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. Author manuscript; available in PMC: 2013 Jan 18.
Published in final edited form as: Circulation. 2012 Aug 28;126(14):1728–1738. doi: 10.1161/CIRCULATIONAHA.112.115089

Figure 4.

Figure 4

Follistatin-like 1 (FSTL1) antagonizes bone morphogenetic protein-4 (BMP-4) signaling in the ischemic heart and cardiac myocytes. A, BMP-4 is upregulated in the heart in response to ischemia/reperfusion (I/R). Representative blots of BMP-4 and phosphorylated Smad1/5/8 (p-Smad1/5/8) at remote nonischemic area (remote) and ischemic area at risk (AAR) of pigs are shown from 4 independent experiments. B, Hypoxia/reoxygenation (H/R) increases the expression of BMP-4 in NRVMs. Representative blots of BMP-4 are shown from 4 independent experiments. C, FSTL1 attenuates the phosphorylation of Smad1/5/8 in neonatal rat ventricular myocytes (NRVMs) under conditions of H/R. NRVMs were treated with FSTL1 (100 or 250 ng/mL) or vehicle under conditions of normoxia or H/R. The phosphorylation levels of Smad1/5/8 (p-Smad1/5/8) were analyzed by Western blotting and expressed relative to β-actin levels (n=3). D, FSTL1 abolishes BMP-4 –induced apoptosis of NRVMs. NRVMs were treated with BMP-4 protein (100 ng/mL) or vehicle along with FSTL1 protein (100 or 250 ng/mL) or vehicle for 18 hours under normoxic conditions. Top, Representative pictures of NRVMs stained with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL; green) and DAPI (blue). Bottom, Quantitative analysis of TUNEL-positive NRVMs (n=4). E, Effect of AMP-regulated protein kinase (AMPK) inactivation on FSTL1-mediated inhibition of BMP4-stimulated apoptosis of NRVMs. NRVMs were transduced with dominant-negative form of AMPK tagged with c-myc (Ad-dn-AMPK) or β-galactosidase (Ad-β-gal) at a multiplicity of infection of 10 for 24 hours and treated with BMP-4 protein (100 ng/mL) or vehicle along with FSTL1 protein (250 ng/mL) or vehicle for 18 hours under normoxic conditions (n=4). F, FSTL1 suppresses BMP-4 –stimulated phosphorylation of Smad1/5/8 in NRVMs. NRVMs were treated with BMP-4 protein (100 ng/mL) or vehicle along with FSTL1 protein (100 or 250 ng/mL) or vehicle for 18 hours under normoxic conditions. The phosphorylation levels of Smad1/5/8 (p-Smad1/5/8) were determined by Western blot analysis and expressed relative to β-actin levels (n=4). G, Intracoronary administration of FSTL1 attenuates the phosphorylation of Smad1/5/8 in the ischemic myocardium in pigs. Top, Representative blots of p-Smad1/5/8, Smad1/5/8, and β-actin. Bottom, Quantitative analysis of relative phosphorylation levels of Smad1/5/8 (n=4). H, Involvement of BMP-4 antagonization and AMPK activation in the inhibitory action of FSTL1 on H/R-induced myocyte apoptosis. NRVMs are transfected with siRNAs targeting BMP-4 or unrelated siRNAs (40 nmol/L) and transduced with an Ad-dn-AMPK or Ad-β-gal followed by treatment with FSTL1 (250 ng/mL) or vehicle (n=4).