Role of the Calcium-Sensing Receptor in Cardiomyocyte Apoptosis via the Sarcoplasmic Reticulum and Mitochondrial Death Pathway in Cardiac Hypertrophy and Heart Failure

Heart failure is a major cause of cardiovascular death in China [1]. Previous work has indicated that dysfunction of the intracellular Ca²⁺ cycle is crucial for the pathogenesis of heart failure [2]. The endoplasmic/sarcoplasmic reticulum (ER/SR) is an intracellular Ca²⁺ storage organelle, which is sensitive to alterations in Ca²⁺ homeostasis and disturbances in its environment. The depletion of Ca²⁺ from the SR lumen can disrupt SR function, resulting in ER stress. Prolonged and severe ER stress results in organelle damage and dysfunction, ultimately leading to apoptosis [3,4]. It has been reported that ER stress is involved in heart diseases that contribute to heart failure, including hypertension, myocardial ischemia, and dilated cardiomyopathy [5]. The heart is the most mitochondria-rich organelle, as mitochondria occupy approximately 30% of the volume of a ventricular cardiomyocyte. Mitochondria are the organelles responsible for the production of ATP and are the gatekeepers of cellular viability. In heart failure, mitochondrial ATP production is decreased and its apoptotic pathway is activated [6,7]. Many studies have confirmed the important role of biochemical cross-talk between the sarcoplasmic reticulum and mitochondria in normal cardiomyocyte survival [8,9,10,11]. Although the SR and mitochondria have different functions, their coordination is necessary to support synchronous cardiomyocyte contraction/relaxation as well as cell viability. SR and mitochondrial dysfunction are associated with heart failure [12]. However, how the interplay between the SR and mitochondria contributes to heart failure is unclear.

The calcium-sensing receptor (CaR) is G protein-coupled receptor that stimulates the phosphatidyl inositol-specific phospholipase C (PLC), leading to the generation of the secondary messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃) [13,14]. The mobilization of Ca²⁺ from intracellular stores by IP₃ via the activation of a specific inositol 1,4,5-trisphosphate receptor (IP₃R) signaling pathway could release Ca²⁺ from the SR to increase the microdomain Ca²⁺ concentration ([Ca²⁺]) at focal contacts, known as mitochondria-associated ER membranes (MAM) between the ER/SR and mitochondria, and then activate the mitochondrial calcium uniporter (MCU). Recent studies have suggested that IP₃Rs are highly compartmentalized in MAMs, providing direct mitochondrial Ca²⁺ signaling [8].

In a previous study, we demonstrated that CaR is involved in apoptosis in isolated adult rat hearts and in rat neonatal cardiomyocytes during hypoxia/reoxygenation (H/Re) [14]. Although CaR is known to elevate the concentration of intracellular calcium, the in-depth mechanism has not been delineated. In the present study, we tested the hypothesis that the SR and mitochondrial apoptotic pathway plays a role in cardiomyocyte apoptosis in heart failure through CaR activation.

Isoproterenol (Iso), calindol, calhex231, sodium tauroursodeoxy cholate were purchased from Sigma-Aldrich (St. Louis, MO,USA). Antibodies for Calcium sensing receptor (CaR), type 3IP₃R, ATF6, GRP78, caspase-12, CHOP (GADD153), cytochrome c, VDAC, β-actin and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horse-radish peroxidase-conjugated secondary antibodies (goat anti-rabbit immunoglobulin G and rabbit anti-mouse immunoglobulin G) and enhanced chemiluminescence reagents were from Pierce Biotechnology (now part of Thermo Fisher Scientific, Rockford, IL). Polyvinylidene difluoride (PVDF) membranes were from Whatman (now part of GE Healthcare Life Sciences, Buckinghamshire, UK). TUNEL kit was from Roche (Darmstadt, Germany). Fluo-5N and X-rhod-1 AM were from Molecular Probe (Molecular Probe, USA).

Male Wistar rats (200-250 g) were obtained from the Experimental Animal Center of Harbin Medical University (Harbin, People's Republic of China). All animal experimental protocols complied with the ‘Guide for the Care and Use of Laboratory Animals' published by the United States National Institutes of Health. The study was approved by the Institutional Animal Research Committee of Harbin Medical University. All animals were housed at the animal care facility of Harbin Medical University at 25°C with 12/12-h light/dark cycles and allowed free access to normal rat chow and water throughout the study period. Rats were randomly assigned to different treatment groups.

