Comparison of waveforms between the Sham (2-d) and Infarct (2-d) groups after bolus injection of epinephrine.

<p>The only morphological differences in peri-infarct MAP were identified in the Infarct (2-d) group. (<b>A</b>) ECG Lead II waveforms showing that both groups had a comparable degree of heart rate increase. (<b>B</b>) MAP recorded at the peri-infarct epicardial zone showing that after the adrenergic challenge, the MAP of the Infarct (2-d) group had a dramatic shortening of MAPD30, whereas the MAPD90 was only minimally changed. (<b>C</b>) Direct overlapping of MAP showing that the MAP of the Infarct (2-d) group demonstrates more prominent triangulation.</p

The effects of the overexpression of miR-497 on the proliferation and apoptosis of HUVECs.

<p>(A) One representative cell cycle profile of HUVECs detected using a flow cytometer. The cells were stained with PI prior to detection. The overexpression of miR-497 reduced the percentage of cells in the S phase. (B) Representative profiles from the EdU cell proliferation assay after transfection with miR-497 for 48 h (magnification 200×). (E) Amount of EdU-positive cells in the different treatment groups. Increased miR-497 expression inhibited cellular DNA replication in HUVECs. (C) Apoptotic status after transfection, as detected by FCM after Annexin V-FITC/PI labeling. (F) Comparison of apoptotic cells in various groups. The percentage of early apoptotic cells was significantly higher in the experimental group. (D) CCK-8 cell proliferation assay for HUVECs. Cell proliferation was significantly inhibited after miR-497 transfection. The data are presented as the mean ± SD. All results are representative of three independent experiments. **P<0.01 versus the control groups.</p

QRT-PCR analysis was used to analyze the levels of miR-497 in HUVECs stimulated with ox-LDL.

<p>HUVECs were exposed to 100 μg/ml ox-LDL for 3, 6, 12, 24, 36, or 48 h. The miR-497 levels were significantly upregulated after 36 h of stimulation with 100 μg/ml ox-LDL; **P<0.01.</p

Divergent molecular mechanisms for potassium channel remodeling in animal models of heart disease.

<p>AV, atrioventricular; ICM, ischemic cardiomyopathy; ND, not determined; -, no change.</p><p>*Weak bands limited the reliability of the measurement.</p>‡<p>KCNQ1.2, a truncated isoform of canine KCNQ1, was increased and may suppress I_Ks in a dominant-negative fashion.</p

Epicardial MAPD90/60/30 and triangulation (MAPD90 - MAPD30) recorded from the remote zone (basal).

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031545#s2" target="_blank">Results</a> were means ± STD. There were no significant differences between groups.</p

MicroRNA-497 Induces Apoptosis and Suppresses Proliferation via the Bcl-2/Bax-Caspase9-Caspase3 Pathway and Cyclin D2 Protein in HUVECs

<div><p>Introduction</p><p>MicroRNAs play crucial roles in various types of diseases. However, to date, no information about the role of miR-497 in the development of atherosclerosis has been reported. This study investigated the possible role of miR-497 in vascular endothelial cell injury during the early stage of atherosclerosis.</p><p>Materials and Methods</p><p>The expression level of miR-497 in human umbilical vein endothelial cells (HUVECs) exposed to ox-LDL was detected using qRT-PCR. To perform gain of function and loss of function analyses, miR-497 mimics were transfected into HUVECs, and miR-497 inhibitors were transfected into HUVECs stimulated with ox-LDL. Flow cytometry was used to analyze cell cycle progression and apoptosis. EdU and CCK-8 assays were employed to detect DNA synthesis and cell proliferation, respectively. After bioinformatics prediction, a dual Luciferase Reporter assay was used to analyze the direct target genes of miR-497. The mRNA and protein levels of the target genes were detected using qRT-PCR and western blot analyses, respectively. Caspase-9/3 activity was analyzed to determine the mechanism of endothelial dysfunction.</p><p>Results</p><p>We showed that miR-497 was significantly upregulated in HUVECs stimulated with ox-LDL. Ectopic expression of miR-497 suppressed cell proliferation, induced apoptosis and increased the activity of caspase-9/3. After verification, Bcl2 and CCND2 were shown to be direct target genes of miR-497 in HUVECs. MiR-497 significantly suppressed cell proliferation by arresting the cell cycle through the CCND2 protein and induced apoptosis through the Bcl2/Bax-caspase9-caspase3 pathway.</p><p>Conclusion</p><p>Overall, our study shows that miR-497 might play a role in the development of atherosclerosis by inducing apoptosis and suppressing the proliferation of vascular endothelial cells. Therefore, miR-497 could be a potential therapeutic target for the treatment of atherosclerosis.</p></div

Study protocol 1 of experiments.

<p>Monophasic action potentials were recorded after the equilibration at both the middle of the infarct zone and the unaffected zone of the epicardium. For L-768,673, The infusion rate of the initial dose was 0.5 µg/kg/h for 30 min, and the maintenance rate was 0.25 µg/kg/h for two hours. MAP duration data were expressed as MAPD90/60/30_{Baseline, Initial Dose, I5, I10, I15, I20, R5, R10, R15, R20, R25, R30, R45, R60, R75, R90, R105, R120}, which stood for MAPD90/60/30 recorded at baseline, after initial dose, at ischemia for 5 min, 10 min, 15 min, 20 min and at reperfusion for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 120 min, respectively.</p

MAPDs, triangulations and MAP waveforms comparison between the IR+L-768,673 and IR+vehicle groups.

<p>(<b>A</b>) Epicardial MAPDs and triangulation recorded from the ischemia/reperfusion zone (apical) in the IR+L-768,673 group and the IR+vehicle group. Triangulations of the IR+L-768,673 group were increased compared with those of the IR+vehicle group by 31.1%, 26.5%, and 19.3% at R45, R60, and R75 respectively. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031545#s2" target="_blank">Results</a> are mean ± standard deviation (STD). * P<0.05 vs. IR+vehicle. (<b>B</b>) Comparison of monophasic action potential (MAP) waveforms between the IR+L-768,673 and IR+vehicle groups at R45, R60, and R75.</p

Details of PCR.

<p>Details of PCR.</p

The effects of miR-497 suppression on the proliferation and apoptosis of ox-LDL-induced HUVECs.

<p>(A) Cell cycle changes detected by a flow cytometer. The cells were stained with PI prior to detection. Suppression of miR-497 could partially increase the percentage of S-phase cells that were reduced by the addition of ox-LDL. (B) EdU cell proliferation analysis after transfection, as detected using a fluorescence microscope (magnification 200×). (C) Apoptotic status after transfection, as detected by FCM after Annexin V-FITC/PI labeling. (D) CCK-8 cell proliferation assay for HUVECs. Suppression of miR-497 could partially reduce the inhibition of cell proliferation induced by ox-LDL. (E) Rate of EdU-positive cells in the different groups. Loss of miR-497 can partially reverse the reduction of cellular DNA replication in HUVECs stimulated with ox-LDL. (F) Comparison of apoptotic cells in the various groups. Inhibition of miR-497 could partially reduce the number of apoptotic cells induced by ox-LDL. The data are presented as the mean ± SD. All results shown are representative of three independent experiments. *P<0.05 and **P<0.01 versus the control groups.</p