Jiening Xiao

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.

miR-605 joins p53 network to form a p53:miR-605:Mdm2 positive feedback loop in response to stress

In cancers with wild‐type (WT) p53 status, the function of p53 is inhibited through direct interaction with Mdm2 oncoprotein, a negative feedback loop to limit the function of p53. In response to cellular stress, p53 escapes the p53:Mdm2 negative feedback to accumulate rapidly to induce cell cycle arrest and apoptosis. We demonstrate herein that an microRNA miR‐605 is a new component in the p53 gene network, being transcriptionally activated by p53 and post‐transcriptionally repressing Mdm2. Activation of p53 upregulated miR‐605 via interacting with the promoter region of the gene. Overexpression of miR‐605 directly decreased Mdm2 expression at the post‐transcriptional level but indirectly increased the transcriptional activity of p53 on miR‐34a via downregulating Mdm2; knockdown of miR‐605 did the opposite. Mdm2 inhibitor upregulated expression of both miR‐34a and miR‐605, which was mitigated by p53 inhibitor. miR‐605 preferentially induced apoptosis in WT p53‐expressing cells, an effect abolished by p53 inhibition. These results indicate that miR‐605 acts to interrupt p53:Mdm2 interaction to create a positive feedback loop aiding rapid accumulation of p53 to facilitate its function in response to stress.

Feedback Remodeling of Cardiac Potassium Current Expression A Novel Potential Mechanism for Control of Repolarization Reserve

Background— Inhibition of individual K+ currents causes functionally based compensatory increases in other K+ currents that minimize changes in action potential duration, a phenomenon known as repolarization reserve. The possibility that sustained K+ channel inhibition may induce remodeling of ion current expression has not been tested. Accordingly, we assessed the effects of sustained inhibition of one K+ current on various other cardiac ionic currents. Methods and Results— Adult canine left ventricular cardiomyocytes were incubated in primary culture and paced at a physiological rate (1 Hz) for 24 hours in the presence or absence of the highly selective rapid delayed-rectifier K+ current (IKr) blocker dofetilide (5 nmol/L). Sustained dofetilide exposure led to shortened action potential duration and increased repolarization reserve (manifested as a reduced action potential duration–prolonging response to IKr blockade). These repolarization changes were accompanied by increased slow delayed-rectifier (IKs) density, whereas IKr, transient-outward (Ito), inward-rectifier (IK1), L-type Ca2+ (ICaL), and late Na+ current remained unchanged. The mRNA expression corresponding to KvLQT1 and minK (real-time polymerase chain reaction) was unchanged, but their protein expression (Western blot) was increased, suggesting posttranscriptional regulation. To analyze possible mechanisms, we quantified the muscle-specific microRNA subtypes miR-133a and miR-133b, which can posttranscriptionally regulate and repress KvLQT1 protein expression without affecting mRNA expression. The expression levels of miR-133a and miR-133b were significantly decreased in cells cultured in dofetilide compared with control, possibly accounting for KvLQT1 protein upregulation. Conclusions— Sustained reductions in IKr may lead to compensatory upregulation of IKs through posttranscriptional upregulation of underlying subunits, likely mediated (at least partly) by microRNA changes. These results suggest that feedback control of ion channel expression may influence repolarization reserve.

Regulation of human cardiac ion channel genes by microRNAs: theoretical perspective and pathophysiological implications.

Excitability is a fundamental characteristic of cardiac cells, which is delicately determined by ion channel activities modulated by many factors. MicroRNA (miRNA) expression is dynamically regulated and altered miRNA expression can render expression deregulation of ion channel genes leading to channelopathies-arrhythmogenesis. Indeed, evidence has emerged indicating the crucial role of miRNAs in controlling cardiac excitability by regulating expression of ion channel genes at the post-transcriptional level. However, the very limited experimental data in the literature hinder our understanding of the role of miRNAs and the often one-to-one interaction between miRNA and ion-channel gene in the published studies also casts a doubt about fullness of our view. Unfortunately, currently available techniques do not permit thorough characterization of miRNA targeting; computational prediction programs remain the only source for rapid identification of a putative miRNA target in silico. We conducted a rationally designed bioinformatics analysis in conjunction with experimental approaches to identify the miRNAs from the currently available miRNA databases which have the potential to regulate human cardiac ion channel genes and to validate the analysis with several pathological settings associated with the deregulated miRNAs and ion channel genes in the heart. We established a matrix of miRNAs that are expressed in cardiac cells and have the potential to regulate the genes encoding cardiac ion channels and transporters. We were able to explain a particular ionic remodeling process in hypertrophy/heart failure, myocardial ischemia, or atrial fibrillation with the corresponding deregulated miRNAs under that pathological condition; the changes of miRNAs appear to have anti-correlation with the changes of many of the genes encoding cardiac ion channels under these situations. These results indicate that multiple miRNAs might be critically involved in the electrical/ionic remodeling processes of cardiac diseases through altering their expression in cardiac cells, which has not been uncovered by previous experimental studies.