β-adrenergic stimulation can be induced by subcutaneous injection of high dose Iso as previously reported [15]. Eighty rats were randomly divided into four groups. In group 1 (n= 20), the rats were subcutaneously injected with saline for 4 days as a control. In group 2 (Iso, n= 20), the rats were subcutaneously injected with Iso (170 mg/kg/d in saline) for 4 days to induce cardiac hypertrophy and heart failure. In group 3 (Iso-A, n= 20), the rats were subcutaneously injected with calindol (10 μmol/kg/d in saline) for 2, 4, 6, or 8 weeks after administration of Iso for 4 days. Calindol is a specific activator of CaR. In group 4 (Iso-I, n= 20), the rats were subcutaneously injected with calhex231 (10 μmol/kg/d in saline) for 2, 4, 6, or 8 weeks after administration of Iso for 4 days. Calhex231 is a specific inhibitor of CaR.

Pressure overload and cardiac hypertrophy were induced by thoracic aorta constriction (TAC) in male Kunming mice according to a previously described method [16]. Thirty-two rats were randomly assigned to four experimental groups. In group 1 (n =20), sham-operated animals served as controls. In group 2 (n= 20), cardiac hypertrophy was induced by TAC without further treatment. In group 3 (n =20), TAC was performed and the rats were treated with calindol (10 μmol/kg/d in saline) for 2 or 4 weeks after the operation. In group 4 (n= 20), TAC was performed, and the rats were treated with calhex231 (10 μmol/kg/d in saline) for 2 or 4 weeks after the operation.

Rats were anesthetized with 2% pentobarbital sodium (50 mg kg^-1, ip). Echocardiographic images were obtained with a Vivid 7 Dimension echocardiographic machine (GE Healthcare, Waukesha, WI, USA). Regional wall thickening, myocardial contractile function and wall motion were determined by a single experienced investigator in a blinded fashion. These images and parameters were recorded for subsequent review and analysis.

Formalin fixed hearts were embedded in paraffin, sectioned into 4 µm slices, and stained with hematoxylin and eosin (H&E). Cardiac myocyte crosssectional diameter was measured. The distance across the myocardial cell at its narrowest plane across the nucleus was measured in 75 cells from each LV. The mean diameter was calculated for the LV in each rat.

The TUNEL assay was used to detect the apoptosis of cardiomyocytes in rat hearts. The heart tissues (n=3 for sham group and n=5 for each HF group) were fixed in 4% paraformaldehyde overnight, dehydrated and then embedded in paraffin. The assay was tested by a TdT- DNA Fragmentation Detection kit acquired from Roche, and the empirical procedure was performed according to the kit's protocol.

Mitochondrial ultrastructure changes of cardiomyocytes in our present study was detected by EM (electron microscope) [14]. Mitochondria isolated from heart tissue was fixed with 2.5% glutaldehyde and 1.5% paraldehyde, washed in PBS, fixed in osmium tetroxide, dehydrated in an ethanol series, embedded in epoxy resin, and then examined with a Philips CM 120 electron microscope.

Primary cultures of neonatal rat cardiomyocytes were performed as previously described [17]. 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 × 10⁵ cells/cm² 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% CO₂ incubator at 37°C. At 72 h post-culturing with 10% FBS/DMEM, the cells were randomly divided into six groups: (1) control group: cells were continuously cultured for 48h with 10% FBS/DMEM; (2) Iso group: cells were placed in culture medium for 48 h with 5 μM Iso; (3) Iso+calindol(Iso-A): cells were placed in culture medium for 48 h with 5μM Iso and 10 μM calindol; (4) Iso+calhex231+calindol (Iso-I): cells were placed in culture medium for 48 h with 5 μM Iso and 10 μM calhex231 and 10 μM calindol; (5) Iso+2-APB+calindol (Iso-2-APB-A): cells were placed in culture medium for 48 h with 5 μM Iso and 20 μM 2-APB and 10 μM calindol. 2-APB is an inhibitor of IP₃Rs; (6): Iso+Rutheium red +calindol (Iso-Ru R-A): cells were placed in culture medium for 48 h with 5 μM Iso and 10 μM Rutheium red and 10 μM calindol. Rutheium red is a blocker of mitochondrial calcium unitransporter

The protein concentration of each sample was quantified using the enhanced BCA (bicinchoninic acid) Protein Assay kit (Beyotime, Nantong, China). Cell lysates and heart extracts were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis(SDS-PAGE) and blotted on PVDF membranes (Millipore). Membranes were incubated with antibodies directed against CaR, type 3 IP₃R, GRP78, ATF6, CHOP and caspase-12 protein. A secondary antibody was AP-IgG (Promega). The volume of the protein bands was quantified by a Bio-Rad Chemi Doc™ EQ densitormeter and a Bio-Rad Qantity One software (Bio-Rad laboratories, Hercules, USA).

