Yunlong Bai

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.

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.

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.

A single anti-microRNA antisense oligodeoxyribonucleotide (AMO) targeting multiple microRNAs offers an improved approach for microRNA interference

Anti-miRNA antisense inhibitors (AMOs) have demonstrated their utility in miRNA research and potential in miRNA therapy. Here we report a modified AMO approach in which multiple antisense units are engineered into a single unit that is able to simultaneously silence multiple-target miRNAs, the multiple-target AMO or MTg-AMO. We validated the technique with two separate MTg-AMOs: anti-miR-21/anti-miR-155/anti-miR-17-5p and anti-miR-1/anti-miR-133. We first verified the ability of the MTg-AMOs to antagonize the repressive actions of their target miRNAs using luciferase reporter activity assays and to specifically knock down the levels of their target miRNAs using real-time RT-PCR methods. We then used the MTg-AMO approach to identify several tumor suppressors—TGFBI, APC and BCL2L11 as the target genes for oncogenic miR-21, miR-155 and miR-17-5p, respectively, and two cardiac ion channel genes HCN2 (encoding a subunit of cardiac pacemaker channel) and CACNA1C (encoding the α-subunit of cardiac L-type Ca2+ channel) for the muscle-specific miR-1 and miR-133. We further demonstrated that the MTg-AMO targeting miR-21, miR-155 and miR-17-5p produced a greater inhibitory effect on cancer cell growth, compared with the regular single-target AMOs. Moreover, while using the regular single-target AMOs excluded HCN2 as a target gene for either miR-1 or miR-133, the MTg-AMO approach is able to reveal HCN2 as the target for both miR-1 and miR-133. Our findings suggest the MTg-AMO as an improved approach for miRNA target finding and for studying function of miRNAs. This approach may find its broad application for exploring biological processes involving multiple miRNAs and multiple genes.

Ionic mechanisms underlying abnormal QT prolongation and the associated arrhythmias in diabetic rabbits: a role of rapid delayed rectifier K+ current.

Abnormal QT prolongation with the associated arrhythmias is considered the major cardiac electrical disorder and a significant predictor of mortality in diabetic patients. The precise ionic mechanisms for diabetic QT prolongation remained unclear. We performed whole-cell patch-clamp studies in a rabbit model of alloxan-induced insulin-dependent diabetes mellitus. We demonstrated that heart rate-corrected QT interval and action potential duration (APD) were prolonged by ñ20% with frequent occurrence of ventricular tachyarrhythmias. Several K+ currents were found decreased in diabetic rabbits including transient outward K+current (Ito) that was reduced by ñ60%, rapid delayed rectifier K+ current (IKr) reduced by ñ70% and slow delayed rectifier K+ current (IKs) reduced by ñ40%. The time-dependent kinetics of these currents remained unaltered. The peak amplitude of L-type Ca% current (ICaL) was reduced by ñ22% and the inactivation kinetics was slowed; the integration of these two effects yielded ñ15% reduction of ICaL. The inward rectifier K+ current (IK1) and fast sodium current (INa) were unaffected. Simulation with LabHEART, a computer model of rabbit ventricular action potentials, revealed that inhibition of Ito or IKs alone fails to alter APD whereas inhibition of IKr alone results in 30% APD prolongation and inhibition of ICaL alone causes 10% APD shortening. Integration of changes of all these currents leads to ñ20% APD lengthening. Protein levels of the pore-forming subunits for these ion channels were decreased to varying extents, as revealed by immunoblotting analysis. Our study represents the first documentation of IKr channelopathy as the major ionic mechanism for diabetic QT prolongation.

A Single Decoy Oligodeoxynucleotides Targeting Multiple Oncoproteins Produces Strong Anticancer Effects

Cancer in general is a multifactorial process. Targeting a single factor may not be optimal in therapy, because single agents are limited by incomplete efficacy and dose-limiting adverse effects. Combination pharmacotherapy or “drug cocktail” therapy has value against many diseases, including cancers. We report an innovative decoy oligodeoxynucleotide (dODN) technology that we term complex decoy oligodeoxynucleotide (cdODNs) in which multiple cis elements are engineered into single dODNs attacking multiple target transcription factors, mimicking the drug cocktail approach. We designed dODNs targeting NF-κB, E2F, and Stat3 separately and a cdODN targeting NF-κB, E2F, and Stat3 concomitantly. We evaluated effects of this cdODN on expression of cancer-related genes, viability of human cancer cell lines, and in vivo tumor growth in nude mice. The cdODN targeting all NF-κB, E2F, and Stat3 together demonstrated enhancement of efficacy of more than 2-fold and increases in potency of 2 orders of magnitude compared with each of the dODNs or the combination of all three dODNs. The cdODN also showed earlier onset and longer-lasting action. Most strikingly, the cdODN acquired the ability to attack multiple molecules critical to cancer progression via multiple mechanisms, leading to elimination of regression. Real-time reverse transcription-polymerase chain reaction revealed that the cdODNs knocked down expression of the genes regulated by the target transcription factors. The cdODN strategy offers resourceful combinations of varying cis elements for concomitantly targeting multiple molecules in cancer biological processes and opens the door to “one-drug, multiple-target” therapy for a broad range of human cancers.

