Baofeng Yang

The muscle-specific microRNA miR-1 regulates cardiac arrhythmogenic potential by targeting GJA1 and KCNJ2

MicroRNAs (miRNAs) are endogenous noncoding RNAs, about 22 nucleotides in length, that mediate post-transcriptional gene silencing by annealing to inexactly complementary sequences in the 3′-untranslated regions of target mRNAs. Our current understanding of the functions of miRNAs relies mainly on their tissue-specific or developmental stage-dependent expression and their evolutionary conservation, and therefore is primarily limited to their involvement in developmental regulation and oncogenesis. Of more than 300 miRNAs that have been identified, miR-1 and miR-133 are considered to be muscle specific. Here we show that miR-1 is overexpressed in individuals with coronary artery disease, and that when overexpressed in normal or infarcted rat hearts, it exacerbates arrhythmogenesis. Elimination of miR-1 by an antisense inhibitor in infarcted rat hearts relieved arrhythmogenesis. miR-1 overexpression slowed conduction and depolarized the cytoplasmic membrane by post-transcriptionally repressing KCNJ2 (which encodes the K+ channel subunit Kir2.1) and GJA1 (which encodes connexin 43), and this likely accounts at least in part for its arrhythmogenic potential. Thus, miR-1 may have important pathophysiological functions in the heart, and is a potential antiarrhythmic target.

Circulating microRNA-1 as a potential novel biomarker for acute myocardial infarction

Recent studies have revealed the role of microRNAs (miRNAs) in a variety of basic biological and pathological processes and the association of miRNA signatures with human diseases. Circulating miRNAs have been proposed as sensitive and informative biomarkers for multiple cancers diagnosis. We have previously documented aberrant up-regulation of miR-1 expression in ischemic myocardium and the consequent slowing of cardiac conduction. However, whether miR-1 could be a biomarker for predicting acute myocardial infarction (AMI) is unclear. In the present study, we recruited 159 patients with or without AMI for quantification of miR-1 level in plasma using real-time RT-PCR method. We performed Wilcoxon rank sum and signed rank tests for comparison. Univariable linear regression and logistics regression analyses were performed to assess the potential correlation between miR-1 and known AMI markers. We also conducted receiver-operator characteristic curve (ROC) analysis to evaluate the diagnostic ability of miR-1. We found that: miR-1 level was significantly higher in plasma from AMI patients compared with non-AMI subjects and the level was dropped to normal on discharge following medication. Increased circulating miR-1 was not associated with age, gender, blood pressure, diabetes mellitus or the established biomarkers for AMI. However, miR-1 level was well correlated with QRS by both univariable linear and logistics regression analyses. The area under ROC curve (AUC) was 0.7740 for separation between non-AMI and AMI patients and 0.8522 for separation AMI patients under hospitalization and discharge. Collectively, our results revealed that circulating miR-1 may be a novel, independent biomarker for diagnosis of AMI.

The muscle-specific microRNAs miR-1 and miR-133 produce opposing effects on apoptosis by targeting HSP60, HSP70 and caspase-9 in cardiomyocytes.

The microRNAs miR-1 and miR-133 are preferentially expressed in cardiac and skeletal muscles and have been shown to regulate differentiation and proliferation of these cells. We report here a novel aspect of cellular function of miR-1 and miR-133 regulation of cardiomyocyte apoptosis. miR-1 and miR-133 produced opposing effects on apoptosis, induced by oxidative stress in H9c2 rat ventricular cells, with miR-1 being pro-apoptotic and miR-133 being anti-apoptotic. miR-1 level was significantly increased in response to oxidative stress. We identified single target sites for miR-1 only, in the 3′-untranslated regions of the HSP60 and HSP70 genes, and multiple putative target sites for miR-133 throughout the sequence of the caspase-9 gene. miR-1 reduced the levels of HSP60 and HSP70 proteins without changing their transcript levels, whereas miR-133 did not affect HSP60 and HSP70 expression at all. By contrast, miR-133 repressed caspase-9 expression at both the protein and mRNA levels. The post-transcriptional repression of HSP60 and HSP70 and caspase-9 was further confirmed by luciferase reporter experiments. Our results indicate that miR-1 and miR-133 are involved in regulating cell fate with increased miR-1 and/or decreased miR-133 levels favoring apoptosis and decreased miR-1 and/or miR-133 levels favoring survival. Post-transcriptional repression of HSP60 and HSP70 by miR-1 and of caspase-9 by miR-133 contributes significantly to their opposing actions.

