Original articleRedistribution of intracellular calcium and its effect on apoptosis in macrophages: Induction by oxidized LDL
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.
References (31)
- et al.
Calcium signalling and the regulation of apoptosis
Toxicol in Vitro
(1998) - et al.
Intracellular-free calcium dynamics and F-actin alteration in the formation of macrophage foam cells
Biochem Biophys Res Commun
(2005) Neuronal calcium signaling
Neuron
(1998)- et al.
Enrichment of endoplasmic reticulum with cholesterol inhibits sarcoplasmic–endoplasmic reticulum calcium atpase-2b activity in parallel with increased order of membrane lipids
J Biol Chem
(2004) - et al.
Mitochondrial calcium uptake from physiological-type pulses of calcium. A description of the rapid uptake mode
J Biol Chem
(1995) - et al.
The mitochondrial permeability transition
Biochim Biophys Acta
(1995) - et al.
Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria
Cell
(1997) - et al.
Stimulation of mitogen activated protein kinase by LDL and oxLDL in human U-937 macrophage-like cells
FEBS Lett
(1996) - et al.
Selective loading of rhod 2 into mitochondria shows mitochondrial Ca2+ transients during the contractile cycle in adult rabbit cardiac myocytes
Biochem Biophys Res Commun
(1997) - et al.
Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence
Biophys J
(1999)
Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals
Cell
Calcium signaling and apoptosis
Biochem Biophys Res Commun
Oxidized low-density lipoprotein-induced apoptosis
Biochim Biophys Acta
Atherosclerosis — an inflammatory disease
N Engl J Med
Consequences and therapeutic implications of macrophage apoptosis in atherosclerosis the importance of lesion stage and phagocytic efficiency
Arterioscler Thromb Vasc Biol
Cited by (10)
PACS2 is required for ox-LDL-induced endothelial cell apoptosis by regulating mitochondria-associated ER membrane formation and mitochondrial Ca <sup>2+</sup> elevation
2019, Experimental Cell ResearchCitation Excerpt :Recently, we found that ox-LDL induced Ca2+ overload prior to mitochondrial membrane potential (MMP) loss and endothelial progenitor cell apoptosis [6]. In ECs, ox-LDL reportedly triggers intracellular Ca2+ overload to induce calpain-mediated mitochondrial permeability transition pore (mPTP) opening and cell apoptosis [7]. Additionally, mitochondrial Ca2+ overload, a critical sensitizing signal of mitochondrial dysfunction, has been reported in EC apoptosis induced by high glucose [8], but as yet, its role and the underlying mechanisms in ox-LDL-induced EC apoptosis have not been fully determined.
ORP4L Facilitates Macrophage Survival via G-Protein-Coupled Signaling
2016, Circulation ResearchMitochondrial permeability transition pore plays a role in the cardioprotection of CB2 receptor against ischemia-reperfusion injury
2014, Canadian Journal of Physiology and Pharmacology