Calcium-sensing receptors regulate cardiomyocyte Ca2+ signaling via the sarcoplasmic reticulum-mitochondrion interface during hypoxia/reoxygenation

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.

abundance of mitochondria, many of which are in close apposition to SR Ca 2+ release sites [4].
The SR is a multifunctional organelle that controls protein translation and Ca 2+ homeostasis. Under SR stress (e.g., SR Ca 2+ depletion), SR chaperone proteins such as Grp78 and Grp94 are up-regulated [5]. Prolonged SR stress will initiate apoptotic signals in the SR, including bax/bak-translocation to the SR to activate the release of Ca 2+ from the SR, cleavage and activation of procaspase 12 and BAP31, and Ire 1-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) [6].
The calcium-sensing receptor (CaR) is a member of the family of G protein-coupled receptors (GPCRs). One of the effects of CaR signal transduction is the activation of phospholipase C, which leads to the generation of the secondary messengers diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP 3 ). IP 3 then mobilizes Ca 2+ from intracellular stores via the activation of specific IP 3 receptors [7]. Wang et al. and Tfelt-Hansen et al. reported that CaR was functionally expressed in rat cardiac tissue and rat neonatal ventricular cardiomyocytes, respectively [8,9]. Later, Berra-Romani et al. showed that cardiac microvascular endothelial cells express a functional CaR [10]. Our group has demonstrated that CaR is involved in apoptosis in isolated adult rat hearts and in rat neonatal cardiomyocytes during ischemia/reperfusion [11]. Although it is known that CaR elevates the intracellular calcium concentration and then induces apoptosis, the in-depth mechanisms are still not known. The aim of this study was to investigate whether [Ca 2+ ] SR would change with CaR activation in response to hypoxia/reoxygenation in cardiomyocytes. We specifically focused on the relationship between SR Ca 2+ depletion, mitochondrial Ca 2+ uptake and cardiomyocyte apoptosis during hypoxia/reoxygenation (H/Re).

Isolation of neonatal rat cardiomyocytes and H/Re experiments
Primary cultures of neonatal rat cardiomyocytes were performed as previously described [12]. Newborn Wistar rats (1-3 days) were used for this study. The rats were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the China National Institutes of Health. Briefly, hearts from male Wistar rats (1-3 days old) were minced and dissociated with 0.25% trypsin. Dispersed cells were seeded at 2 × 10 5 cells/cm 2 in 60-mm culture dishes with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and then cultured in a 5% CO 2 incubator at 37°C. Hypoxic conditions were produced using D-Hanks solution (mM: 5.37 KCl, 0.44 KH 2 PO 4 , 136.89 NaCl, 4.166 NaHCO 3 , 0.338 Na 2 HPO 4 , 5 D-glucose, pH 7.3-7.4 at 37°C) saturated with 95% N 2 and 5% CO 2 . The pH was adjusted to 6.8 with lactate to mimic ischemic conditions. The dishes were put into a hypoxic incubator that was equilibrated with 1% O 2 /5%CO 2 / 94%N 2 . After hypoxic treatment, the culture medium was rapidly replaced with fresh DMEM with 10% FBS (10% FBS/DMEM) to initiate reoxygenation [13].

3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyltetrazolium bromide(MTT) assay
In the current study, cardiomyocytes were planted in 96well plates. The MTT assay was performed as described previously [10]. Briefly, MTT (Sigma) was added into the cell cultures at a final concentration of 0.5 mg/mL and the mixture was incubated for 4 h at 37°C. Subsequently, the culture medium was removed and DMSO was added to each well to dissolve the resulting formazan crystals. The absorbance was measured at a wavelength of 570 nm using a microplate reader (Bio-Tek Instruments Inc., Richmond, Va). Background absorbance of medium in the absence of cells was subtracted [14]. Percent viability was defined as the relative absorbance of treated versus untreated control cells.

Hoechst staining
Apoptotic cells were identified by the distinctive condensed or fragmented nuclear structure in cells stained with the chromatin dye Hoechst 33342 (Sigma). Cells were fixed with 4% paraformaldehyde for 10 min at room temperature and were washed twice with phosphate buffer solution (PBS). Cells were then incubated with 5 μg/ mL Hoechst 33342 for 15 min. Next, the cells were washed three times and photographed using fluorescence microscope (Leica DFC500 System; Leica Microsystems, Bannockburn, Ill). At least 500 nuclei from randomly selected fields in each group were analyzed for each experiment, and the percentage of apoptotic cells was calculated as the ratio of the number of apoptotic cells versus the total cells counted.

