Original article
Redistribution of intracellular calcium and its effect on apoptosis in macrophages: Induction by oxidized LDL

https://doi.org/10.1016/j.biopha.2008.04.008Get rights and content

Abstract

Calcium signaling, as a key to early step of the elementary intracellular events, has been implicated in controlling the development of atherosclerosis. We have shown previously that oxidized low density lipoprotein OxLDL-induced spatiotemporal increases of intracellular free calcium ([Ca2+]i) in the early formation of macrophage foam cells. Here, we evaluated how spatiotemporal redistribution of intracellular calcium occurs and would affect OxLDL-induced apoptosis. Confocal laser scanning microscopy and flow cytometry showed the time-dependent increase of mitochondrial Ca2+ ([Ca2+]m) in acute and chronic exposure of U937-derived macrophages to OxLDL (100 μg/ml). Independent of the presence or absence of external Ca2+, OxLDL-induced a peak of [Ca2+]m in acute exposure, whose amplitude in the absence of extracellular Ca2+ was obviously lower than the presence of extracellular Ca2+. In addition, the thapsigargin-mediated increase of [Ca2+]i, through endoplasmic reticulum (ER) Ca2+ pump depletion, was obviously reduced by 1-h pretreatment of OxLDL. OxLDL also caused a time-dependent opening of mitochondrial permeability transition pores (PTPs). EGTA/AM, an intracellular Ca2+ chelator, significantly reduced OxLDL-induced apoptosis and failed to prevent OxLDL-induced necrosis at 6 h. In contrast to control cells, chelation of cytosolic Ca2+ by EGTA/AM at 6 h did not completely reverse OxLDL-induced apoptosis. OxLDL stimulated depolarization of mitochondrial membrane potentialψ) in time-dependent manner. Our data demonstrated that OxLDL-induced spatiotemporal Ca2+ redistribution in appropriate organelles and mediated Ca2+-dependent apoptosis in relation to depolarization of Δψ. These findings suggested that manipulation of the intracellular calcium balance may be a useful strategy to limit the loss of macrophages in early atherosclerosis.

Introduction

The accumulation of cholesterol-loaded macrophages in lesions is an important measure of atherosclerotic burden. Macrophage death has been regarded as an important player in the development of early atherosclerotic lesions, and oxidized low density lipoprotein (OxLDL) is implicated as a major risk factor for the progression of this disease [1], [2], [3]. Therefore, the relationship between OxLDL and macrophage apoptosis has received widespread attention, and there are multifactor correlations with macrophage apoptosis in atherosclerotic lesions. It has been found that a perturbation of intracellular Ca2+ homeostasis triggers cell apoptosis [4], [5], [6], and we recently showed that OxLDL caused a dynamic imbalance of [Ca2+]i during macrophage foam cells formation [7]. However, little is known about the precise mechanism of how intracellular Ca2+ stores are involved in the OxLDL-toxicity effect.

As an ubiquitous intracellular messenger, Ca2+ creates a wide range of spatial and temporal signals via amplitude and spatiotemporal fashion [8], [9]. In mammalian cells, cytoplasmic Ca2+ mainly originates from two sources: (1) Ca2+ ingress through voltage- and ligand-gated Ca2+ channels in the plasma membrane and (2) Ca2+ release from internal stores, mainly the endoplasmic reticulum (ER) [8]. It has been reported that apoptosis is triggered by the enrichment of free cholesterol (FC) in the endoplasmic reticulum (ER), resulting in depletion of ER calcium stores [10]. Recent studies also indicated that depletion of ER Ca2+ stores plays an important role in apoptosis [11], [12]. Mitochondrial Ca2+ uptake could modulate the amplitude and spatiotemporal organization of [Ca2+]i. Recent studies suggested that [Ca2+]m might function as a metabolic mediator to control the cellular metabolic rate including the Krebs cycle, pyruvate dehydrogenase and a-ketoglutarate dehydrogenase [13], [14], [15]. Moreover, Ca2+ is the single most important factor for opening of the mitochondrial permeability transition pore (PTP), and addition of Ca2+ alone is sufficient to induce a mitochondrial permeability transition [16]. The mitochondrial perturbations are often associated with apoptosis. As a consequence of both the dysfunction of the electrochemical gradient caused by pore opening and rupture of the outer mitochondrial membrane, the mitochondrial membrane potential (ΔΨm) generally collapses [17], [18], [19], [20]. But, there is limited information on the effect of OxLDL on spatiotemporal redistribution of intracellular calcium, and the potential roles of OxLDL-mediated calcium flows in mitochondrial dysfunction and apoptosis are unknown.

Our aim with this study was to shed light on the effect of OxLDL on dynamics of mitochondrial and ER calcium, mitochondrial dysfunction and apoptosis in macrophages. Based on our observations, this study suggests that OxLDL could significantly induce mitochondrial Ca2+ transient, release of ER Ca2+, opening of mitochondrial PTP, depolarization of mitochondrial membrane potential in early macrophage foam cells. These results indicated that regulating Ca2+ imbalance and mitochondrial dysfunction might lessen the early atherosclerosis damage.

Section snippets

Reagents

Culture media and reagents were purchased from Invitrogen Co. (N.Y., USA). Phorbol 12-myristate 13-acetate (PMA), thapsigargin (Tg) and EGTA/AM were purchased from Sigma Chemical Co. (St. Louis, USA). Fluo-3/AM, JC-1 and calcein/AM were obtained from Molecular Probes (USA), and annexin V-FITC apoptosis detection kit (contained with FITC-conjugated annexin V, PI and 4× binding buffer) was purchased from CALTAG Laboratories (Netherlands). All other chemicals were of the highest grade of purity

Mitochondrial calcium transients caused by OxLDL

Rhod-2/AM, a derivative of rhodamine 123, contains one net positive charge and accumulates into mitochondria, where mitochondrial esterases cleave the acetoxymethyl (AM) ester to liberate Rhod-2 free acid [20], [22]. We have previously shown that increases in OxLDL-induced [Ca2+]i were different in the presence or absence of extracellular Ca2+ (Fig. 1A) [7]. Therefore, the study was designed to confirm how OxLDL would alter [Ca2+]m in response to calcium ions entering the cytoplasm from

Discussion and conclusion

Previous studies demonstrated that OxLDL elicits a high and sustained rise in cytosolic calcium, which can then activate calcium-dependent enzymes involved in the cellular events leading to necrosis or apoptosis [26], [28]. The redistribution of intracellular calcium and mitochondrial dysfunction may be a pivotal event in the process. In the present study, acute exposure to OxLDL (100 μg/ml) resulted in the marked increase of mitochondrial Ca2+ with time. Our results also demonstrated OxLDL

Acknowledgements

This work was supported by Nature Science Foundation of Zhejiang Province (NO. Y206643) and the Key Laboratory for Biomedical Engineering of Ministry of China, the Economic and Trade commission of Zhejiang Province, and the Key Laboratory of Chinese Medicine Screening, Exploitation and Medicinal Effectiveness Appraise for Cardio-cerebral Vascular and Nervous System of Zhejiang Province.

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