Unique mechanistic insights into the beneficial effects of angiotensin-(1-7) on the prevention of cardiac fibrosis: A metabolomic analysis of primary cardiac fibroblasts

Cell metabolic pathways are highly conserved among species and change rapidly in response to drug stimulation. Therefore, we explore the effects of angiotensin-(1-7) in a primary cell model of cardiac fibrosis established in angiotensin II-stimulated cardiac fibroblasts via metabolomics analysis and further clarify the potential protective mechanism of angiotensin-(1-7).After exposing cardiac fibroblasts to angiotensin II and/or angiotensin-(1-7), 172 metabolites in these cells were quantified and identified by gas chromatography-mass spectrometry. The data were subsequently analyzed by orthogonal partial least squares discriminant analysis to shortlist biochemically significant metabolites associated with the antifibrotic action of angiotensin-(1-7). Seven significant metabolites were identified: 10,13-dimethyltetradecanoic acid, arachidonic acid, aspartic acid, docosahexaenoic acid (DHA), glutathione, palmitelaidic acid, and pyroglutamic acid. By metabolic network analysis, we found that these metabolites were involved in six metabolic pathways, including arachidonic acid metabolism, leukotriene metabolism, and the γ-glutamyl cycle. Since these metabolic pathways are related to calcium balance and oxidative stress, we further verified that angiotensin-(1-7) suppressed the abnormal extracellular calcium influx and excessive accumulation of intracellular reactive oxygen species (ROS) in angiotensin II-stimulated cardiac fibroblasts. Furthermore, we found that angiotensin-(1-7) suppressed the abnormal calcium- and ROS-dependent activation of calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ), the increased expression of CaMKIIδ-related proteins (NADPH oxidase 4 (Nox4), cellular communication network factor 2 (CTGF), and p-ERK1/2), and excessive collagen deposition in vitro and in vivo.Angiotensin-(1-7) can ameliorate the angiotensin II-stimulated metabolic perturbations associated with cardiac fibroblast activation. These metabolic changes indicate that modulation of calcium- and ROS-dependent activation of CaMKIIδ mediates the activity of angiotensin-(1-7) against cardiac fibrosis. Moreover, pyroglutamic acid and arachidonic acid may be potential biomarkers for monitoring the antifibrotic action of angiotensin-(1-7).