Abstract 1220: Thioredoxin 1 Negatively Regulates Angiotensin II-Induced Cardiac Hypertrophy through Upregulation of miR-98

Abstract

Thioredoxin 1 (Trx1) is a 12 kD antioxidant, which protects the heart through multiple mechanisms, including suppression of hypertrophy. We examined the effect of Trx1 on angiotensin II (AII)-induced cardiac hypertrophy (CH). Continuous infusion of a subpressor dose (200 ng/kg/min) of AII by osmotic pump for 2 weeks induced CH (+10% in left ventricular weight/body weight, p<0.05) in mice. AII-induced CH was significantly attenuated in cardiac specific Trx1 transgenic mice (Tg-Trx1) (+1%, ns), whereas it was significantly enhanced in dominant negative Trx1 mice (Tg-DN-Trx1) (+26%, p<0.05). These results suggest that Trx1 is a negative regulator of AII-induced CH. MicroRNAs are naturally existing small noncoding RNA molecules that downregulate posttranscriptional gene expression. In order to examine the involvement of microRNA in the anti-hypertrophic effects of Trx1, total RNA was extracted from Tg-Trx1 hearts, enriched for small RNA, and analyzed by microarrays of mature rodent microRNA. Both miR1 and miR98 are upregulated in the Tg-Trx1 heart. Since miR1 negatively regulates CH, we here examined the role of miR98 in regulation of CH. qRT-PCR showed that miR98 is upregulated in both Tg-Trx1 and Trx1 adenovirus transduced cardiac myocytes (CM) (+15 fold, 3.2 fold). Adenovirus-mediated expression of miR98 in CM reduced cell size both at basal levels (−14%, p<0.05) and in response to AII (−20%, p<0.05), suggesting that miR98 negatively regulates CH. Cyclin D2 (ClnD2) is one of the predicted targets of miR98 and has been shown to play an essential role in mediating AII-induced CH. AII significantly upregulated ClnD2 protein (+34%, p<0.05) at 3 to 6 h in CM. Overexpression of Trx1 downregulated, while expression of DN-Trx1 augmented the AII-induced increase in ClnD2 in vitro (+17.4%, +45.3 %). Similarly ClnD2 was downregulated in Tg-Trx1 and upregulated in Tg-DN-Trx1 mouse hearts (−70.2%, +86.7%). Expression of miR98 in CM decreased expression of ClnD2 by 27% as determined by immunoblotting and reduced nuclear immunostaining of ClnD2, suggesting that both Trx1 and miR98 negatively regulate ClnD2 in CM. These results suggest that Trx1 upregulates miR98, which in turn inhibits CH, possibly through negative regulation of ClnD2 in CM.