Abstract 13141: Cellular Microrna 449b Upregulation Sensitizes Ischemia/reperfusion Injury via Inhibition of Nrf-1, Increasing Ros Production and Apoptosis in the Diabetic Heart

Abstract

Myocardial ischemic/reperfusion (MI/R) injury is significantly enhanced in diabetes by incompletely understood mechanisms. Recent clinical and experimental studies demonstrate that hypoadiponectinemia during diabetes enhances oxidative stress and exaggerates MI/R injury. However, molecular mechanisms responsible for hypoadiponectinemia-induced oxidative stress remain unknown. In a discovery-driven fashion, we determined the role of cardiac microRNAs in the MI/R response in adiponectin knockout (APNKO) mice. From 68 total miRNAs differentially expressed between APNKO and wild type (WT) mice, miRNA 449b was identified as the microRNA most relevant to oxidative stress and apoptosis. In cultured neonatal cardiomyocytes, miRNA 449b silencing inhibited hypoxia/reoxygenation-induced apoptosis, whereas miR-449b overexpression significantly increased oxidative stress and cardiomyocyte apoptosis. In APNKO mice, administration of anti-miR-449b decreased oxidative stress (-17.2±3.8%, p<0.05), reduced caspase-3 activity (-21.3±4.2%, p<0.05), attenuated myocardial apoptosis (-16.3±4.1%, p<0.05), and improved myocardial function (1.4±0.3 fold). To identify the downstream molecule regulated by miRNA 449b, we integrated transcriptomics and proteomics data with computational annotation data, and identified Nrf-1 as a miRNA 449b target. A luciferase reporter gene assay demonstrated that miRNA 449b inhibited Nrf-1 expression via Nrf-1 mRNA 3’UTR region binding. Finally, we demonstrated that miRNA 449b was significantly upregulated, Nrf-1 expression was significantly decreased, and the anti-oxidative molecule metallothionein (MT) was significantly inhibited in the diabetic heart subjected to MI/R. Administration of anti-miR-449b in diabetic animals upregulated Nrf-1 and MT expression, reduced oxidative stress, and improved cardiac function (P<0.01) after MI/R. Taken together, this study provides the first evidence that hypoadiponectinemia during diabetes causes cardiac miRNA-449b upregulation and subsequent downregulation of Nrf-1 and MT, thus enhancing oxidative stress and MI/R injury. MicroRNA 449b may represent a potential therapeutic target against diabetic heart disease.