Benzhi Cai

Downregulation of miR-133 and miR-590 contributes to nicotine-induced atrial remodelling in canines

AIMS The present study was designed to decipher molecular mechanisms underlying nicotines promoting atrial fibrillation (AF) by inducing atrial structural remodelling. METHODS AND RESULTS The canine model of AF was successfully established by nicotine administration and rapid pacing. The atrial fibroblasts isolated from healthy dogs were treated with nicotine. The role of microRNAs (miRNAs) on the expression and regulation of transforming growth factor-beta1 (TGF-beta1), TGF-beta receptor type II (TGF-betaRII), and collagen production was evaluated in vivo and in vitro. Administration of nicotine for 30 days increased AF vulnerability by approximately eight- to 15-fold in dogs. Nicotine stimulated remarkable collagen production and atrial fibrosis both in vitro in cultured canine atrial fibroblasts and in vivo in atrial tissues. Nicotine produced significant upregulation of expression of TGF-beta1 and TGF-betaRII at the protein level, and a 60-70% decrease in the levels of miRNAs miR-133 and miR-590. This downregulation of miR-133 and miR-590 partly accounts for the upregulation of TGF-beta1 and TGF-betaRII, because our data established TGF-beta1 and TGF-betaRII as targets for miR-133 and miR-590 repression. Transfection of miR-133 or miR-590 into cultured atrial fibroblasts decreased TGF-beta1 and TGF-betaRII levels and collagen content. These effects were abolished by the antisense oligonucleotides against miR-133 or miR-590. The effects of nicotine were prevented by an alpha7 nicotinic acetylcholine receptor antagonist. CONCLUSION We conclude that the profibrotic response to nicotine in canine atrium is critically dependent upon downregulation of miR-133 and miR-590.

Tanshinone IIA protects against sudden cardiac death induced by lethal arrhythmias via repression of microRNA-1

Background and purpose:  Tanshinone IIA is an active component of a traditional Chinese medicine based on Salvia miltiorrhiza, which reduces sudden cardiac death by suppressing ischaemic arrhythmias. However, the mechanisms underlying the anti‐arrhythmic effects remain unclear.

microRNA-124 Regulates Cardiomyocyte Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells Via Targeting STAT3 Signaling†‡§

Accumulating evidence demonstrated that bone marrow‐derived mesenchymal stem cells (BMSCs) may transdifferentiate into cardiomyocytes and replace apoptotic myocardium so as to improve functions of damaged hearts. However, little information is known about molecular mechanisms underlying myogenic conversion of BMSCs. microRNAs as endogenous noncoding small molecules function to inhibit protein translation post‐transcriptionally by binding to complementary sequences of targeted mRNAs. Here, we reported that miR‐124 was remarkably downregulated during cardiomyocyte differentiation of BMSCs induced by coculture with cardiomyocytes. Forced expression of miR‐124 led to a significant downregulation of cardiac‐specific markers—ANP, TNT, and α‐MHC proteins as well as reduction of cardiac potassium channel currents in cocultured BMSCs. On the contrary, the inhibition of endogenous miR‐124 with its antisense oligonucleotide AMO‐124 obviously reversed the changes of ANP, TNT, and α‐MHC proteins and increased cardiac potassium channel currents. Further study revealed that miR‐124 targeted the 3′UTR of STAT3 gene so as to suppress the expression of STAT3 protein but did not affect its mRNA level. STAT3 inhibitors AG490, WP1066, and S3I‐201 were shown to attenuate the augmented expression of ANP, TNT, α‐MHC, GATA‐4 proteins, and mRNAs in cocultured BMSCs with AMO‐124 transfection. Moreover, GATA‐4 siRNA reduced the expression of ANP, TNT, α‐MHC, and GATA‐4 proteins but did not impact STAT3 protein in cocultured BMSCs, indicating GATA‐4 serves as an effector of STAT3. In summary, we found that miR‐124 regulated myogenic differentiation of BMSCs via targeting STAT3 mRNA, which provides new insights into molecular mechanisms of cardiomyogenesis of BMSCs. STEM CELLS2012;30:1746–1755

