Effects of homocysteine on MAPKs and apoptotic proteins expression in BMSCs.

<p>(a) The total and phosphorylated JNK, p38 and ERK1/2 protein was detected by western blotting in BMSCs after treatment with homocysteine at the time point of 0, 2, 4, 8, 12 and 24 h. Homocysteine effectively activated phosphorylated JNK expression after treatment with homocysteine. But homocysteine did not increase the expression of total JNK protein in BMSCs. (b) Influences of homocysteine on the expression of Bcl-2, caspase-3, cleaved caspase-3, and p-p53 proteins in BMSCs. n = 3 independent experiments.</p

JNK signal is involved in the apoptosis of BMSCs induced by homocysteine.

<p>JNK specific inhibitor effectively attenuated the apoptosis induced by homocysteine 300 µM in BMSCs. However, p38 and ERK specific inhibitors did not affect homocysteine-induced apoptotic morphological changes in BMSCs.</p

Effects of homocysteine on intracellular ROS and mitochondrial membrane potential of BMSCs.

<p>(a) Intracellular ROS level was measured in BMSCs treated with homocysteine 30, 100 and 300 µM for 24 h by DCFH-DA staining. The ROS level was gradually increased with the increase of homocysteine concentration. (b) Homocysteine induced an obvious depolarization of mitochondrial membrane potential in BMSCs apoptosis by JC-1 staining.</p

Effects of difference concentrations of homocysteine on the morphological appearance and cellular viability of BMSCs.

<p>(a) The morphology of cultured BMSCs was observed after treatment with homocysteine 30, 100, 300 and 1000 µM for 24 h. Homocysteine caused aberrant morphological appearance of BMSCs. (b) Homocysteine significantly decreased the cellular viability of BMSCs in a concentration-dependent manner. * p<0.05 versus Control.</p

Increased ROS is required for homocysteine-induced apoptosis of BMSCs.

<p>(a) DMTU and NAC attenuated the increase of ROS level by homocysteine in BMSCs. (b) Effects of DMTU and NAC on the apoptotic appearance of BMSCs. The inhibition of ROS with DMTU and NAC abolished the apoptosis of BMSCs induced by homocysteine. (c) Homocysteine-induced depolarization of mitochondrial membrane potential was also effectively reserved by DMTU and NAC in BMSCs.</p

Homocysteine induced apoptotic cellular changes of BMSCs.

<p>(a) AO/EB double staining demonstrated the effects of difference concentrations of homocysteine on the apoptosis of BMSCs. BMSCs were incubated with homocysteine for 24 h. (b) Hoeschest33342 staining detected the changes in the nucleus of BMSCs after treatment with homocysteine 30, 100 and 300 µM (scale bar, 20 µm). (c, e) The inhibitory effects of homocysteine on BMSCs were determined by Live/Dead staining. (d, f) TUNEL was used to determine the effects of homocysteine on BMSCs apoptosis (n = 3 independent experiments). * p<0.05 versus Control.</p

Effects of miR-1 on cardiac infarct area of mice after ischemia/reperfusion injury in mice.

<p>A. Representative images showing infarct areas; B. Statistical analysis of IA/AAR ratio. LNA-1, LNA-antimiR-1; Scramble, scramble locked nucleic acid; IA, infarct area; AAR, area at risk. Data are expressed as mean±SEM; n = 8; *P<0.05 <i>vs</i> WT.</p

Effects of miR-1 on serum creatinine kinase, lactate dehydrogenase level and cardiac caspase-3 activity after IR injury in mice.

<p>A, serum creatine kinase; B, lactate dehydrogenase; C, caspase-3 activity. Data are expressed as mean±SEM; n = 8 for LDH and CK, n = 5 for caspase-3; *P<0.05 <i>vs</i> WT, #P<0.05 <i>v</i>s IR.</p

Echocardiography of wild type (WT) and miR-1 transgenic (miR-1 Tg) mice.

<p>EF, eject fraction; FS, fractional shortening; LVDd, left ventricle diastolic diameter; LVSd, left ventricle systolic diameter; IVSd, interventricular septum diastolic thickness; IVSs interventricular septum systolic thickness. Data are expressed as mean±SD; n = 6 for each group.</p

Downregulation of miR-151-5p Contributes to Increased Susceptibility to Arrhythmogenesis during Myocardial Infarction with Estrogen Deprivation

<div><p>Estrogen deficiency is associated with increased incidence of cardiovascular diseases. But merely estrogen supplementary treatment can induce many severe complications such as breast cancer. The present study was designed to elucidate molecular mechanisms underlying increased susceptibility of arrhythmogenesis during myocardial infarction with estrogen deprivation, which provides us a new target to cure cardiac disease accompanied with estrogen deprivation. We successfully established a rat model of myocardial ischemia (MI) accompanied with estrogen deprivation by coronary artery ligation and ovariectomy (OVX). Vulnerability and mortality of ventricular arrhythmias increased in estrogen deficiency rats compared to non estrogen deficiency rats when suffered MI, which was associated with down-regulation of microRNA-151-5p (miR-151-5p). Luciferase Reporter Assay demonstrated that miR-151-5p can bind to the 3′-UTR of <i>FXYD1</i> (coding gene of phospholemman, PLM) and inhibit its expression. We found that the expression of PLM was increased in (OVX+MI) group compared with MI group. More changes such as down-regulation of Kir2.1/I_K1, calcium overload had emerged in (OVX+MI) group compared to MI group merely. Transfection of miR-151-5p into primary cultured myocytes decreased PLM levels and [Ca²⁺]_i, however, increased Kir2.1 levels. These effects were abolished by the antisense oligonucleotides against miR-151-5p. Co-immunoprecipitation and immunofluorescent experiments confirmed the co-localization between Kir2.1 and PLM in rat ventricular tissue. We conclude that the increased ventricular arrhythmias vulnerability in response to acute myocardial ischemia in rat is critically dependent upon down-regulation of miR-151-5p. These findings support the proposal that miR-151-5p could be a potential therapeutic target for the prevention of ischemic arrhythmias in the subjects with estrogen deficiency.</p></div