Neonatal rat cardiomyocytes loaded with Fluo-4 AM, Fluo-5N AM and X-rhod-1 AM and cell permeabilization
[Ca 2+ ]i was determined as previously described [15]. Briefly, cells were seeded on the culture slides. After experimentation, cells were loaded with fluo-4 AM in 1% working solution at 37°C for 1 h, washed three times with Ca 2+ -free PBS to remove extracellular fluo-4 AM, and diluted to the required concentration. The reagents were added in Ca 2+ -free solution (145 mM NaCl, 5 mM KCl, 1.0 mM EGTA, 1 mM MgCl 2 , 10 mM HEPES-Na, 5.6 mM glucose, pH 7.4). Fluorescence measurement of Ca 2+ was performed using a laser confocal scanning microscope To determine [Ca 2+ ] SR , cardiomyocytes were treated with Fluo-5N acetoxymethylester (10 μM) for 2 h and deesterified for 1.5 h. For intact myocytes, the superfusate contained (in mM) 140 NaCl, 4 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 HEPES, and 10 glucose (pH 7.4, 23°C). For permeabilization, myocytes were exposed to solution (in mM: 0.1 EGTA, 10 HEPES, 120 K-aspartate, 1 free MgCl 2 , 5 ATP, 10 reduced glutathione, and 5 phosphocreatine; pH 7.4) and then permeabilized using saponin (50 μg/ml) for 20 seconds. Excitation was set at 488 nm and emission was measured at 530 nm at room temperature [15]. Images of fluorescence reflecting [Ca 2+ ] i and [Ca 2+ ] SR were recorded using a laser confocal scanning microscope (Olympus, LSM, Japan). There were more than 10 cells to be analyzed in each view and quantified using the analysis software for the microscope.
Recent study showed that the mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) consistently increases during reoxygenation [12]. Therefore, [Ca 2+ ] m was measured at 60 min post-reoxygenation. [Ca 2+ ] m was determined according to the manufacturer's instructions (Molecular Probes). In brief, the cultured cardiomyocytes (1 × 10 6 cells/sample) were initially washed with HEPES buffer containing (in mM) 130 NaCl, 4.7 KCl, 1.2 MgSO 4 , 1.2 KH 2 PO 4 , 10 HEPES, 11 glucose, and 0.2 CaCl 2 at pH 7.4 and then stained with 5 μmol/L X-rhod-1 AM for 30 min at room temperature. To avoid deesterification of intracellular Xrhod-1 AM in the cytosolic compartment, which would interfere with the detection of [Ca 2+ ] m , the cardiomyocytes were rinsed and incubated with 100 μM MnCl 2 -HEPES for an additional 20 min to quench the cytosolic Ca 2+ signal [16]. Fluorescence measurement was determined using a fluorescence plate reader (CytoFluor II; PerSeptive Biosystems; Framingham, MA) at an excitation wavelength of 580 nm and an emission wavelength of 645 nm for [Ca 2+ ] m . To validate the measurement of [Ca 2+ ] m , the cultured cardiomyocytes were transferred into a slide chamber after X-rhod-1 AM staining and were placed on the stage of a fluorescence microscope (×50 objective; Olympus). The images from the slides were captured using a digital camera connected to Image-Pro Plus software (Media Cybernetics; Silver Spring, MD). There were more than 10 cells to be analyzed in each view.

Measurement of mitochondrial membrane potential
Mitochondrial membrane potential (nψ m ) was measured with a unique cationic dye of 5,5',6,6'-tetrachloro 1,1'3,3'tetraethylbenzimidazolcarbocyanine iodide (JC-1), as previously described [12]. Briefly, cells were seeded on culture slides and treated according to experimental protocols. Previous data demonstrated that [Ca 2+ ] m might continuously increase during the process of reoxygenation and result in mitochondrial nψ m collapse [12], so we detected nψ m at 1 h after reoxygenation. At the end of the above-described treatments, cells were stained with JC-1 (1 μg/ml) at 37°C for 15 min and then rinsed three times with PBS. Observations were immediately made using a laser confocal scanning microscope. In live cells, the mitochondria appear red due to the aggregation of accumulated JC-1, which has absorption/emission maxima of 585/590 nm (red). In apoptotic and dead cells, the dye remains in its monomeric form, which has absorption/emission maxima of 510/530 nm (green). More than 100 areas were selected from each image. The average intensity of red and green fluorescence was determined. The ratio of JC-1 aggregate (red) to monomer (green) intensity was calculated. A decrease in this ratio was interpreted as a decrease in the nψ m , whereas an increase in this ratio was interpreted as a gain in the nψ m .

