Huixian Lin

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.

Choline produces cytoprotective effects against ischemic myocardial injuries: evidence for the role of cardiac m3 subtype muscarinic acetylcholine receptors.

Background/Aims: Accumulating evidence indicates the presence of functional M3 subtype of acetylcholine muscarinic receptors (M3-mAChR), in addition to the well-recognized M2-mAChR, in the heart of various species including man. However, the pathophysiological role of the cardiac M3-mAChR remain undefined. This study was designed to explore the possible role of M3-mAChR in cytoprotection of myocardial infarction and several related signaling pathways as potential mechanisms. Methods: Studies were performed in a rat model of myocardial infarction and in isolated myocytes. Results: We found that choline relieved myocardial injuries during ischemia or under oxidative stress, which was achieved by correcting hemodynamic impairment, diminishing ventricular arrhythmias and protecting cardiomyocytes from apoptotic death. The beneficial effects of choline were reversed by the M3-selective antagonists but not by the M2-selective antagonist. Choline/M3-mAChR activated several survival signaling molecules (antiapoptotic proteins Bcl-2 and ERKs), increased endogenous antioxidant reserve (SOD), and reduced apoptotic mediators (proapoptotic proteins Fas and p38 MAPK) and intracellular Ca2+ overload. Conclusion: Choline improves cardiac function and reduces ischemic myocardial injuries via stimulating the cardiac M3-mAChRs which in turn result in alterations of multiple signaling pathways leading to cytoprotection. The findings suggest M3-mAChR as a new target for drug development for improving cardiac function and preventing cardiac injuries during ischemia/reperfusion.

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.

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.

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.