MicroRNA-328 Contributes to Adverse Electrical Remodeling in Atrial Fibrillation

Background— A characteristic of both clinical and experimental atrial fibrillation (AF) is atrial electric remodeling associated with profound reduction of L-type Ca2+ current and shortening of the action potential duration. The possibility that microRNAs (miRNAs) may be involved in this process has not been tested. Accordingly, we assessed the potential role of miRNAs in regulating experimental AF. Methods and Results— The miRNA transcriptome was analyzed by microarray and verified by real-time reverse-transcription polymerase chain reaction with left atrial samples from dogs with AF established by right atrial tachypacing for 8 weeks and from human atrial samples from AF patients with rheumatic heart disease. miR-223, miR-328, and miR-664 were found to be upregulated by >2 fold, whereas miR-101, miR-320, and miR-499 were downregulated by at least 50%. In particular, miR-328 level was elevated by 3.9-fold in AF dogs and 3.5-fold in AF patients relative to non-AF subjects. Computational prediction identified CACNA1C and CACNB1, which encode cardiac L-type Ca2+ channel &agr;1c- and &bgr;1 subunits, respectively, as potential targets for miR-328. Forced expression of miR-328 through adenovirus infection in canine atrium and transgenic approach in mice recapitulated the phenotypes of AF, exemplified by enhanced AF vulnerability, diminished L-type Ca2+ current, and shortened atrial action potential duration. Normalization of miR-328 level with antagomiR reversed the conditions, and genetic knockdown of endogenous miR-328 dampened AF vulnerability. CACNA1C and CACNB1 as the cognate target genes for miR-328 were confirmed by Western blot and luciferase activity assay showing the reciprocal relationship between the levels of miR-328 and L-type Ca2+ channel protein subunits. Conclusions— miR-328 contributes to the adverse atrial electric remodeling in AF through targeting L-type Ca2+ channel genes. The study therefore uncovered a novel molecular mechanism for AF and indicated miR-328 as a potential therapeutic target for AF.

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.

Retracted: Novel approaches for gene‐specific interference via manipulating actions of microRNAs: Examination on the pacemaker channel genes HCN2 and HCN4

Recent evidence has suggested microRNAs as viable therapeutic targets for a wide range of human disease. However, lack of gene‐specificity of microRNA actions may hinder this application. Here we developed two new approaches, the gene‐specific microRNA mimic and microRNA‐masking antisense approaches, to explore the possibility of using microRNAs principle of actions in a gene‐specific manner. We examined the value of these strategies as rational approaches to develop heart rate‐reducing agents and “biological pacemakers” by manipulating the expression of the cardiac pacemaker channel genes HCN2 and HCN4. We showed that the gene‐specific microRNA mimics, 22‐nt RNAs designed to target the 3′untranslated regions (3′UTRs) of HCN2 and HCN4, respectively, were efficient in abrogating expression and function of HCN2 and HCN4. The gene‐specific microRNA mimics repressed protein levels, accompanied by depressed f‐channel conductance and the associated rhythmic activity, without affecting mRNA levels of HCN2 and HCN4. Meanwhile, we also designed the microRNA‐masking antisense based on the miR‐1 and miR‐133 target sites in the 3′UTRs of HCN2 and HCN4 and found that these antisense oligodeoxynucleotides markedly enhanced HCN2/HCN4 expression and function, as reflected by increased protein levels of HCN2/HCN4 and If conductance, by removing the repression of HCN2/HCN4 expression induced by endogenous miR‐1/miR‐133. The experimental examination of these techniques and the resultant findings not only indicate feasibility of interfering miRNA action in a gene‐specific fashion but also may provide a new research tool for studying function of miRNAs. The new approaches also have the potential of becoming alternative gene therapy strategies. J. Cell. Physiol. 212: 285–292, 2007.

MicroRNA-101 Inhibited Postinfarct Cardiac Fibrosis and Improved Left Ventricular Compliance via the FBJ Osteosarcoma Oncogene/Transforming Growth Factor-β1 Pathway

