Abstract 1987: Cardiomyocyte Protein Turnover, Growth and Secretory Activity Are modulated by Stat3 Dependent Expression of Microrna-199a
Background: Cardiomyocyte (CM) STAT3 plays a pivotal role in various forms of cardiac pathophysiology affecting CMs and, in a paracrine manner non-cardiomyocytes.
Objective: We evaluated the role of putative STAT3-dependent miRNAs with regard to CM phenotype and secretome.
Methods and Results: miCHIP and quantitative real-time PCR analysis revealed elevated cardiac expression of miR-199a in mice with a CM restricted knock out of STAT3 (qRT-miR-PCR: + 7.4 fold). Lentiviral-mediated RNAi against STAT3 in CMs increased pri-miR-199a (16-fold; p<0.01) and miR-199a (46-fold, P<0.01) clearly documenting repression of miR-199a by STAT3. Transfection with pre-miR-199a resulted in a significant hypertrophic growth of CMs. Electron microscopy detected massively enlarged nuclei of cardiomyocytes, myofibrillar derangement of the sarcomeres and prominent swelling of the mitochondria. In silico analysis identified ubiquitin-conjugating enzyme E2i (Ube2i) as a target of miR-199a which was confirmed by immunoblotting: antimir-199a transfection enhanced (+49±11%, p<0.05), whereas pre-miR-199a transfection silenced its expression (−74±11%, p<0.05). Liquid chromatography tandem mass spectrometry displayed regulatory properties of miR-199a for ubiquitination and degradation of sarcomeric proteins. Gas chromatography detected increased levels of the endogenous NO-synthase inhibitor asymmetric dimethylarginine (ADMA) in supernatants (SN) of pre-miR-199a (839±6 nM; contr.: 231±8 nM; p<0.01) and decreased levels in antimir-199a (45±4 nM; contr.: 259±22 nM, p<0.01) transfected CMs. SN-pre-miR-199a enhanced proliferation (+120±13%; p<0.01) and reactive oxygen species (ROS) production (+803±180%; p<0.05) of cardiac fibroblasts. SN-pre-miR-199a reduced NO-bioavailability (−49±3%, p<0.01) and enhanced ROS production (+133±21%, p<0.01) in endothelial cells while this was prevented by SN-antimir-199a.
Conclusion: Protein turnover, growth and secretory activity of CMs are modulated by STAT3 dependent expression of microRNA-199a. The miR-199a-induced alteration of the cardiomyocytic secretome causes fibroblast proliferation and endothelial dysfunction in a paracrine fashion, most likely by enhancing endogenous ADMA levels.