Original article
β-Blocker carvedilol protects cardiomyocytes against oxidative stress-induced apoptosis by up-regulating miR-133 expression

https://doi.org/10.1016/j.yjmcc.2014.07.009Get rights and content

Highlights

  • Carvedilol reduced oxidative stress-induced cardiomyocyte apoptosis.

  • Carvedilol up-regulated miR-133 expression in the presence of oxidative stress.

  • miR-133 antisense inhibitor abolished the cytoprotective action of carvedilol.

  • Forced expression of miR-133 mimicked the anti-apoptotic effects of carvedilol.

  • Repression of caspase-9 and -3 may be a mechanism for the action of carvedilol.

Abstract

Oxidative stress is a causal factor and key promoter of a variety of cardiovascular diseases associated with apoptotic cell death by causing deregulation of related genes. Though carvedilol, a β-adrenergic blocker, has been shown to produce cytoprotective effects against cardiomyocyte apoptosis, the mechanisms are not fully understood. The present study was designed to investigate whether the beneficial effects of carvedilol are related to microRNAs which have emerged as critical players in cardiovascular pathophysiology via post-transcriptional regulation of protein-coding genes. In vivo, we demonstrated that carvedilol ameliorated impaired cardiac function of infarct rats and restored miR-133 expression. In vitro, carvedilol protected cardiomyocytes from H2O2 induced apoptosis detected by TUNEL staining and MTT assays, and increased miR-133 expression in cardiomyocytes. Overexpression of miR-133, a recognized anti-apoptotic miRNA, produced similar effects to carvedilol: reduction of reactive oxygen species (ROS) and malondialdehyde (MDA) content and increment of superoxide dismutase (SOD) activity and glutathione peroxidase (GPx) level, so as to protect cardiomyocytes from apoptosis by downregulating caspase-9 and caspase-3 expression in the presence of H2O2. Transfection with AMO-133 (antisense inhibitor oligodeoxyribonucleotides) alone abolished the beneficial effects of carvedilol. Caspase-9-specific inhibitor z-LEHD-fmk, caspase-3-specific inhibitor z-DEVD-fmk, caspase-9 siRNA and caspase-3 siRNA were used to establish caspase-3 as a downstream target of miR-133. In conclusion, our data indicated that carvedilol protected cardiomyocytes by increasing miR-133 expression and suppressing caspase-9 and subsequent apoptotic pathways.

Introduction

Cardiovascular disease is the main cause of death and birth defects all over the world [1]. Oxidative stress is one of the common responses to cardiac injuries in a variety of cardiovascular diseases [2]. Over-production of oxidative stress causes damage to a variety of cellular macromolecules, including lipids, DNA and proteins [3]. Increased ROS level contributes critically to the advance of atherosclerosis, hypertension, heart failure and acute myocardial infarction (MI) [4]. Oxidative stress is a major inducer of cardiomyocyte apoptosis [5]. However, the mechanisms underlying oxidative stress-induced apoptosis in cardiomyocytes are poorly understood.

It has been well recognized that microRNAs (miRNAs, miRs) play a central role in regulating some key protein-coding genes related to cardiovascular disease [6], [7], [8], [9], [10]. Among the known miRNAs, miR-133 is found to be specifically expressed in adult cardiac and skeletal muscles [11], [12]. Recent functional studies indicate that miR-133 produces an anti-apoptotic effect, protecting cardiomyocytes against apoptotic cell death under stress [13].

β-Adrenergic blockers have been widely used for the treatment of cardiovascular diseases, such as hypertension, hyperlipidemia, and coronary heart disease, especially for preventing sudden cardiac death in patients suffering acute or chronic MI [14], [15], [16]. Carvedilol, a nonselective β-adrenoceptor antagonist, is a multiple-action drug that has potent antioxidant properties [17]. Several experimental models of ischemia–reperfusion (IR) have already underlined the cardioprotective effects of carvedilol [18]. Although studies have shown that carvedilol also inhibits cardiomyocyte apoptosis [19], the exact mechanism is still unknown. In this study, we investigated whether carvedilol protects cardiomyocyte from apoptosis by influencing miRNA under pathological conditions relevant to human cardiac disease.

Section snippets

Ethics statement

This study was approved by the Ethic Committees of the Harbin Medical University. Experimental procedures were approved by the Animal Care and Use Committee of Harbin Medical University, and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Rat model of myocardial infarction (MI) and drug administration

Healthy male Wistar rats (200–250 g) were used and kept under standard animal room conditions at room temperature (23 ± 1 °C), with constant humidity of 55

Carvedilol ameliorates impaired cardiac function of infarct rats

To explore the potential role of carvedilol in MI, we evaluated cardiac function by echocardiography as shown in Fig. 1A. The ejection fraction (EF%) and fractional shortening (FS%) were significantly declined to 38.4 ± 5.3% and 19.4 ± 3.0%, respectively, in the MI group, while they were increased to 60.7 ± 3.7% and 33.8 ± 2.8%, respectively, in the MI + Car group (Figs. 1B and C). The results strongly indicated that carvedilol plays a protective role in MI rats.

MiR-24, -133, -145, -210, -494, and -499

Discussion

In this study, we made an effort to understand whether miRNA is involved in the long recognized cytoprotective effects of carvedilol, a β-blocker. The main findings of this study include: (1) carvedilol ameliorated the impaired cardiac function of infarct rats and up-regulated miR-133 expression in vivo; (2) carvedilol markedly up-regulated miR-133 expression in the presence of oxidative stress and reduced oxidative stress-induced cardiomyocyte apoptosis; and (3) knockdown of miR-133 by its

Conclusions

As a whole, our study revealed that carvedilol produced cardioprotective effects by preventing oxidative stress-induced apoptosis of cardiomyocytes. The beneficial effects were at least partially mediated by up-regulating miR-133, resulting in caspase-9/caspase-3 downregulation.

Disclosures

All authors declare that they have no competing interests for this study.

Acknowledgments

We thank Prof Baofeng Yang for his constructive suggestions to improve the quality of the paper. This work was supported in part by the Funds for Creative Research Groups [81121003] and the Major Program [81230081] of National Natural Science Foundation of China, the National Nature Science Foundation of China [grant numbers 81001434, 81270042, 30901208], and the Funds for Harbin Science and Technology Foundation [2009RFQXS006].

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