Beta-Adrenoceptor activation increases cardiac galectin-3 levels via the hippo signaling pathway

Background: Galectin-3 (Gal-3) is a clinical biomarker for risk of cardiovascular disease and a disease mediator forming a therapeutic target. However, the mechanism(s) that regulate cardiac expression of Gal-3 remains unknown. Activation of the sympatho-β-adrenergic system is a hallmark of heart disease, but the relationship of βAR activation and cardiac content of Gal-3 remains unknown. Purpose: To determine the role of βAR activation in regulating cardiac Gal3 level and the responsible mechanism focusing on the Hippo signalling pathway. Methods: Wild-type and Gal-3 gene deleted (Gal3-KO) mice were used. To test the role of the Hippo pathway, we used transgenic (TG) mouse strains with cardiac overexpression of mammalian-20-like sterile kinase 1 (Mst1, mammalian orthology of Drosophila Hippo kinase) either in wildtype form (TG-Mst1) or dominative-negative kinase dead mutant form (TGdnMst1). Effects of β-antagonist (isoprenaline, ISO) and antagonists were determined. We measured phosphorylation (Ser127) of YAP as a transcription co-regulator acting as the main signal output of the Hippo pathway. Results: In wild-type mice, treatment with ISO led to a time- and dosedependent increase in cardiac expression of Gal-3 (Fig. A) accompanied by elevated circulating Gal-3 levels (Fig. B). ISO treatment stimulated cardiac expression of Mst1 and YAP hyper-phosphorylation (i.e. inactivation, Fig. C), indicating activation of the Hippo signalling. These effects of ISO were inhibited by β-blockers (propranolol, Prop; carvedilol, Carv; Fig. D,E). Relative to non-TG controls, ISO-induced expression of Gal-3 was inhibited by 75% in TG-dnMst1 mice (inactivated Mst1), but exaggerated by 7-fold in TG-Mst1 mice (activated Mst1). Mst1-TG mice had a 45- fold increase in Gal-3 content, YAP hyper-phosphorylation and enhanced pro-fibrotic signaling. In Mst1-TG mice, whilst blood Gal-3 level was unchanged, treatment with ISO (6 mg, 2 days) evoked a marked increase in cardiac and blood Gal-3 levels. Using rat cardiomyoblasts, we showed that ISO-mediated Mst1 expression and YAP phosphorylation were PKAdependent and that siRNA-mediated YAP knockdown led to Gal-3 upregulation. The role of Gal-3 in mediating ISO-induced cardiomyopathy was examined by treating wild-type and Gal3-KO mice with ISO (30 mg/kg, 7 days). ISO-treated wild-type mice had 8-fold increase in cardiac Gal-3, ventricular dysfunction, fibrosis, hypertrophy and activated inflammatory or fibrotic signalling. All these changes, except hypertrophy, were abolished by Gal3-KO. Conclusion: βAR stimulation increases cardiac expression of Gal-3 through activation of the Hippo signalling pathway. This is accompanied by elevated circulating Gal-3 level. βAR antagonists inhibited βAR-Mst1 (Hippo) signalling and cardiac Gal-3 expression, actions likely contributing to the overall efficacy of β-blockers