Na+–Ca2+ exchanger targeting miR-132 prevents apoptosis of cardiomyocytes under hypoxic condition by suppressing Ca2+ overload

https://doi.org/10.1016/j.bbrc.2015.03.129Get rights and content

Highlights

  • miR-132 targets NCX1.

  • miR-132 prevents Ca2+ overload under hypoxic condition.

  • miR-132 attenuates hypoxia-induced cardiomyocyte apoptosis.

Abstract

During ischemia-reperfusion (IR) injury of the heart, Ca2+ overload occurs, leading to cardiomyocyte dysfunction and eventual cell death by apoptosis. Since preventing Ca2+ overload during IR injury has been reported to protect cardiomyocytes, interrupting Ca2+ signaling cascades leading to Ca2+ overload may exert protective effect on cardiomyocytes under hypoxic condition. One of the key regulators of the intracellular Ca2+ level during IR injury is Na+–Ca2+ exchanger 1 (NCX1), whose down-regulation during IR injury conferred protection of heart. In the present study, we examined whether down-regulation of NCX1 using exogenous microRNA ameliorates apoptosis of cardiomyocytes under hypoxic condition. Here, we identified miR-132 as a novel microRNA targeting the NCX1, whose expression increased during hypoxia. Delivery of miR-132 suppressed the increase of intracellular Ca2+ in cardiomyocytes under hypoxia, and the expressions of apoptotic molecules, such as Bax, cytochrome C, and caspase 3, and the number of apoptotic cells were also decreased by exogenous miR-132 treatment. These results suggest the potential of miR-132 as an effective therapeutic agent against IR damage to heart by preventing Ca2+ overload during hypoxic condition and warrant further studies to validate its anti-apoptotic effect in vivo.

Introduction

Severe damage to heart, such as ischemia-reperfusion (IR) injury and myocardial infarction, has been associated with apoptosis of cardiomyocyte [1], [2], [3]. Myocardial ischemia, which premises absence of oxygen, changes cell metabolism to anaerobic respiration, and this decreases intracellular pH by producing lactate [4]. To cope with the accumulation of intracellular H+, Na+–H+ exchanger (NHE) is activated to extrude H+, resulting in intracellular Na+ overload. In turn, to compensate the intracellular Na+ overload, Na+–Ca2+ exchanger (NCX) goes into reverse mode to extrude excessive Na+, eventually leading to intracellular Ca2+ overload [5]. Furthermore, a burst of oxidative stress is produced during reperfusion [6], and this stimulates Ca2+ release channel of the sarcoplasmic reticulum further exacerbating Ca2+ overload [7], which can lead to cytotoxicity and trigger eventual cell death [8]. The sustained rise of Ca2+ has been associated with irreversible cell injury, and interventions that reduced the rise in Ca2+ during IR injury attenuated cell death [9]. Thus, it may be possible to prevent IR-induced cardiomyocyte death by interrupting IR-induced signaling cascade leading to Ca2+ overload, and one of the candidate molecules whose suppression may prevent IR-induced Ca2+ overload is NCX.

NCX plays a crucial role in maintaining Ca2+ homeostasis in the heart under physiologic condition, but in its reverse mode during reperfusion, it facilitates and exacerbates Ca2+ overload [10], [11]. Furthermore, different from NHE whose inhibition failed to prevent Ca2+ overload [12], cardiac specific ablation of NCX protected heart from IR injury [13], suggesting down-regulation of NCX may confer protection against IR injury to cardiomyocytes. In fact, there are previous studies demonstrated that inhibition of NCX using chemical compounds protected cardiomyocytes from IR injury [14], [15], [16]. In addition, recent studies have indicated that microRNAs can be used instead of chemical compounds for regulating expressions of NCX [17], [18]. MicroRNAs (miRNAs or miR) are a class of endogenously expressed noncoding RNAs that control the stability and translation of protein-coding mRNAs [19]. A number of studies have reported that miRNAs as effective regulators in IR injured heart [20], [21], suggesting miRNAs as potent therapeutic targets and tools for preventing IR damage to heart. Currently, there is a no single most effective way to treat/manage IR-induced heart damage. Thus, until an optimized therapeutic strategy is established, the efforts to find alternative means (i.e., different miRNAs) can be justified. In the present study, we examined the feasibility of ameliorating apoptosis of cardiomyocytes using exogenous miRNA targeting NCX1. We first screened miRNAs for targeting NCX1 on miRNA target prediction program, and the effect of selected miRNA on hypoxia-induced cardiomyocyte apoptosis was further examined.

Section snippets

Isolation of rat ventricular cardiomyocytes

All experimental procedures for animal studies were approved by the Committee for the Care and Use of Laboratory Animals, Yonsei University College of Medicine, and performed in accordance with the Committee's Guidelines and Regulations for Animal Care. Neonatal rat cardiomyocytes from 1 to 2 day-old Sprague Dawley rat pups were isolated. Detailed methods are presented in the Supplementary Materials.

Hypoxia and cell viability assay

Cardiomyocytes with serum free α-MEM were incubated in hypoxic chamber maintained 1% O2, 5% CO2,

Hypoxia-induced death of cardiomyocytes is associated with Ca2+ overload and increased NCX1 expression

To examine the effect of hypoxia on cardiomyocyte viability, cardiomyocytes were exposed to hypoxic condition for up to 12 h and cell viability was measured. The number of viable cells decreased as time increased (Fig. 1A), while hypoxia increased the intracellular Ca2+ level in cardiomyocytes in a time-dependent manner, indicating hypoxia-induced Ca2+ overload (Fig. 1B). To further examine whether the increase of intracellular Ca2+ was associated with Ca2+ handling proteins, we examined the

Discussion

Ca2+ has been recognized as an important regulator of cardiomyocytes function in physiologic, as well as pathologic, conditions [33], [34]. Accumulating evidence indicates that miRNAs are involved in the Ca2+-mediated signaling in cardiomyocytes [24]. In the present study, we demonstrate that miR-132 is a novel NCX1-targeting miRNA that has potential as a therapeutic tool for preventing Ca2+ overload-mediated apoptosis of cardiomyocytes. The expression of NCX1 has been known to increase both at

Conflict of interest

None.

Acknowledgments

This study was supported by a Korea Science and Engineering Foundation grant funded by the Korean government (MEST) (2014030459) and a grant from the Korea Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea (A120478).

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