Isolation of mitochondria protein from left ventricular tissue was performed according to the manufacturer's protocol (Beyotime, Nantong, China) [18]. Protein concentrations were determined using bicinchoninic acid Protein Assay kit (Beyotime, Nantong, China).

To determine [Ca²⁺]_SR, cardiomyocytes were loaded with Fluo-5N acetoxymethyl ester (10 μM) were performed as previously described [19]. For intact myocytes, the superfusate contained (in mM) 140 NaCl, 4 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES and 10 glucose (pH 7.4, 23˚C). For permeabilization, myocytes were exposed to a solution containing (in mM) 0.1 EGTA, 10 HEPES, 120 K-aspartate, 1 free MgCl₂, 5 ATP, 10 reduced glutathione, 5 phosphocreatine [pH 7.4] and then permeabilized with saponin (50 μg ⁄ ml) for 20 sec. Excitation was set at 488 nm and emission was measured at 530 nm at room temperature.

[Ca²⁺]_m was determined according to the manufacturer‘s instructions (Molecular Probes). In brief, the cultured cardiomyocytes (1 × 10⁶ cells/sample) were initially washed with HEPES buffer and then stained with 5 μmol/L X-rhod-1 AM for 30 min at room temperature. To avoid deesterification of intracellular Xr-hod-1 AM in the cytosolic compartment, which would interfere with the detection of [Ca²⁺]m, the cardiomyocytes were rinsed and incubated with 100 μM MnCl₂- HEPES for an additional 20 min to quench the cytosolic Ca²⁺ 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 [Ca²⁺]m. The images from the slides were captured using a digital camera connected to Image- Pro Plus software (Media Cybernetics; Silver Spring, MD).

Mitochondrial membrane potential (Δψm) was measured with a unique cationic dye of 5,5‘,6,6‘-tetra-chloro 1,1‘3,3‘-tetraethylbenzimidazolcarbocyanine iodide (JC-1), as previously described [18]. Briefly, cells were seeded on culture slides and treated according to experimental protocols. 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. The ratio of JC-1 aggregate (red) to monomer (green) intensity was calculated.

All values are expressed as mean ± S.E. unless noted otherwise. Differences between data groups were evaluated for significance using Student t-test of unpaired data or one-way analysis of variance (ANOVA). P<0.05 was accepted as statistically significant.

To investigate the in vivo effect of CaR on ER stress in failing hearts, we first established a cardiac hypertrophy model by injecting the rats with a high dose of Iso for 4 days. Morphological analyses showed that Iso induced myocyte hypertrophy, i.e., significantly increased the cardiomyocyte cross-sectional diameter in the HCM 2w and 4w and HCM 2w-A and 4w-A groups, respectively, and this parameter was significantly reduced in the hearts of rats treated with the CaR inhibitor calhex231 (Fig. 1a). Myocardial function was examined by echocardiography. Compared to the sham group, the interventricular septal diastolic thickness (IVSd) was increased in HCM 2w group. Increases in the left ventricular ejection fraction (LVEF) and the ratio between the early and atrial peak filling velocities (Ev/Av) showed that cardiac systolic and diastolic function was improved in the HCM 2w and HCM 2w-A groups. However, the LVEF and Ev/Av were decreased after Iso administration for 4 weeks compared to the sham group (Fig. 1b).

In contrast, 6 weeks after Iso administration, the LVEF and Ev/Av were decreased up to 60%, indicating the occurrence of heart failure. In the HF 2w, HF 4w, HF 2w-A and HF 4w-A groups, the LVEF and Ev/Av were significantly decreased compared to those in the HF-I 2w and 4w groups. These data suggest that CaR could be involved in cardiac hypertrophy and aggregated heart function in rats with heart failure (Fig. 1c).