Background— Cardiac interstitial fibrosis is a major cause of the deteriorated performance of the heart in patients with chronic myocardial infarction. MicroRNAs (miRs) have recently been proven to be a novel class of regulators of cardiovascular diseases, including those associated with cardiac fibrosis. This study aimed to explore the role of miR-101 in cardiac fibrosis and the underlying mechanisms. Methods and Results— Four weeks after coronary artery ligation of rats, the expression of miR-101a and miR-101b (miR-101a/b) in the peri-infarct area was decreased. Treatment of cultured rat neonatal cardiac fibroblasts with angiotensin II also suppressed the expression of miR-101a/b. Forced expression of miR-101a/b suppressed the proliferation and collagen production in rat neonatal cardiac fibroblasts, as revealed by cell counting, MTT assay, and quantitative reverse transcription–polymerase chain reaction. The effect was abrogated by cotransfection with AMO-101a/b, the antisense inhibitors of miR-101a/b. c-Fos was found to be a target of miR-101a because overexpression of miR-101a decreased the protein and mRNA levels of c-Fos and its downstream protein transforming growth factor-&bgr;1. Silencing c-Fos by siRNA mimicked the antifibrotic action of miR-101a, whereas forced expression of c-Fos protein canceled the effect of miR-101a in cultured cardiac fibroblasts. Strikingly, echocardiography and hemodynamic measurements indicated remarkable improvement of the cardiac performance 4 weeks after adenovirus-mediated overexpression of miR-101a in rats with chronic myocardial infarction. Furthermore, the interstitial fibrosis was alleviated and the expression of c-Fos and transforming growth factor-&bgr;1 was inhibited. Conclusion— Overexpression of miR-101a can mitigate interstitial fibrosis and the deterioration of cardiac performance in postinfarct rats, indicating the therapeutic potential of miR-101a for cardiac disease associated with fibrosis.Background —Cardiac interstitial fibrosis is a major cause of the deteriorated performance of the heart in patients with chronic myocardial infarction. MicroRNAs have recently been proven a novel class of regulators of cardiovascular diseases, including those associated with cardiac fibrosis. This study aimed to explore the role of miR-101 in cardiac fibrosis and the underlying mechanisms. Methods and Results —Four weeks after coronary artery ligation of rats, the expression of miR-101a and miR-101b (miR-101a/b) in the peri-infarct area was decreased. Treatment of cultured rat neonatal cardiac fibroblasts (CFs) with angiotensin II (AngII) also suppressed the expression of miR-101a/b. Forced expression of miR-101a/b suppressed the proliferation and collagen production in rat neonatal CFs, as revealed by cell counting, MTT assay and qRT-PCR. The effect was abrogated by co-transfection with AMO-101a/b, the antisense inhibitors of miR-101a/b. c-Fos was found to be a target of miR-101a as overexpression of miR-101a decreased the protein and mRNA levels of c-Fos and its downstream protein TGFβ1. Silencing c-Fos by small interfering RNA mimicked the anti-fibrotic action of miR-101a, whereas forced expression of c-Fos protein canceled the effect of miR-101a in cultured CFs. Strikingly, echocardiography and hemodynamic measurements indicated remarkable improvement of the cardiac performance 4-weeks after adenovirus mediated overexpression of miR-101a in rats with chronic myocardial infarction. Furthermore, the interstitial fibrosis was alleviated and the expression of c-Fos and TGFβ1 was inhibited. Conclusions —Overexpression of miR-101a can mitigate interstitial fibrosis and the deterioration of cardiac performance in post-infarct rats, indicating the therapeutic potential of miR-101a for cardiac disease associated with fibrosis.

miR-101 Inhibited Post-Infarct Cardiac Fibrosis and Improved Left Ventricular Compliance via FOS/TGFβ1 Pathway

Background— Cardiac interstitial fibrosis is a major cause of the deteriorated performance of the heart in patients with chronic myocardial infarction. MicroRNAs (miRs) have recently been proven to be a novel class of regulators of cardiovascular diseases, including those associated with cardiac fibrosis. This study aimed to explore the role of miR-101 in cardiac fibrosis and the underlying mechanisms. Methods and Results— Four weeks after coronary artery ligation of rats, the expression of miR-101a and miR-101b (miR-101a/b) in the peri-infarct area was decreased. Treatment of cultured rat neonatal cardiac fibroblasts with angiotensin II also suppressed the expression of miR-101a/b. Forced expression of miR-101a/b suppressed the proliferation and collagen production in rat neonatal cardiac fibroblasts, as revealed by cell counting, MTT assay, and quantitative reverse transcription–polymerase chain reaction. The effect was abrogated by cotransfection with AMO-101a/b, the antisense inhibitors of miR-101a/b. c-Fos was found to be a target of miR-101a because overexpression of miR-101a decreased the protein and mRNA levels of c-Fos and its downstream protein transforming growth factor-&bgr;1. Silencing c-Fos by siRNA mimicked the antifibrotic action of miR-101a, whereas forced expression of c-Fos protein canceled the effect of miR-101a in cultured cardiac fibroblasts. Strikingly, echocardiography and hemodynamic measurements indicated remarkable improvement of the cardiac performance 4 weeks after adenovirus-mediated overexpression of miR-101a in rats with chronic myocardial infarction. Furthermore, the interstitial fibrosis was alleviated and the expression of c-Fos and transforming growth factor-&bgr;1 was inhibited. Conclusion— Overexpression of miR-101a can mitigate interstitial fibrosis and the deterioration of cardiac performance in postinfarct rats, indicating the therapeutic potential of miR-101a for cardiac disease associated with fibrosis.Background —Cardiac interstitial fibrosis is a major cause of the deteriorated performance of the heart in patients with chronic myocardial infarction. MicroRNAs have recently been proven a novel class of regulators of cardiovascular diseases, including those associated with cardiac fibrosis. This study aimed to explore the role of miR-101 in cardiac fibrosis and the underlying mechanisms. Methods and Results —Four weeks after coronary artery ligation of rats, the expression of miR-101a and miR-101b (miR-101a/b) in the peri-infarct area was decreased. Treatment of cultured rat neonatal cardiac fibroblasts (CFs) with angiotensin II (AngII) also suppressed the expression of miR-101a/b. Forced expression of miR-101a/b suppressed the proliferation and collagen production in rat neonatal CFs, as revealed by cell counting, MTT assay and qRT-PCR. The effect was abrogated by co-transfection with AMO-101a/b, the antisense inhibitors of miR-101a/b. c-Fos was found to be a target of miR-101a as overexpression of miR-101a decreased the protein and mRNA levels of c-Fos and its downstream protein TGFβ1. Silencing c-Fos by small interfering RNA mimicked the anti-fibrotic action of miR-101a, whereas forced expression of c-Fos protein canceled the effect of miR-101a in cultured CFs. Strikingly, echocardiography and hemodynamic measurements indicated remarkable improvement of the cardiac performance 4-weeks after adenovirus mediated overexpression of miR-101a in rats with chronic myocardial infarction. Furthermore, the interstitial fibrosis was alleviated and the expression of c-Fos and TGFβ1 was inhibited. Conclusions —Overexpression of miR-101a can mitigate interstitial fibrosis and the deterioration of cardiac performance in post-infarct rats, indicating the therapeutic potential of miR-101a for cardiac disease associated with fibrosis.

