Extracellular matrix secretion by cardiac fibroblasts: role of microRNA-29b and microRNA-30c.

Rationale: MicroRNAs (miRNAs), in particular miR-29b and miR-30c, have been implicated as important regulators of cardiac fibrosis. Objective: To perform a proteomics comparison of miRNA effects on extracellular matrix (ECM) secretion by cardiac fibroblasts (CFs). Methods and Results: Mouse CFs were transfected with pre-/anti-miR of miR-29b and miR-30c and their conditioned medium was analysed by mass spectrometry. MiR-29b targeted a cadre of proteins involved in fibrosis, including multiple collagens, matrix metalloproteinases, and leukemia inhibitory factor (LIF), insulin-like growth factor-1 (IGF-1) and pentraxin-3, three predicted targets of miR-29b. MiR-29b even attenuated the CF response to TGFβ. In contrast, miR-30c had little effect on ECM production, but opposite effects with regards to LIF and IGF-1. Both miRNAs indirectly affected cardiac myocytes: Upon transfection with pre-miR-29b, the conditioned medium of CFs lost its ability to support adhesion of rat ventricular myocytes and led to a significant reduction of cardiac myocyte proteins (alpha-actinin, cardiac myosin-binding protein C and cardiac troponin I). Similarly, cardiomyocytes derived from mouse embryonic stem cells atrophied under pre-miR-29 conditioned medium whereas pre-miR-30c conditioned medium had a prohypertrophic effect. Levels of miR-29a, miR-29c and miR-30c but not miR-29b were significantly reduced in a mouse model of pathological but not physiological hypertrophy. Treatment with antagomiRs to miR-29b induced excess fibrosis after aortic constriction without overt deterioration in cardiac function. Conclusions: Our proteomic analysis revealed novel molecular targets of miRNAs that are linked to a fibrogenic cardiac phenotype. Such comprehensive screening methods are essential to define the concerted actions of miRNAs in cardiovascular disease.