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.

Genomic structure, transcriptional control, and tissue distribution of HERG1 and KCNQ1 genes.

The long QT syndrome genes human ether-a-go-go-related gene (HERG1) and voltage-gated K+ channel, KQT-like subfamily, member 1, gene (KCNQ1), encoding K+ channels critical to the repolarization rate and repolarization reserve in cardiac cells, and thereby the likelihood of arrhythmias, are both composed of two isoforms: HERG1a and HERG1b and KCNQ1a and KCNQ1b, respectively. Expression of these genes is dynamic, depending on the differentiation status and disease states. We identified their core promoter regions and transcription start sites. Our data suggest that HERG1a and HERG1b, and KCNQ1a and KCNQ1b, represent independent transcripts instead of being alternatively spliced variants of the same gene, for they each have their own transcription start sites and their own promoter regions. We obtained data pointing to the potential role of stimulating protein 1 (Sp1) in the transactivation of these genes. We compared expression profiling of these genes across a variety of human tissues. Consistent with the general lack of cis elements for cardiac-specific transcription factors and the presence of multiple sites for ubiquitous Sp1 sites in the core promoter regions of HERG1a/HERG1b and KCNQ1a/KCNQ1b genes, the transcripts demonstrated widespread distribution across a variety of human tissues. We further revealed that the mRNA levels of all HERG1 and KCNQ1 isoforms were asymmetrically distributed within the heart, being more abundant in the right atria and ventricles relative to the left atria and ventricles. These findings open up an opportunity for studying interventricular gradients of slow and rapid delayed rectifier K+ current and of cardiac repolarization as well. Our study might help us understand the molecular mechanisms for arrhythmias since heterogeneity of ion channel activities is an important substrate for arrhythmogenesis.

Transcriptional and post-transcriptional mechanisms for oncogenic overexpression of ether à go-go K+ channel.

The human ether-à-go-go-1 (h-eag1) K+ channel is expressed in a variety of cell lines derived from human malignant tumors and in clinical samples of several different cancers, but is otherwise absent in normal tissues. It was found to be necessary for cell cycle progression and tumorigenesis. Specific inhibition of h-eag1 expression leads to inhibition of tumor cell proliferation. We report here that h-eag1 expression is controlled by the p53−miR-34−E2F1 pathway through a negative feed-forward mechanism. We first established E2F1 as a transactivator of h-eag1 gene through characterizing its promoter region. We then revealed that miR-34, a known transcriptional target of p53, is an important negative regulator of h-eag1 through dual mechanisms by directly repressing h-eag1 at the post-transcriptional level and indirectly silencing h-eag1 at the transcriptional level via repressing E2F1. There is a strong inverse relationship between the expression levels of miR-34 and h-eag1 protein. H-eag1antisense antagonized the growth-stimulating effects and the upregulation of h-eag1 expression in SHSY5Y cells, induced by knockdown of miR-34, E2F1 overexpression, or inhibition of p53 activity. Therefore, p53 negatively regulates h-eag1 expression by a negative feed-forward mechanism through the p53−miR-34−E2F1 pathway. Inactivation of p53 activity, as is the case in many cancers, can thus cause oncogenic overexpression of h-eag1 by relieving the negative feed-forward regulation. These findings not only help us understand the molecular mechanisms for oncogenic overexpression of h-eag1 in tumorigenesis but also uncover the cell-cycle regulation through the p53−miR-34−E2F1−h-eag1 pathway. Moreover, these findings place h-eag1 in the p53−miR-34−E2F1−h-eag1 pathway with h-eag as a terminal effecter component and with miR-34 (and E2F1) as a linker between p53 and h-eag1. Our study therefore fills the gap between p53 pathway and its cellular function mediated by h-eag1.

