The role of Follistatin-like 3 (Fstl3) in cardiac hypertrophy and remodelling

Abstract

Follistatins are extracellular inhibitors of TGF-β family ligands like activin A, myostatin and bone morphogenetic proteins. Follistatin-like 3 (Fstl3) is a potent inhibitor of activin signalling and antagonises the cardioprotective role of activin A in the heart. The expression of Fstl3 is elevated in patients with heart failure and upregulated by hypertrophic stimuli in cardiomyocytes. However its role in cardiac remodelling is largely unknown. The aim of this thesis was to analyse the function of Fstl3 in the myocardium in response to hypertrophic stimuli using both in vivo and in vitro approaches. To explore the role of Fstl3 in cardiac hypertrophy, cardiac-specific Fstl3 knock-out mice (Fstl3 KO) were subjected to pressure overload induced by trans-aortic constriction. They showed attenuated cardiac hypertrophy and improved cardiac function compared to wild type littermate control mice. Knock-out of Fstl3 specifically from cardiomyocytes was sufficient to reduce the total expression of Fstl3 in the heart following pressure overload, implying that cardiomyocytes are the major source of Fstl3 in the heart after TAC. Fstl3 KO mice also showed reduced expression of typical hypertrophic markers including ANP, BNP, α-skeletal actin and β-MHC. Similarly, treatment of neonatal rat cardiomyocytes with Fstl3 resulted in hypertrophy as measured by increased cell size and protein synthesis. This was also accompanied by activation of p38 and JNK signalling pathways. Microarray analysis of ventricular samples from these mice indicated reduced expression of genes involved in protein binding and extracellular matrix. Verification by quantitative real-time RT-PCR revealed reduced expression of collagens and TGF- β1 in the myocardium, indicating reduced fibrosis. This was supported by histological analysis showing reduced interstitial fibrosis. As an in vitro model of pressure overload, cardiomyocytes were subjected to mechanical stretch, which elevated the expression of Fstl3. In order to explore the role of cardiomyocyte-derived Fstl3 in modulating fibroblast function, cardiac fibroblasts were treated with the conditioned medium from stretched cardiomyocytes and collagen synthesis was measured. Fstl3 was found to be necessary for conditioned medium to induce an increase in collagen synthesis in fibroblasts. Fstl3 was also shown to induce low levels of cell death in cardiac fibroblasts. In order to identify stretched cardiomyocyte derived factors necessary for Fstl3 action on fibroblast collagen synthesis, a yeast two hybrid analysis was undertaken. Results indicated that Fstl3 may act, at least in part, through binding to proteins of the extracellular matrix as well as pro-fibrotic factors, including connective tissue growth factor (CTGF). While CTGF did not affect fibroblast collagen synthesis in the presence of Fstl3, it inhibited Fstl3 induced cell death, indicating a protective role. In summary, data presented in this thesis demonstrates that Fstl3 is upregulated after cardiac injury and functions by activating the pro-hypertrophic and pro-fibrotic responses in the myocardium, making it an attractive therapeutic target for intervention of cardiac pathologies.