nM isoproterenol and ROS levels were assessed by dihydroethidium fluorescence. The expression level of target genes and proteins was assessed by quantitative real-time PCR and Western blot, respectively. siRNA approaches were used to down-regulate either the NADPH oxidase subunit p22phox or SGLT2. Results The isoproterenol treatment increased LV and RV systolic pressures in wild-type mice but not in p22phox ecKO mice. p22phox ecKO mice were protected against isoproterenol-induced fibrosis, cardiac remodeling characterized by upregulation of mRNA levels of ANP, BNP and β-MHC, and pulmonary congestion. LV remodeling was associated with upregulation of the NADPH oxidase subunits p22phox, Nox2, and Nox4 as well as of SGLT2 in wild-type mice, however no such effects were observed in p22phox ecKO mice. Exposure of HMEC-1 to isoproterenol stimulated the formation of ROS and caused an upregulation of p22phox and SGLT2 protein levels in a time- and concentration-dependent manner. No such effects were observed following silencing of either p22phox or SGLT2, or use of a selective SGLT2 inhibitor. Conclusion Deletion of the NADPH oxidase subunit p22phox in the endothelium protected against SNS hyperactivity induced LV cardiac dysfunction and remodeling, and prevented upregulation of SGLT2. Since depletion of SGLT2 prevented the pro-oxidant response to isoproterenol in endothelial cells, the endothelial NADPH oxidase/SGLT2 pathway seems to have a prominent role in promoting cardiac remodeling and dysfunction in SNS hyperactivity. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Deutsches Herzzentrum München