Background: Cardiac fibrosis is an integral part of myocardial remodelling in response to stress. Mice with cardiac myocyte-specific overexpression of beta3-adrenergic receptor (β3-AR) are protected not only from the development of hypertrophy but also cardiac fibrosis. As overexpression was restricted to cardiac myocytes (CM), we postulated that the protection involved a paracrine cross-talk to cardiac fibroblasts (CF).
Methods: We developed a model of superfusion of CF in serum-free conditions with media conditioned by cultured CM with adenoviral expression of the human β3AR and treated or not with phenylephrine (PE). Cardiac extracts were analysed in wild type (WT) and heterozygote transgenic mice with cardiac myocyte-specific overexpression of human β3AR (hβ3TG) submitted to transaortic constriction (TAC) for 9 weeks.
Results: Incubation of CF with medium conditioned by control CM treated with PE stimulated their proliferation, migration, myofibroblats differentiation (α-SMA expression) and pro-collagen I expression compared with medium from CM not treated with PE. This was associated with activation of the ERK pathway in CF. All these effects were abolished by heat inactivation of the media, suggesting implication of paracrinally secreted peptides. The effects of medium conditioned by untreated β3AR overexpressing CM were similar to those untreated controls. However, incubation of CF with medium of β3AR overexpressing CM treated with PE did not activate their differentiation into myofibroblats or pro-collagen I expression. Activation of the ERK pathway was also absent. To unravel the idendity of paracrine factors, the secretome of CM was submitted to shotgun proteomic analysis by LC tandem MS. Top candidates significantly downregulated in β3AR-PE conditioned medium compared with GFP-PE included CTGF, galectin-3 binding protein, plasminogen activator inhibitor 1, collagen I and III, fibronectin, thrombospondin-1. Accordingly, β3AR was associated with decreased mRNA expression of CTGF in CM in vitro. These results were confirmed in vivo after TAC stress in mice at mRNA and proteins levels (cTGF protein expression: 1.03±0.1 (WT) vs 0.51±01 (β3AR), p<0.01).
Conclusion: We conclude that β3AR decreases the expression of pro-fibrotic paracrine factors such as CTGF, resulting in protection from myocardial fibrosis in response to neurohormonal stimulation.