Scutellarin-induced endothelium-independent relaxation in rat aorta

Scutellarin is a flavonoid extracted from the traditional Chinese herb, Erigeron breviscapus Hand Mazz. In the present study, the vasorelaxant effects of scutellarin and the underlying mechanism were investigated in isolated rat aorta. Scutellarin (3, 10, 30, 100 µm) caused a dose‐dependent relaxation in both endothelium‐intact and endothelium‐denuded rat aortic rings precontracted with noradrenaline bitartrate (IC50 = 7.7 ± 0.6 µm), but not with potassium chloride. Tetraethylammonium, glibenclamide, atropine, propranolol, indomethacin and N(G)‐nitro‐l‐arginine methyl ester had no influence on the vasorelaxant effect of scutellarin, which further excluded the involvement of potassium channels, muscarinic receptor, nitric oxide pathway and prostaglandin in this effect. Pretreatment with scutellarin decreased the tonic phase, but not the phasic phase of the noradrenaline bitartrate induced tension increment. Scutellarin also alleviated Ca2+‐induced vasoconstriction in Ca2+‐depleted/noradrenaline bitartrate pretreated rings in the presence of voltage‐dependent calcium channel blocker verapamil. The noradrenaline bitartrate evoked intracellular calcium increase was inhibited by scutellarin. Scutellarin had no effect on phorbol‐12,13‐diacetate induced contraction in a calcium‐free bath solution. These results showed that scutellarin could relax thoracic artery rings in an endothelium‐independent manner. The mechanism seems to be the inhibition of extracellular calcium influx independent of the voltage‐dependent calcium channel. Copyright

Exosomes in mesenchymal stem cells, a new therapeutic strategy for cardiovascular diseases?

Cardiovascular diseases (CVDs) are still a major cause of people deaths worldwide, and mesenchymal stem cells (MSCs) transplantation holds great promise due to its capacity to differentiate into cardiovascular cells and secrete protective cytokines, which presents an important mechanism of MSCs therapy for CVDs. Although the capability of MSCs to differentiate into cardiomyocytes (CMCs), endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) has been well recognized in massive previous experiments both in vitro and in vivo, low survival rate of transplanted MSCs in recipient hearts suggests that therapeutic effects of MSCs transplantation might be also correlated with other underlying mechanisms. Notably, recent studies uncovered that MSCs were able to secret cholesterol-rich, phospholipid exosomes which were enriched with microRNAs (miRNAs). The released exosomes from MSCs acted on hearts and vessels, and then exerted anti-apoptosis, cardiac regeneration, anti-cardiac remodeling, anti-inflammatory effects, neovascularization and anti-vascular remodeling, which are considered as novel molecular mechanisms of therapeutic potential of MSCs transplantation. Here we summarized recent advances about the role of exosomes in MSCs therapy for CVDs, and discussed exosomes as a novel approach in the treatment of CVDs in the future.

Upregulation of microRNA-1 and microRNA-133 contributes to arsenic-induced cardiac electrical remodeling.

BACKGROUND A large body of evidence showed that arsenic trioxide (As2O3), a front-line drug for the treatment of acute promyelocytic leukemia, induced abnormal cardiac QT prolongation, which hampers its clinical use. The molecular mechanisms for this cardiotoxicity remained unclear. This study aimed to elucidate whether microRNAs (miRs) participate in As2O3-induced QT prolongation. METHODS A guinea pig model of As2O3-induced QT prolongation was established by intravenous injection with As2O3. Real-time PCR and Western blot were employed to determine the expression alterations of miRs and mRNAs, and their corresponding proteins. RESULTS The QT interval and QRS complex were significantly prolonged in a dose-dependent fashion after 7-day administration of As2O3. As2O3 induced a significant upregulation of the muscle-specific miR-1 and miR-133, as well as their transactivator serum response factor. As2O3 depressed the protein levels of ether-a-go-go related gene (ERG) and Kir2.1, the K(+) channel subunits responsible for delayed rectifier K(+) current IKr and inward rectifier K(+) current IK1, respectively. In vivo transfer of miR-133 by direct intramuscular injection prolonged QTc interval and increased mortality rate, along with depression of ERG protein and IKr in guinea pig hearts. Similarly, forced expression of miR-1 widened QTc interval and QRS complex and increased mortality rate, accompanied by downregulation of Kir2.1 protein and IK1. Application of antisense inhibitors to knockdown miR-1 and miR-133 abolished the cardiac electrical disorders caused by As2O3. CONCLUSIONS Deregulation of miR-133 and miR-1 underlies As2O3-induced cardiac electrical disorders and these miRs may serve as potential therapeutic targets for the handling of As2O3 cardiotoxicity.

Arsenic trioxide induces the apoptosis in vascular smooth muscle cells via increasing intracellular calcium and ROS formation

The present study was designed to investigate whether arsenic trioxide induced the apoptosis in rat mesenteric arterial smooth muscle cells (SMCs), which provides new insights into mechanisms of arsenic-related vascular diseases. Here, we found that arsenic trioxide significantly decreased the viability of SMCs in a dose-dependent manner. In addition, higher level of arsenic trioxide directly caused cellular necrosis. The Hoechst and AO/EB staining demonstrated that apoptotic morphological change was presented in SMCs exposed to arsenic trioxide. The TUNEL assay displayed that more positive apoptotic signal appeared in SMCs treated with arsenic trioxide. The following result showed that ROS formation was markedly increased in arsenic trioxide-treated SMCs. Pretreatment with N-acetylcysteine, an anti-oxidant reagent, obviously attenuated the enhancement of ROS production and the reduction of cell viability induced by arsenic trioxide in SMCs. Arsenic trioxide also enhanced free intracellular Ca2+ level in SMCs. BAPTA also significantly prevented the increased intracellular Ca2+ and decreased cell viability induced by arsenic trioxide in SMCs. These results suggested that arsenic trioxide obviously induced apoptosis in SMCs, and its mechanism was partially associated with intracellular ROS formation and free Ca2+ increasing.

