MicroRNA-99b-3p promotes angiotensin II-induced cardiac fibrosis in mice by targeting GSK-3ß.

Cardiac fibrosis is a typical pathological change in various cardiovascular diseases. Although it has been recognized as a crucial risk factor responsible for heart failure, there is still a lack of effective treatment. Recent evidence shows that microRNAs (miRNAs) play an important role in the development of cardiac fibrosis and represent novel therapeutic targets. In this study we tried to identify the cardiac fibrosis-associated miRNA and elucidate its regulatory mechanisms in mice. Cardiac fibrosis was induced by infusion of angiotensin II (Ang II, 2 mg·kg-1·d-1) for 2 weeks via osmotic pumps. We showed that Ang II infusion induced cardiac disfunction and fibrosis accompanied by markedly increased expression level of miR-99b-3p in heart tissues. Upregulation of miR-99b-3p and fibrotic responses were also observed in cultured rat cardiac fibroblasts (CFs) treated with Ang II (100 nM) in vitro. Transfection with miR-99b-3p mimic resulted in the overproduction of fibronectin, collagen I, vimentin and α-SMA, and facilitated the proliferation and migration of CFs. On the contrary, transfection with specific miR-99b-3p inhibitor attenuated Ang II-induced fibrotic responses. Similarly, intravenous injection of specific miR-99b-3p antagomir could prevent Ang II-infused mice from cardiac dysfunction and fibrosis. We identified glycogen synthase kinase-3 beta (GSK-3ß) as a direct target of miR-99b-3p. In CFs, miR-99b-3p mimic significantly reduced the expression of GSK-3ß, leading to activation of its downstream profibrotic effector Smad3, whereas miR-99b-3p inhibitor caused anti-fibrotic effects. GSK-3ß knockdown ameliorated the anti-fibrotic role of miR-99b-3p inhibitor. These results suggest that miR-99b-3p contributes to Ang II-induced cardiac fibrosis at least partially through GSK-3ß. The modulation of miR-99b-3p may provide a new approach for tackling fibrosis-related cardiomyopathy.

EGCG inhibits pressure overload-induced cardiac hypertrophy via the PSMB5/Nmnat2/SIRT6-dependent signalling pathways.

AIM: Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea, exerts multiple protective effects against cardiovascular diseases, including cardiac hypertrophy. However, the molecular mechanism underlying its anti-hypertrophic effect has not been clarified. This study revealed that EGCG could inhibit pressure overload-induced cardiac hypertrophy by regulating the PSMB5/Nmnat2/SIRT6-dependent signalling pathway. METHODS: Quantitative real-time polymerase chain reaction and western blotting were used to determine the expression of mRNA and protein respectively. A fluorometric assay kit was used to determine the activity of SIRT6, a histone deacetylase. Luciferase reporter gene assay and electrophoretic mobility shift assay were employed to measure transcriptional activity and DNA binding activity respectively. RESULTS: EGCG could significantly increase Nmnat2 protein expression and enzyme activity in cultured neonatal rat cardiomyocytes stimulated with angiotensin II (Ang II) and heart tissues from rats subjected to abdominal aortic constriction. Nmnat2 knockdown by RNA interference attenuated the inhibitory effect of EGCG on cardiac hypertrophy. EGCG blocked NF-κB DNA binding activity induced by Ang II, which was dependent on Nmnat2 and the subsequent SIRT6 activation. Moreover the activation of PSMB5 (20S proteasome subunit ß-5, chymotrypsin-like) was required for EGCG-induced Nmnat2 protein expression. Additionally, we demonstrated that EGCG might interact with PSMB5 and inhibit the activation of the proteasome. CONCLUSIONS: These findings serve as the first evidence that the effect of EGCG against cardiac hypertrophy may be, at least partially, attributed to the modulation of the PSMB5/Nmnat2-dependent signalling pathway, suggesting the therapeutic potential of EGCG in the prevention and treatment of cardiac hypertrophy.