Retraction: Overexpression HERG K+ channel gene mediates cell‐growth signals on activation of oncoproteins SP1 and NF‐κB and inactivation of tumor suppressor Nkx3.1

The long QT syndrome gene human ether‐a‐go‐go related gene (HERG) encodes a K+ channel critical to cardiac repolarization. It peculiarly overexpresses in cancer cells of different histogenesis and promotes tumorigenesis. To decipher the molecular mechanisms for HERG overexpression, we identified and characterized the promoter region of the HERG gene, which contains cis‐elements for multiple oncoproteins and tumor suppressors. Oncoprotein Sp1 was found to be essential to driving the HERG promoter thereby transcription. Another oncoprotein NF‐κB transactivated, while tumor suppressor Nkx3.1 repressed HERG promoter activity and endogenous HERG transcription. Loss‐of‐function mutations in the corresponding cis‐elements rendered a loss of the ability of the oncoproteins Sp1 and NF‐κB to transactivate, and of the tumor repressor Nkx3.1 to repress, HERG transcription. Either activation of Sp1 and NF‐κB or silencing of Nkx3.1 promoted tumor cell growth, and the effects were abrogated by HERG inhibition or knockdown, but facilitated by overexpression of HERG, indicating that HERG mediates the cell growth signals generated by activation of oncoproteins or inactivation of tumor suppressors. Binding of Sp1, NF‐κB, and Nkx3.1 to their respective cis‐elements in the HERG promoter in vitro and their presence on the HERG promoter in vivo were confirmed. Therefore, the HERG promoter region is characterized by multiple Sp1 binding sites that are responsible for transcription initiation of the HERG gene and by binding sites for multiple other oncogenes and tumor suppressor genes being important for regulating HERG expression. The HERG K+ channel is likely a mediator of growth‐promoting processes induced by oncoproteins and/or by silencing of tumor suppressors. J. Cell. Physiol. 212: 137–147, 2007.

Detailed characterization of microRNA changes in a canine heart failure model: Relationship to arrhythmogenic structural remodeling

Heart failure (HF) causes left-atrial (LA) and left-ventricular (LV) remodeling, with particularly-prominent changes in LA that create a substrate for atrial fibrillation (AF). MicroRNAs (miRs) are potential regulators in cardiac remodeling. This study evaluated time-dependent miR expression-changes in LA and LV tissue, fibroblasts and cardiomyocytes in experimental HF. HF was induced in dogs by ventricular tachypacing (varying periods, up to 2weeks). Following screening-microarray, 15 miRs were selected for detailed real-time qPCR assay. Extracellular matrix mRNA-expression was assessed by qPCR. Tachypacing time-dependently reduced LV ejection-fraction, increased LV-volume and AF-duration, and caused tissue-fibrosis with LA changes greater than LV. Tissue miR-expression significantly changed in LA for 10 miRs; in LV for none. Cell-selective analysis showed significant time-dependent changes in LA-fibroblasts for 10/15 miRs, LV-fibroblasts 8/15, LA-cardiomyocytes in 6/15 and LV-cardiomyocytes 3/15. Cell-expression specificity did not predict cell-specificity of VTP-induced expression-changes, e.g. 4/6 cardiomyocyte-selective miRs changed almost exclusively in fibroblasts (miR-1, miR-208b, miR133a/b). Thirteen miRs directly implicated in fibrosis/extracellular-matrix regulation were prominently changed: 9/13 showed fibroblast-selective alterations and 5/13 LA-selective. Multiple miRs changed in relation to associated extracellular-matrix targets. Experimental HF causes tissue and cell-type selective, time-dependent changes in cardiac miR-expression. Expression-changes are greater in LA versus LV, and greater in fibroblasts than cardiomyocytes, even for most cardiomyocyte-enriched miRs. This study, the first to examine time, chamber and cell-type selective changes in an experimental model of HF, suggests that multiple miR-changes underlie the atrial-selective fibrotic response and emphasize the importance of considering cell-specificity of miR expression-changes in cardiac remodeling paradigms.