We then created another animal model of heart failure by performing TAC and concurrently treated them with or without the CaR activator calindol or the CaR inhibitor cal-hex231 for 2 or 4 weeks. Similar results were observed from these TAC mice. TAC and TAC with calindol treatment for 2 weeks induced significant cardiac hypertrophy. The left ventricle function of the TAC 2w and TAC 2w-I and TAC 4w-I animals was slightly improved, as assessed by echocardiography, compared to the control and TAC 2w-A, TAC 4w, and TAC 4w-A groups (Fig. 1d).

We also noticed that the cardiomyocyte expression of CaR was significantly increased in the progression of heart failure (Fig. 2a). However, type 3 IP₃R expression was not changed significantly during heart failure induced by Iso (Fig. 2b).

To explore the in vivo effect of CaR on ER stress in failing hearts, we detected the expression of several molecular indicators of ER stress in hypertrophic and failing hearts in rats. ER stress responses were observed in the process of cardiac hypertrophy and heart failure, as assessed by the increased expression of GRP78 in the HCM, HCM-A, HCM 2w-I, HF and HF-A groups. The CaR inhibitor calhex231 significantly suppressed ER stress responses in HCM4w-I and HF4w-I groups (Fig. 3a,c). Similar results were also found in TAC mice. GRP78 expression in the TAC and TAC-A groups was increased at both 2 and 4 weeks (Fig. 3b).

The ATF6 is a transcription factor that is retained in the ER with GRP78. When unfolded proteins are accumulated, GRP78 is sequestered away, resulting in the translocation of ATF6 to the Golgi apparatus, where it is cleaved and activated. A 60-kDa form of ATF6 (p60ATF6) is released into the cytoplasm, and a 36-kDa form of this protein (p36ATF6) is translocated to the nucleus. Western blot analysis showed that p60ATF6 was significantly increased in the HF 4w and HF 2w-A animals compared with the HF 4w-A, HF 2w-I and HF 4w-I groups (Fig. 3d).

Caspase-12 and CHOP are characteristic markers of ER stress-induced apoptosis [20,21]. We examined the expression of caspase-12 and CHOP in hearts subjected to Iso and either calindol or calhex231. Our results showed that the expression of CHOP and cleavage of caspase-12 increased dramatically in the HF 4w and HF 4w-A groups. Inhibition of CaR abolished the expression of CHOP and cleavage of caspase-12, while activation of CaR increased their expression (Fig. 4a, b). In accordance with the above results, the level of apoptotic cardiomyocytes, as examined by TUNEL, markedly increased in the HF 2w, HF 4w, HF2w -A, and HF 4w-A groups compared to the HF 2w-I and HF 4w-I groups (Fig. 4e).

The cleavage of caspase-12 and CHOP expression levels were significantly increased in the TAC 4w and TAC 4w-A groups compared to the control and TAC 4w-I groups (Fig. 4c,d). Taken together, CaR induces sustained ER stress, which initiates its apoptotic pathway.

In the sham group, mitochondria showed regular cristae and a normal arrangement of sarcomeres. In the HF 4w and HF-A 4w groups, mitochondrial derangements were always present. The mitochondria appeared swollen, with disorganized cristae and the formation of wrinkled bodies, and some mitochondria had a ruptured outer membrane. The mitochondria in the HF-I 4w group had slightly swollen mitochondria and disorganized cristae (Fig. 5a). The mitochondrial release of cytochrome c was analyzed to prove the involvement of the mitochondrial apoptotic pathway in heart failure. We found that the release of cytochrome c from the mitochondria in the HF 2w, HF 4w, HF 2w-A, and HF 4w-A groups was significantly decreased compared with the control, HF 2w-I, and HF 4w-I groups (Fig. 5b).

To further investigate the induction of the ER stress-mediated apoptotic pathway by CaR, we examined [Ca²⁺]_SR by Fluo-5N staining in the rat neonatal ventricular cardiomyocytes (NVCMs). Fluo-5N is a low-affinity Ca²⁺ indicator (Kd = 400 μmol/L), which is only bright where [Ca²⁺] is very high, such as in the SR [22]. NVCMs were loaded with Fluo-5N and permeabilized with saponin. After treatment with Iso for 48 h, the Fluo-5N signal was detected in the SR. We found that the fluorescence intensity in the SR in the Iso-A group was significantly decreased compared to the control, Iso plus calhex231, 2-APB (IP₃Rs inhibitor) groups. Some studies have confirmed that 2-APB inhibits IP₃Rs and prevents the PE-induced enhancement of Ca²⁺ sparks in neonatal cardiomyocytes [23]. Our study also suggests that 3 μM 2-APB maintains [Ca²⁺]_SR through the inhibition of Ca²⁺ release from the SR via IP₃Rs. These results suggest that the activation of CaR by calindol induces the release of Ca²⁺ from the SR (Fig. 6a).

