Abstract
While restoration of coronary blood flow to the ischemic heart is the most effective strategy for reducing infarct size, reperfusion injury represents a significant limiting factor on clinical outcomes in myocardial infarction patients. Ischemic preconditioning (IPC) has been shown to inhibit reperfusion injury and represents an attractive model for studying cardioprotective signal transduction pathways. Long non-coding RNAs (lncRNAs) are a structurally and functionally heterogenous class of RNA transcripts with unknown roles in IPC-induced cardioprotection. Through microarray-based expression profiling of 31,423 lncRNAs in cardiac tissue from IPC mice, we identified the nuclear transcript Neat1 to be rapidly and robustly decreased in response to IPC. siRNA-mediated knock down of Neat1 reduced apoptosis and necrosis in murine cardiomyocytes (CM) and human iPS-derived CMs in response to prolonged hypoxia and hypoxia-reoxygenation, assessed with Annexin V/propidium iodide-staining, a Caspase 3/7 activity assay, LDH release, and western blot for cleaved Caspase 3. Mechanistically, Neat1 was shown to regulate processing of pro-apoptotic microRNA-22 (miR-22) in murine and human CM nuclei using a luciferase reporter assay. Hypoxia-induced downregulation of Neat1 was shown to result in accumulation of unprocessed pri-miRNA and decreased availability of biologically active miRNA, including miR-22. Addition of exogenous synthetic miR-22 reversed the protective effect of Neat1 knock down in human iPS-CM. In conclusion, we have identified the nuclear lncRNA Neat1 as part of a conserved oxygen-sensitive feedback mechanism by regulation of miRNA processing and a potential target in cardioprotection.
Highlights
Restoration of coronary circulation is vital in limiting ischemic injury and improving outcomes after myocardial infarction (MI), but the act of reperfusion does in itself cause irreversible injury to the myocardium[1]
Induced cardioprotection, we performed an exploratory profiling of 31,423 Long non-coding RNAs (lncRNAs) in the area at risk (AAR) and remote myocardium (RM) from mice 30 min after Ischemic preconditioning (IPC) (n = 2) with microarray (Fig. 1a)
Neat[1], a nuclear RNA and an essential component of paraspeckles[24] was one of the most distinctly downregulated lncRNAs in the AAR
Summary
Restoration of coronary circulation is vital in limiting ischemic injury and improving outcomes after myocardial infarction (MI), but the act of reperfusion does in itself cause irreversible injury to the myocardium[1]. In the context of cardiac IPC, a number of studies points to distinct roles for the microRNA (miRNA) class of non-coding RNAs. Yin et al reported that miRNAs play a role in activating endothelial nitric oxide synthase and the heat shock pathway in late phase IPC cardioprotection[13] whereas Cheng et al showed that miR-21 is triggered by IPC and confers a cardioprotective effect through repression of the PDCD4 gene[14]. The long non-coding RNA (lncRNA) class comprise a group of structurally and functionally diverse transcripts with known roles in cardiac development[15,16] and disease[17,18] but evidence of a mechanistic link between cardiac IPC and lncRNA is still elusive
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