Abstract 17156: Extracellular Matrix Secretion by Cardiac Fibroblasts: Proteomics for MicroRNA Target Identification of MiR-29b and MiR-30c
Background: Fibrosis is a critical feature of structural myocardial remodeling in many cardiac diseases. MicroRNAs (miRNAs) have been implicated as important regulators of cardiac fibrosis. Objective: We used a proteomics approach to directly compare the effects of miR-29b and miR-30c on extracellular matrix secretion by cardiac fibroblasts.
Methods and Results: Mouse cardiac fibroblasts were transfected with pre- and anti-miRs of miR-29b or miR-30c. A total of 173 extracellular proteins were identified by mass spectrometry. MiR-29b targeted a cadre of proteins involved in fibrosis, including multiple collagens and matrix metalloproteinases. In contrast, miR-30c had little effect on extracellular matrix production. However, both miRNAs affected the cross-talk between fibroblasts and cardiomyocytes: Upon transfection with pre-miR-29b, the conditioned medium of cardiac fibroblasts lost its ability to support adhesion of neonatal and adult rat ventricular myocytes leading to a significant reduction of cardiomyocyte markers. Similarly, transfection of cardiac fibroblasts with anti-miR-30c impacted on cardiac marker expression (alpha-actinin and cardiac troponin I) but without affecting adhesion. Interestingly, our proteomic analysis confirmed three predicted targets of miR-29b: leukaemia inhibitory factor (LIF), insulin-like growth factor-1 (IGF-1) and pentraxin-3 (PTX3). Even in the presence of TGF-beta, miR-29b attenuated the secretion of LIF, PTX3 and IGF-1 suggesting that miR-29b controls the fibroblast response to this master regulator of fibrosis. Surprisingly, overexpression of pre-miR-30c upregulated LIF and IGF-1 although both lack a seed-matching sequence for miR-30c in their 3' untranslated region. For in vivo validation, mice were injected with antagomirs to miR-29b. Besides increasing IGF-1 expression in the heart, antagomir treatment raised plasma levels of IGF-1 by 25%.
Conclusions: Our proteomic analyses suggest novel molecular mechanisms for miR-29b and miR-30c in regulating fibroblast-cardiomyocyte interactions. However, the observed systemic effect of antagomir treatment also challenges our reductionist approach in explaining cardiovascular phenotypes upon miRNA manipulation.
- © 2012 by American Heart Association, Inc.