Elsevier

Gene

Volume 590, Issue 2, 30 September 2016, Pages 201-209
Gene

Research paper
Valsartan ameliorates KIR2.1 in rats with myocardial infarction via the NF-κB-miR-16 pathway

https://doi.org/10.1016/j.gene.2015.11.047Get rights and content

Highlights

  • We focused on miR-16, a cardiac muscle enriched miRNA received less attention.

  • We confirmed that miR-16 is a direct regulator of Kir2.1 for the first time.

  • Valsartan protects Kir2.1 protein possibly via the mechanism of NF-kB-mir16.

Abstract

Background

MicroRNAs have an important role in regulating arrhythmogenesis. MicroRNA-16 (miR-16) is predicted to target KCNJ2. The regulation of miR-16 is primarily due to NF-κB. Whether valsartan could downregulate miR-16 via the inhibition of NF-κB after MI and whether miR-16 targets KCNJ2 remain unclear.

Methods

MI rats received valsartan or saline for 7 days. The protein levels of NF-κB p65, inhibitor κBα (IκBα), and Kir2.1 were detected by Western blot analysis. The mRNA levels of Kir2.1 and miR-16 were examined by quantitative real-time PCR. Whole cell patch-clamp techniques were applied to record IK1.

Results

MiR-16 expression was higher in the infarct border, and was accompanied by a depressed IK1/KIR2.1 level. Additionally, miR-16 overexpression suppressed KCNJ2/KIR2.1 expression. In contrast, miR-16 inhibition or binding-site mutation enhanced KCNJ2/KIR2.1 expression, establishing KCNJ2 as a miR-16 target. In the MI rats, compared to saline treatment, valsartan reduced NF-κB p65 and miR-16 expression and increased IκBα and Kir2.1 expression. In vitro, angiotensin II increased miR-16 expression and valsartan inhibited it. Overexpressing miR-16 in cells treated with valsartan abrogated its beneficial effect on KCNJ2/Kir2.1. NF-κB activation directly upregulates miR-16 expression.

Conclusions

miR-16 controls KCNJ2 expression, and valsartan ameliorates Kir2.1 after MI partly depending on the NF-κB-miR-16 pathway.

Introduction

Ventricular arrhythmias following myocardial infarction (MI) remain a major causes of mortality (Jugdutt, 2006). Numerous studies have confirmed that a decreased inward-rectifier K + current (IK1), along with decreased expression of the principal underlying subunit KCNJ2 mRNA and its encoded Kir2.1 protein, is a prominent feature of ventricular electrical remodeling after MI (Li et al., 2009). IK1 is the key K + current contributing to the resting membrane potential, controlling cardiac excitability and modulating late-phase repolarization and action potential duration (APD) in cardiac cells. Furthermore, IK1 is a main factor for cardiac excitability and arrhythmogenesis and a promising target for new antiarrhythmic approaches (Noujaim et al., 2010).

The mechanism of IK1 dysregulation after MI is mostly through the intracellular signaling pathway. However, the gene regulation is poorly understood. Recent studies have discovered that microRNAs (miRNAs) have an important role in regulating cardiac excitability and arrhythmogenesis in various cardiac diseases, such as myocardial infarction (Yang et al., 2007), atrial fibrillation (Girmatsion et al., 2009, Lu et al., 2010), cardiac hypertrophy (Carè et al., 2007), diabetic cardiomyopathy (Feng et al., 2010), and other cardiac diseases (Matkovich et al., 2010, Terentyev et al., 2009, Zhao et al., 2007). Luo X performed a computational analysis of miRNAs to identify miRNA candidates for IK1 dysregulation and found that miR-16 has the potential to repress KCNJ2/Kir2.1/IK1 (Luo et al., 2010). Furthermore, miR-16 is cardiac-enriched (Liang et al., 2007), but not cardiac-specific (Ye and Steinle, 2015, Huang et al., 2015). MiR-16 sequences are highly conserved across species. The 3′ UTR of KCNJ2 mRNA contains 3 binding sites for miR-16 (Fig. 3A). Therefore, we hypothesized that miR-16 regulates KCNJ2/Kir2.1/IK1 expression.

The regulation of miR-16 is primarily due to NF-κB. NF-κB-binding sites are located in miR-16 gene transcriptional elements (Shin et al., 2011). NF-κB DNA-binding activity reached its peak value at day 7 in the infarct region and was suppressed by AT1 receptor blocker (ARB), candesartan (Yoshiyama et al., 2001). Previous studies have reported that ARBs have significant effects on kinds of ion channels (Ushio-Yamana et al., 2012, Imai et al., 2001, Han et al., 2013). Therefore, we hypothesized that valsartan, one of ARBs, could also regulate NF-κB DNA-binding activity, which have further effect on the downstream gene–miR-16 expression. In this study, we used a rat model of acute myocardial infarction (AMI) to test whether valsartan regulates miR-16 via the inhibition of NF-κB after MI and whether miR-16 targets KCNJ2 after MI.

Section snippets

Ethics statement

The use of animals and all procedures were performed in accordance with the regulations of the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH publication no. 85–23, revised 1996) and approved by the Animal Care and Use Committee of Shandong University.

Cell culture

The H9c2 (rat embryonic ventricle) cell line used in this study was purchased from ATCC (Zhongyuan Ltd., Beijing, China) and cultured in DMEM. CoCl2 (300 μM) (Sigma, St. Louis, MO,

Dysregulation of miR-16 and KCNJ2/Kir2.1 in the MI rats

In an effort to explore the roles of miRNAs in AMI, we found that miR-16 was significantly upregulated, lasting 1 week, in a rat AMI model (Fig. 2A), indicating possible involvement in AMI pathophysiology. Moreover, in line with previous studies (Li et al., 2009, Lu et al., 2009), we found Kir2.1 protein and KCNJ2 mRNA expression to be downregulated in MI rats (Fig. 2B and C). These results are consistent with the notion that miR-16 might contribute to KCNJ2 dysregulation in MI, a possibility

Discussion

Taken together, our results identify miR-16 as a potentially important regulator of KCNJ2 gene expression. In addition, our findings identify miR-16 as a potential mediator of the electrophysiological effects of NF-κB signaling pathway and provide a basis for the anti-electrical irritability function of valsartan. Therefore, our study reveals what we believe to be a novel molecular control mechanisms for ion channel remodeling after MI at the miRNA level (Fig. 6).

Cardiac IK1 current is a

Conclusions

In conclusion, we have discovered that miR-16 regulates the KCNJ2 gene and its encoded channel IK1. Moreover, valsartan regulates miR-16 through inhibiting the activation of NF-κB after MI to improve the heart's electrical remodeling. These findings provide what we believe are new insights into the molecular mechanisms underlying a common and important cardiac ion channel remodeling and may provide a basis for the anti-electrical irritability function of valsartan.

Funding

This work was supported by the National Natural Science Foundation of China [81070088] (in study design); the Specialized Research Fund for the Doctoral Program of Higher Education of China [20130131110069] (in the collection of data); the National Natural Science Foundation of Shandong Province [2009HW074] (in the analysis of data); the Science and Technology Foundation of Shandong Province [2009GG10002049] (in the writing of the manuscript); and the Shandong Taishan Scholarship (in the

Acknowledgments

We thank for professor Liu Jun. for helping us with the conduction of patch clamp and professor Liu Ju for helping us with the manuscript writing.

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