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.

Sphingolipid Metabolite Ceramide Causes Metabolic Perturbation Contributing to HERG K+ Channel Dysfunction

Ceramide, a sphingolipid metabolite, has emerged as a key second messenger molecule that mediates multiple cellular functions. Its de nova synthesis and accumulation in ischemic myocardium, congestive heart failure and diabetic cardiomyopathy is associated with the abnormalities such as abnormal QT prolongation and increased risk of arrhythmias. To investigate how ceramide is involved in modulating cardiac repolarization, we performed whole-cell patch-clamp studies on HERG current (IHERG), a critical determinant of cardiac repolarization, expressed in HEK293 cells. Acute application (superfusion for 25min) of membrane permeable ceramide (C2, 5 µM) did not alter IHERG. Prolonged incubation with C2 for 10hrs caused pronounced IHERG inhibition in a concentration-dependent and voltage-independent fashion and positive shift of voltage-dependent HERG activation. The IC50 for IHERG suppression was 19.5 µM. C2 did not affect the inactivation property and time-dependent kinetics of IHERG. Similar effects were observed with production of endogenous ceramide catalyzed by sphingomyelinase. Tyrosine kinase inhibitors failed to reverse C2-induced suppression of HERG function, and PKA and PKC inhibitors only slightly reversed the IHERG depression. Western blotting and immunocytochemical analyses indicate that C2 does not alter HERG protein expression on the cytoplasmic membrane. The inhibitory effect of C2 on IHERG was reversed by antioxidants vitamin E or MnTBAP. C2 caused considerable production of intracellular reactive oxygen species (ROS), which was prevented by vitamin E or MnTBAP. We conclude that ceramide depresses IHERG mainly via ROS overproduction and ceramide-induced IHERG impairment may contribute to QT prolongation in prolonged myocardial ischemia, heart failure and diabetic cardiomyopathy.

HIV Tat protein inhibits hERG K+ channels: a potential mechanism of HIV infection induced LQTs.

HIV-infected patients have a high prevalence of long QT syndrome (LQTs). hERG K(+) channel encoded by human ether-a-go-go related gene contributes to IKr K(+) currents responsible for the repolarization of cardiomyocytes. Inhibition of hERG K(+) channels leads to LQTs. HIV Tat protein, the virus transactivator protein, plays a pivotal role in AIDS. The aim of the present study is to examine the effects of HIV Tat protein on hERG K(+) channels stably expressed in HEK293 cells. The hERG K(+) currents were recorded by whole-cell patch-clamp technique and the hERG channel expression was measured by real-time PCR and Western blot techniques. HIV Tat protein at 200 ng/ml concentration showed no acute effect on hERG currents, but HIV Tat protein (200 ng/ml) incubation for 24 h significantly inhibited hERG currents. In HIV Tat incubated cells, the inactivation and the recovery time from inactivation of hERG channels were significantly changed. HIV Tat protein incubation (200 ng/ml) for 24h had no effect on the hERG mRNA expression, but dose-dependently inhibited hERG protein expression. The MTT assay showed that HIV Tat protein at 50 ng/ml and 200 ng/ml had no effect on the cell viability. HIV Tat protein increased reactive oxygen species (ROS) generation and the inhibition of hERG channel protein expression by HIV Tat protein was prevented by antioxidant tempol. HIV Tat protein in vivo treatment reduced IKr currents and prolonged action potential duration of guinea pig cardiomyocytes. We conclude that HIV Tat protein inhibits hERG K(+) currents through the inhibition of hERG protein expression, which might be the potential mechanism of HIV infection induced LQTs.

L-type Calcium Current (ICa,L) and Inward Rectifier Potassium Current (IK1) are Involved in QT Prolongation Induced by Arsenic Trioxide in Rat

The present study was designed to study the effects of As₂O₃ on QT interval prolongation and to explore the potential ionic mechanisms in isolated rat ventricular cardiomyocytes. The rats of As₂O₃ group were treated with 0.8 mg·kg^-1·d^-1 As₂O₃ intravenously for 7 days consecutively and the control group with saline. The ECG was recorded to calculate heart rate-corrected QT interval (QTc). Single cardiomyocytes were isolated by using collagenase II, and the action potential duration (APD) and ion currents were recorded by whole-cell patch clamp. [Ca²⁺]_>i was examined by confocal laser scanning microscopy. Our data showed that both QTc and APD were prolonged significantly after As₂O₃treatment. Meanwhile, As₂O₃ suppressed I _K1 and shifted the reversal potential to more positive direction. Moreover, the density of I _Ca,L was augmented significantly, and the steady-state activation curve became more negative, whereas, the inactivation and reactivation of I _Ca,L were not changed notably after As₂O₃ administration. Furthermore, the maximal [Ca²⁺] _i was enhanced obviously by either KCl or caffeine stimulation in As₂O₃-treated cardiomyocytes. Our results show that the potential mechanism of As₂O₃-induced QT interval prolongation in rat might be relative to disturbing the fine balance of transmembrane currents ( increasing I _Ca,L and decreasing I _K1) and causing APD prolongation.