Identification of bax/bak translocation to the mitochondria and assay for cytochrome c release from mitochondria
Western blotting of cellular fractions was used to quantify changes in cytochrome c, bax and bak distribution within cells, as previously described [17]. Briefly, 1 × 10 7 rat cardiomyocytes were homogenized in ice-cold Trissucrose buffer (in mM: 350 sucrose, 10 Tris-HCl, 1 ethylenediaminetetraacetic acid, 0.5 dithiothreitol, and 0.1 phenylmethanesulfonylfluoride; pH 7.5). After 10 min of incubation, cardiomyocyte homogenates were initially centrifuged at 1000 × g for 5 min at 4°C, and the supernatant was further centrifuged at 40,000 × g for another 30 min at 4°C. The supernatant was saved as the cytosolic fraction. The precipitate was re-suspended in the above buffer (containing 0.5% v/v Nonidet P-40) and saved as the mitochondrial fraction. The mitochondrial fractions were blotted with a primary rat anti-bax, bak and cytochrome c monoclonal antibody (Santa Cruz Inc.). The volume of specific bands was measured using a Bio-Rad Chemi EQ densitometer and Bio-Rad QuantityOne software (Bio-Rad laboratories, Hercules, USA).

Western blotting
Western blot analyses were performed as previously described [18]. In brief, the protein concentration of samples was first determined using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). A total of 20 μg of protein was electrophoresed on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Amersham International, Amersham, UK). The membranes were blocked with 10% skim milk in TBST buffer (10 mM Tris, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature and then incubated with a rabbit anti-BAP31 polyclonal antibody (1:500 dilution, sc-48766, Santa Cruz Biotechnology) overnight at 4°C. HRP-conjugated anti-rabbit IgG (1:3000 dilution, Bio-Rad Laboratories) was used as a secondary antibody. Specific bands were visualized with a chemiluminescent substrate (ECL kit, Amersham International).

Statistical analyses
Significance was evaluated using student's t-test, and p < 0.05 was considered statistically significant. Data are expressed as mean ± standard error of the mean (S.E.M.) and are representative of at least three independent experiments. [Ca 2+ ] i data were obtained from 2-3 experiments, and 10-12 images were analyzed in each group.

Asymmetric subcellular distribution of IP 3 R subtypes in cardiomyocytes
Western blot results showed that type 2 and 3 IP 3 Rs were expressed in cardiomyocytes, while type 1 IP 3 R expression was undetectable (Fig. 1A). Similar to the results of the Western blot analysis, type 3 IP 3 R was distributed in the cytoplasm and intense perinuclear and intranuclear staining was evident for type 2 IP 3 R in immunofluorescence study, while type 1 IP 3 R was undetectable.   (Fig. 2).
To further determine whether the cell death induced by H/Re and activation of CaR was mediated by apoptosis, the nuclear morphology was analyzed using the Hoechst staining assay. The apoptotic cells exhibited typical fragmented nuclei and condensed chromatin on staining with Hoechst 33342 (Fig. 3). The percentage of apoptotic cells relative to the total number of cells was increased to H/Re (33 ± 6%), Ca + Ni + Cd-H/Re (31 ± 5%) and Gd + Ni + Cd-H/Re (34 ± 3%) compared with the NPS-2390 + Ca + Ni + Cd-H/Re (20 ± 4%), 2-APB + Ca + Ni + Cd-H/Re (18 ± 4%) and Ru + Ca + Ni + Cd-H/Re (23 ± 5%) groups. Therefore, these data show that the activation of CaR is involved in H/Re -induced cardiomyocyte apoptosis.

