P401AMP-activated protein kinase activation is associated with an inhibition of fibrotic properties of cardiac fibroblasts

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Abstract

Purpose

A stimulation of cardiac fibroblasts (CF) proliferation, migration and myodifferentiation after myocardial infarction, leads to concomitant increased biosynthesis of extracellular matrix components and cardiac fibrosis in the remodelling heart. As cardiac fibrosis leads to ventricular dysfunction, molecular mechanisms involved in the modulation of the fibroblastic response are potent therapeutic targets. We asked whether CF myodifferentiation, proliferation and migration might be processes regulated by AMP-activated protein kinase (AMPK). We also investigated cardiac fibrosis in AMPK knockout mice after myocardial infarction.

Methods

For in vitro studies, human CF were treated by Transforming growth factor-beta-1 (TGF-beta-1), serum or platelet derived growth factor (PDGF-BB) to stimulate myodifferentiation, proliferation and migration respectively. AMPK was activated prior to the different treatments by preincubating cells for 30 min with thienopyridone derivative A-769662, a direct pharmacological activator of AMPK. For in vivo studies, AMPK knockout (KO) mice and their wild-type (WT) littermate were submitted to a ligation of the coronary artery to mimic myocardial infarction. Gene expression in the left ventricule was measured by qRT-PCR, 30 days later.

Results

Administration of TGF-beta-1 to human CF for 48 hours significantly increased expression of alpha-smooth muscle actin (alpha-SMA), a marker of myodifferentiation, and changed drastically the morphology into more spread and larger cells. A pre-treatment of cells with the A-769662 compound dose-dependently prevented induction of alpha-SMA and morphologic changes in cells exposed to TGF-beta-1. In addition, A-769662 decreased by 50% the basal and serum- induced proliferation of human CF and completely prevented the PDGF-induced migration of these cells, without affecting their mortality. Molecular mechanisms responsible for these inhibitions are currently under study. The anti-fibrotic action of AMPK was supported by studies in KO mice. Our data show that RNA expression for collagen I and III was significantly increased in the infarct area of KO mice compared to WT mice, after myocardial infarction.

Conclusion

Our data indicate that AMPK could be a new negative regulator of CF myodifferentiation, proliferation and migration, and by this way could decrease the fibrotic component of ventricular remodelling which takes place after myocardial infarction.

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