The heart cell secretome, comprising soluble proteins and membranous particles called extracellular vesicles (EVs), mediates essential intercellular signalling to regulate homeostasis and response to pathological remodelling. Cardiac fibroblasts are enigmatic cells which perform central roles in the development of cardiac pathologies and fibrosis, however many mechanisms behind their actions remain unknown.
To investigate intracardiac signalling and its effect on cardiac fibroblasts, in vitro derived cardiomyocyte secretome and ex vivo derived hypertensive-heart EVs (cEVs) were treated onto primary cardiac fibroblasts. Remodelled fibroblasts underwent solid-phase-enhanced sample preparation (SP3) and data dependent acquisition (DDA) MS/MS analysis. Parallel functional and proteomic characterisations revealed signalling from dysfunctional cardiomyocytes increased oxidative stress and fibroblast activation, and dysregulated protein networks associated with antioxidant activity, lysosomes, glycosylation, and translation. TiO2-based phosphoproteomic analyses of cardiomyocyte-secretome remodelled cardiac fibroblasts further delineated a potential role for casein kinase II (CK2) activity in pathological cellular remodelling. cEVs isolated from Angiotensin II induced hypertensive mice resulted in sex and pathology dependent effects on the proteome, altering expression of components involved in antioxidant activity, RNA metabolism, fatty acid metabolism, and glycosylation processing. Moreover, regulation of many components in fibroblasts, including CK2, were associated with a significant interaction between disease and sex influences.
Here, we demonstrate a proteomics and phosphoproteomics pipeline to understand inter- and intra-cellular signalling, including through EV and secretome mediated communications, and provide molecular insights into their contribution to cardiac remodelling through regulation of fibroblasts.