Discussion
This study was designed to address the potential involvement of the sarcoplasmic reticulum and mitochondria in The membrane receptor CaR couples to the enzyme PLC, which liberates IP 3 from phosphatidylinositol 4,5bisphosphate (PIP 2 ). The major function of IP 3 is to induce endogenous Ca 2+ release through IP 3 Rs [23]. Ca 2+ is the primary agonist of CaRs. The EC50 for Ca 2+ activation of the CaR is 3-4 mM [24]. CaCl 2 was chosen as an agonist to activate CaR, and was shown to increase the expression of CaR (Additional file 1). NPS-2390 was chosen as an antagonist of CaR. In previous study, NPS-2390 is an allosteric antagonist of the group 1 metabotropic   [25]. It has been reported that IP 3 Rs play an important role in establishing macromolecular complexes on the surface of the SR membranes and in modulating the linkage between the SR and mitochon-drial membranes. Mitochondria respond rapidly to physiological increases in [Ca 2+ ]e, and stimulation with Gqcoupled receptor agonists, which induce IP 3 production and the subsequent release of Ca 2+ from ER, causes a rapid rise in [Ca 2+ ] m [26]. This effect has been detected in many cells types: HeLa cells, fibroblasts, endothelial and epithelial cells, cardiac and skeletal muscle cells, neurons and pancreatic β cells [27,28]. CaR, as a Gq-coupled receptor, could be involved in promoting Ca 2+ release from ER and then in induced the [Ca 2+ ] m rise. Our results suggest that [Ca 2+ ] m was elevated and mitochondrial membrane potential collapsed in the Ca + Ni + Cd-H/Re group, whereas [Ca 2+ ]m and mitochondrial membrane potentials were maintained in the 2-APB + Ca + Ni + Cd-H/Re group. The rapid mitochondrial Ca 2+ uptake is related to the low affinity of the Ca 2+ transport system. Therefore, Ruthenium red, an inhibitor of the mitochondrial calcium transporter, was used in our experiment. The results reveal that [Ca 2+ ] m and mitochondrial potentials were maintained in the Ru + Ca + Ni + Cd-H/Re group. These results suggest that both the SR and the mitochondria orchestrate the regulation of Ca 2+ signaling between these two organelles. Although a role for the SR in the mitochondrial redistribution of Ca 2+ has been implicated in many models of apoptosis, a primary role for IP 3 generation and the activation of IP 3 Rs in this process has been examined in only a few instances. Caspase-8 cleavage of BAP31 at the SR leads to the generation of a p20 fragment, which directs pro-apoptotic signals between the SR and mitochondria, resulting in early discharge of Ca 2+ from the SR and its concomitant uptake into the mitochondria. Early and critical events in apoptosis occur in mitochondria and in the ER, and the release of elements acting as caspase cofactors, such as cytochrome c (from mitochondria) and Ca 2+ (from the ER), into the cytosol are requisites for cell death in many cases [29]. The mitochondrial pathway of apoptosis is regulated by members of the Bcl-2 protein family, subdivided into two groups: anti-apoptotic (Bcl-2) and pro-apoptotic (Bax, Bak). The link between Bcl-2 (localized in several intracellular membranes including those of mitochondria and the ER) and Ca 2+ homeostasis has been established by showing that Bcl-2 reduces the steady state Ca 2+ levels in the ER, thereby dampening the apoptotic signal [30,31]. Jiang et al. showed that CaR was involved in neonatal cardiomyocyte apoptosis in ischemia/reperfusion injury. They suggested that [Ca 2+ ]i was increased, inhibiting the expression of Bcl-2 and elevating the expression of the pro-apoptotic protein caspase-3 in cytoplasm [32]. However, the Ca 2+ -dependent model of apoptosis was subsequently supported by a series of observations with the pro-apoptotic Bcl-2 family members Bax and Bak. Cells deriving from knockout mice lacking Bax and Bak that are very resistant to apoptotic death have a dramatic reduction in the [Ca 2+ ] within the ER and a drastic reduction in the transfer of Ca 2+ from the ER to mitochondria [33].This change prompts mitochondrial fission and cytochrome c release into the cytosol. Green et al. demonstrated that [Ca 2+ ] SR depletion caused bax-and bak-mediated permeability of the outer mitochondrial membrane, thereby releasing pro-apoptotic factors and particularly cytochrome c [34]. Our present data show that CaR activation induced the cleavage of BAP31 with the formation of the pro-apoptotic p20 fragment, causing bax and bak translocation to the mitochondria and cytochrome c release from the mitochondria during H/Re.
In conclusion, our results constitute the first report that CaR plays an important role in the SR-mitochondrial inter-organelle Ca 2+ signaling through the IP 3 Rs, which are also involved in apoptosis during H/Re.