Abstract 20624: Existence of microRNA in Heart-Derived Mitochondria
Cardiovascular disease is one of the major causes of human morbidity and mortality. To develop innovative new therapies and diagnostics for heart disease, it is very important to understand the underlying mechanism. MicroRNAs (miRNA) are involved in the pathogenesis of heart diseases, but their role in the regulation of mitochondrial function is unclear, and even whether they are present in the mitochondria is unclear. Our goal in this study was to determine if there are miRNAs in the mitochondria that might play a role in mitochondrial function or dysfunction. To explore this possibility, it is very important to isolate pure mitochondrial RNA with little or no cytosolic or nuclear contamination. We prepared miRNA enriched total RNA, which was extracted after perfusion from whole rat hearts; and we extracted total RNAs from the mitochondrial fraction, which were isolated by differential centrifugation from the whole heart homogenate after perfusion to remove the blood from the heart. The purity of the mitochondrial RNA was assessed by the absence of 18S and 26S ribosomal RNA (rRNAs), as detected by column electrograph as well as RNA gel electrophoresis. Both approaches showed RNA derived from the mitochondria lack 18S and 26S rRNA, but contain large amounts of 12S and 16S rRNAs compared to RNA derived from total heart tissue. We identified the presence of miRNAs by running a denatured, 15% Urea gel with miRNA enriched fraction from both the groups, followed by ethidium bromide staining. We used microarray to profile the miRNAs in the mitochondrial fraction and identified at least 9 different miRNAs, which are present. To validate our microarray data we used qRT-PCR and used 12S rRNA as our internal control. qRT-PCR not only validated the presence of miR-181c in the mitochondria, but also showed that miR-181c is mainly localized in the mitochondria. This unique observation suggests that microRNA may regulate mitochondrial function and may play a role in the pathogenesis of heart disease.
- Mitochondrial energetics, heart failure, arrhythmias
- Cardiovascular disease prevention
- © 2010 by American Heart Association, Inc.