Studies of miR-21 regulation in wiltype and <i>Per2</i>^<i>-/-</i> mice after cardiac ischemic preconditioning.

<p>Wildtype (<b>A</b>) or <i>Per2</i>^<i>-/-</i> mice (<b>B</b>) were exposed to cardiac IPC, consisting of 4 x 5 minutes of ischemia followed by 5 minutes of reperfusion each, followed by a final reperfusion time of 120 min. Heart tissue was snap-frozen with clamps pre-cooled to the temperature of liquid nitrogen. Total RNA was isolated from murine heart tissue using Qiazol Reagent and separated into mRNA and miRNA components following manufactures instructions (SA-Biosciences, Qiagen). cDNA from miRNA was generated using miScript RT II kits (Qiagen) and transcript levels were determined by real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.; mean±SD, n = 3).</p

Light elicited cardioprotection in wildtype and <i>miR-21</i>^<i>-/-</i> mice.

<p><b>(A-E)</b> Mice underwent 60 min of ischemia and 120 min of reperfusion at room light (200LUX) or after exposure to 3 hours of intense light (10,000 LUX). Infarct sizes were measured by double staining with Evan’s blue and triphenyl-tetrazolium chloride. Infarct sizes are expressed as the percent of the area at risk (AAR) that underwent infarction. (<b>A</b>) Infarct sizes in wildtype or <i>miR-21</i>^<i>-/-</i> mice at room light conditions (mean±SD, n = 4, p<0.05). (<b>B, C</b>) Infarct sizes in wildtype mice after exposure to intense light for 3 h compared to room light conditions. (mean±SD, n = 4, p<0.05). (<b>C</b>) Representative infarct staining in hearts from wildtype mice exposed to intense light or room light prior to <i>in situ</i> myocardial ischemia and reperfusion (blue, retrograde Evan’s blue staining; red and white, area at risk; white, infarcted tissue). (<b>D, E</b>) Infarct sizes in <i>miR-21</i>^<i>-/-</i> mice exposed to intense light or room light prior to <i>in situ</i> myocardial ischemia followed by reperfusion (mean±SD, n = 4, <i>not significant</i>). (<b>E</b>) Representative infarct staining in hearts from <i>miR-21</i>^<i>-/-</i> mice exposed to intense light or room light prior to <i>in situ</i> myocardial ischemia reperfusion (blue, retrograde Evan’s blue staining; red and white, area at risk; white, infarcted tissue).</p

Effects of intense light on miR-21 regulation in mice and human subjects.

<p><b>(A-C)</b> Wildtype mice were exposed to broad spectrum intense light (10,000 lux) for 7 days (LD 14:10) and compared to controls that were maintained at room light (200 lux, LD 14:10). Total RNA was isolated from murine hearts using Qiazol Reagent and separated into mRNA and miRNA components following manufactures instructions (SA-Biosciences, Qiagen). cDNA from miRNA was generated using miScript RT II kits (Qiagen) and miR-21 or Per2 transcript levels were determined by real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.; mean±SD, n = 3, p<0.05). (<b>D-F</b>) 7 Healthy human volunteers were exposed to 30 minutes of intense blue light (Square One Wake Up Light, NatureBright, Day-Light 10,000 Lux) in the morning at 8:30 AM on 5 consecutive days. A blood draw was performed before light exposure on the first day (8:30 AM) and 5 days after light exposure (9.00 AM). Plasma samples were analyzed for miR-21 levels and PFK (phosphofructokinase) activity (mean±SD, n = 7, p<0.05).</p

Per2 dependent micro RNAs during cardiac ischemic preconditioning (IPC).

<p>Shown are the 65 differentially regulated and Per2 dependent micro RNAs identified after IPC treatment of wildtype and <i>Per2</i>^<i>-/-</i> mice.</p

Diurnal expression of miR-21 in murine hearts and lungs.

<p>Analysis of cardiac (<b>A</b>) or lung (<b>B</b>) mir-21 and Per2 levels from wildtype mice at Zeitgeber Time (ZT) 3 or ZT15. Total RNA was isolated from murine heart or lung tissue using Qiazol Reagent and separated into mRNA and miRNA components following manufactures instructions (SA-Biosciences, Qiagen). cDNA from miRNA was generated using miScript RT II kits (Qiagen) and transcript levels were determined by quantitative real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.; mean±SD, n = 3, p<0.05).</p

miR-21 expression in different cardiac tissues at baseline and during hypoxia.

<p>Fibroblasts or myocytes were isolated from C57BL6/J mouse hearts and endothelial cells isolated from C57/BL6 mice were purchased from Cell Biologics for analyzing miR-21 expression at baseline or hypoxic (1% oxygen) conditions. miRNA was isolated using RNeasy Mini Kit (Qiagen), cDNA was generated using miScript RT II kits (Qiagen), and transcript levels were determined by quantitative real-time RT-PCR (iCycler; Bio-Rad Laboratories Inc.). (<b>A</b>) Relative miR-21 expression levels in C57BL6/J mouse isolated cardiac fibroblasts, myocytes, and endothelia at baseline (mean±SD, n = 3, <i>not significant</i>). (<b>B</b>) miR-21 expression in cardiac fibroblasts subjected to normoxia or hypoxia for 6 h (mean±SD, n = 6, <i>not significant</i>). (<b>C</b>) miR-21 expression in cardiac myocytes subjected to normoxia or hypoxia for 1 h (mean±SD, n = 3, <i>not significant</i>). (<b>D</b>) miR-21 expression in cardiac endothelia subjected to normoxia or hypoxia for 6 h (mean±SD, n = 6, p<0.05). (<b>E</b>) miR-21 expression in human endothelia (HMEC-1) subjected to normoxia or hypoxia for 6 h (mean±SD, n = 6, p<0.05).</p

Glycolysis in miR-21 gain or loss of function human endothelial cells.

<p><b>(A)</b> Knockdown confirmation in anti-mir-21 (loss of function, LOF) treated human endothelial cells (HMEC-1). (<b>B-E</b>) Glucose metabolism from control (miScript Inhibitor Neg. Control, scrambled [SCR]) and anti-mir-21 (LOF) treated HMEC-1. (<b>F</b>) Overexpression in miR-21Mimic (gain of function [GOF]) treated HMEC-1. (<b>G-J</b>) Glucose metabolism from control (miScript miRNA Mimic Neg. Control, SCR) and miR-21Mimic (GOF) treated human endothelial cells (HMEC-1). Cells were seeded at a density of 100,000 cells/well. Glycolysis assay was performed using glycolysis stress test kit from Seahorse Biosciences according to manufacturer’s protocol using the XF24 instrument. The extracellular acidification rate (ECAR) response to glucose, oligomycin and 2-DG was measured (mean±SD, n = 6, p<0.05).</p

Proposed model of IPC or light induced miR-21 and glycolysis.

<p>Both IPC and light can induce Per2 in human or murine tissues. In a Per2 dependent manner miR-21 and phosphofructokinase (PFK) are transcriptionally induced which finally leads to increased PFK activity, glycolytic capacity and cardioprotection.</p