Doxorubicin Caused Apoptosis of Mesenchymal Stem Cells via p38, JNK and p53 Pathway

Background/Aims: Doxorubicin is a widely used chemotherapeutic agent, but its clinical use is restricted because of a high risk of cardiotoxicity. Bone marrow-derived mesenchymal stem cells (BMSCs) may repair ischaemically damaged myocardium through transdifferentiation and paracrine action. The aim of this study is to investigate if doxorubicin causes the apoptosis of BMSCs and in turn impairs its healing ability. Methods: BMSCs were exposed to doxorubicin, and cell apoptosis was determined by western blot and stainings. Results: Doxorubicin reduced the survival ratio and caused the apoptosis of BMSCs, with the increase of intracellular ROS level and depolarization of mitochondrial membrane potential. The ROS scavenger NAC abrogated these consequences. Moreover, doxorubicin markedly activated phosphorylated ERK, p38 and JNK proteins in BMSCs. The specific inhibitors for p38 (SB203580) and JNK (SP600125) may abolish doxorubicin-induced apoptosis of BMSCs but the specific ERK inhibitor (PD98059) not, indicating p38 and JNK activation contribute to BMSCs apoptosis. Also, the phosphorylated and total p53 proteins were increased in doxorubicin-treated BMSCs. Proapoptotic cleaved caspases-3 was upregulated and antiapoptotic Bcl-2 protein was reduced in doxorubicin-treated BMSCs. At last, ELISA assay showed that doxorubicin treatment reduced the VEGF and IGF-1 released by BMSCs. Conclusion: Taken together, doxorubicin caused BMSCs apoptosis associated with p38, JNK and p53 pathways.

Arsenic trioxide induces the apoptosis in bone marrow mesenchymal stem cells by intracellular calcium signal and caspase-3 pathways

It was previously reported that excessive arsenic trioxide would produce cardiovascular toxicity. Bone marrow mesenchymal stem cells (BMSCs) have been shown to play a supporting role in cardiovascular functions. The increasing apoptosis of BMSCs commonly would promote the development of cardiovascular diseases. Thus we hypothesize that arsenic trioxide caused apoptosis in BMSCs, which provided a better understanding of arsenic toxicity in hearts. The present study was designed to investigate the proapoptotic effects of arsenic trioxide on BMSCs and explore the mechanism underlying arsenic trioxide-induced BMSCs apoptosis. We demonstrate that arsenic trioxide significantly inhibited survival ratios of BMSCs in a concentration-dependent and time-dependent manner. The Annexin V/PI staining and terminal deoxynucleotidyl transferasemediated dUTP nick-end labelling (TUNEL) assay also showed that arsenic trioxide markedly induced the apoptosis of BMSCs. The caspase-3 activity was obviously enhanced in the presence of arsenic trioxide in a concentration-dependent manner in BMSCs. Additionally, arsenic trioxide caused the increase of intracellular free calcium ([Ca(2+)](i)) in rat BMSCs. BAPTA pretreatment may attenuate the apoptosis of BMSCs induced by arsenic trioxide. Taken together, arsenic trioxide could inhibit the proliferation and induce the apoptosis of BMSCs by modulating intracellular [Ca(2+)](i), and activating the caspase-3 activity.

Homocysteine inhibits potassium channels in human atrial myocytes.

1 A large body of evidence indicates that elevated homocysteine (Hcy) levels portend an increased risk for atrial fibrillation. However, little is known about the electrophysiological effects of Hcy on atrial myocytes. The present study was conducted to investigate the direct effects of Hcy on ion channels in human atria. 2 Whole‐cell patch‐clamp techniques were used to record potassium currents in human atrial cells. 3 In human atrial myocytes, transient outward potassium currents were significantly decreased by 24.8 ± 5.9 and 38.4 ± 10.4% in the presence of 50 and 500 µmol/L Hcy, respectively. The ultrarapid delayed rectifier potassium currents were decreased by approximately 30% when exposed to 500 µmol/L Hcy. The inward rectifier potassium currents were increased by approximately 40% in the presence of 500 µmol/L Hcy. 4 The results of the present study indicate that Hcy, an important risk factor for atrial fibrillation, could cause electrophysiological disturbances of potassium currents in human atrial myocytes.