Abstract Body (Do not enter title and authors here): Background: Myocardial ischemia-reperfusion injury (IRI) refers to the tissue damage that occurs when blood supply returns to the heart after a period of ischemia and the sudden reintroduction of oxygen and nutrients paradoxically triggers a cascade of inflammation and cell death in both cardiomyocytes and coronary artery endothelial cells (EC). Our lab was the first to show Intralipid (ILP), the first safe lipid emulsion for human use, confers robust cardioprotection against IRI in rodents. However, the full downstream mechanisms remain unknown.
Hypothesis: We hypothesize that ILP attenuates IRI-induced inflammation, and apoptosis in the myocardium via downregulation of N-myc downstream regulated gene 1 (NDRG1).
Methods: We performed RNA Sequencing on the left ventricle (LV) of male Sprague-Dawley rats subjected to LAD ligation to induce IRI (30 min ischemia, 180 min reperfusion) or sham surgery. Rats received either one bolus of ILP (20% IV) or saline 5 minutes before the onset of reperfusion. Pathway enrichment analysis was performed using transcriptomics data. We performed qPCR to validate our transcriptomics data. We subjected H9C2 cardiac myoblasts and coronary artery endothelial cells (EC) to hypoxia (4h)/reoxygenation (2h) and ILP (20%) treatment during reoxygenation. We tested the functional role of NDRG1 in EC by silencing NDRG1 via siRNA.
Results: Our transcriptomic pathway enrichment analysis revealed hypoxia and inflammatory response genes were significantly increased in the LV of rats subjected to IRI compared to sham but showed no significant difference between ILP-treated IRI hearts and sham controls. Our transcriptomics analysis identified seven significantly differentially-expressed genes, including the hypoxia molecular switch N-myc downstream-regulated gene 1 (NDRG1). We validated that NDRG1 mRNA in the LV in rats is upregulated by IRI and attenuated by ILP treatment at the onset of reperfusion to a similar level as sham rats. Our in-vitro data also showed that ILP significantly reduced hypoxia-reoxygenation induced upregulation of NDRG1 transcript levels in H9C2 and EC. Functionally, knockdown of NDRG1 in coronary EC using siNRGD1 reduced markers of EC activation (ICAM1), inflammation (IL-6) and apoptosis (cleaved caspase 3) compared to control siRNA.
Conclusion: Taken together, our data suggests that ILP confers cardioprotection against IRI via downregulation of NDRG1 expression, leading to reduced inflammation, and apoptosis.