MicroRNA-26 governs profibrillatory inward-rectifier potassium current changes in atrial fibrillation

Atrial fibrillation (AF) is a highly prevalent arrhythmia with pronounced morbidity and mortality. Inward-rectifier K+ current (IK1) is believed to be an important regulator of reentrant-spiral dynamics and a major component of AF-related electrical remodeling. MicroRNA-26 (miR-26) is predicted to target the gene encoding KIR2.1, KCNJ2. We found that miR-26 was downregulated in atrial samples from AF animals and patients and this downregulation was accompanied by upregulation of IK1/KIR2.1 protein. miR-26 overexpression suppressed expression of KCNJ2/KIR2.1. In contrast, miR-26 knockdown, inhibition, or binding-site mutation enhanced KCNJ2/KIR2.1 expression, establishing KCNJ2 as a miR-26 target. Knockdown of endogenous miR-26 promoted AF in mice, whereas adenovirus-mediated expression of miR-26 reduced AF vulnerability. Kcnj2-specific miR-masks eliminated miR-26-mediated reductions in Kcnj2, abolishing miR-26s protective effects, while coinjection of a Kcnj2-specific miR-mimic prevented miR-26 knockdown-associated AF in mice. Nuclear factor of activated T cells (NFAT), a known actor in AF-associated remodeling, was found to negatively regulate miR-26 transcription. Our results demonstrate that miR-26 controls the expression of KCNJ2 and suggest that this downregulation may promote AF.

MicroRNA-1 downregulation by propranolol in a rat model of myocardial infarction: a new mechanism for ischaemic cardioprotection

AIMS The present study was designed to investigate whether the beneficial effects of beta-blocker propranolol are related to regulation of microRNA miR-1. METHODS AND RESULTS We demonstrated that propranolol reduced the incidence of arrhythmias in a rat model of myocardial infarction by coronary artery occlusion. Overexpression of miR-1 was observed in ischaemic myocardium and strikingly, administration of propranolol reversed the up-regulation of miR-1 nearly back to the control level. In agreement with its miR-1-reducing effect, propranolol relieved myocardial injuries during ischaemia, restored the membrane depolarization and cardiac conduction slowing, by rescuing the expression of inward rectifying K(+) channel subunit Kir2.1 and gap junction channel connexin 43. Our results further revealed that the beta-adrenoceptor-cAMP-Protein Kinase A (PKA) signalling pathway contributed to the expression of miR-1, and serum response factor (SRF), which is known as one of the transcriptional enhancers of miR-1, was up-regulated in ischaemic myocardium. Moreover, propranolol inhibited the beta-adrenoceptor-cAMP-PKA signalling pathway and suppressed SRF expression. CONCLUSION We conclude that the beta-adrenergic pathway can stimulate expression of arrhythmogenic miR-1, contributing to ischaemic arrhythmogenesis, and beta-blockers produce their beneficial effects partially by down-regulating miR-1, which might be a novel strategy for ischaemic cardioprotection.