We found that CaR expression was increased in the Iso and Iso-A groups compared to the control group (Fig. 6b). To investigate whether CaR participates in ER stress, NVCMs were pretreated with Iso and calindol. We found that the expression of GRP78 was significantly increased; however, pretreatment of the NVCMs with calhex231 before Iso treatment had little effect on GRP78 expression (Fig. 6c).

As shown Fig. 6d and e, the levels of cleaved caspase-12 and CHOP were increased by CaR activation in NVCMs treated with Iso, whereas they were reduced by CaR inhibition. In accordance with these results, pretreatment of the cells with calhex231 significantly reduced the cell apoptosis induced by Iso, as determined by TUNEL. These results indicate that the activation of CaR increases ER stress responses and subsequent apoptosis in cardiomyocytes (Fig. 6f).

Although calindol-activated CaR significantly reduced [Ca²⁺]_SR, the role of type 3 IP₃Rs in the MAM in mediating Ca²⁺ uptake by mitochondria is less clear. To address this question, [Ca²⁺]_m was measured by X-rhod-1 AM staining after pretreatment with Iso for 48 h. [Ca²⁺]_m was markedly low in the control group. [Ca²⁺]_m was significantly higher in the Iso and Iso-A groups than in the Iso-I and Iso-Ru R groups (Fig. 7a). The mitochondrial membrane potential was detected with JC-1 staining. The ratio of the intensity of JC-1 aggregates (red) to monomers (green) was reduced in the Iso and Iso-A groups compared with the control, Iso-I, and Iso-Ru R groups (Fig. 7b).

To demonstrate that [Ca²⁺]_SR depletion induced by CaR activation causes apoptosis via a mitochondrial mediated pathway, the mitochondrial release of cytochrome c was analyzed to prove the involvement of the mitochondrial apoptotic pathway. The level of cytochrome c in the mitochondria in the Iso and Iso-A groups was significantly decreased compared with the control (1.0 ± 0.1), Iso-I, and Iso-Ru R groups (Fig. 7c).

The present study describes the role of CaR in inducing SR- and mitochondria-dependent apoptosis in heart failure. This role appears to be dependent on the anatomic functional interplay between the SR and mitochondria and involves Ca²⁺ transfer in these two organelles. Our study shows that the activation of CaR induces the release of Ca²⁺ from the SR, which causes ER stress and apoptosis. Simultaneously, an increase in Ca²⁺ uptake into the mitochondria through the MAM causes an increase in [Ca²⁺]_m and initiates the mitochondrial apoptotic pathway in the failing heart.

Heart failure is characterized by a decline in the force of contraction and dysregulation in intracellular Ca²⁺ homeostasis within myocytes [24]. Under physiological conditions, Ca²⁺ enters cardiomyocytes through the L-type Ca²⁺ channel (LTCC) and β-adrenergic receptor (β-AR), which causes the release of a large amount of Ca²⁺ from the SR by activating the RyR, leading to contraction. However, it has been confirmed that this Ca²⁺ influx is induced by a persistent increase of intracellular calcium concentration through LTCC and the activation of β-AR in failing hearts, which can lead to cellular necrosis and apoptosis [25]. Whether CaR plays a role in this process remains unclear. CaR, a G protein-coupled receptor, initiates complicated intracellular signals through the modulation of a series of intracellular signaling proteins, including G proteins and PLC, and in turn, stimulates IP₃ production and promotes intracellular Ca²⁺ release. CaR is important for the maintenance and regulation calcium homeostasis [26]. Although CaR is also widely expressed in tissues involved in calcium homeostasis, it modulates various cellular functions, including peptide secretion, ion channel/transporter activity, gene expression, proliferation, differentiation and apoptosis [27]. In our study, we chose calindol, a specific agonist of CaR, and calhex231, a specific inhibitor of CaR, to regulate CaR activity and confirm the role of CaR in failing hearts. Our data show that the expression of CaR in the hearts of rats subjected to Iso and calindol was significantly increased. These results reveal that CaR could be involved in the development of heart failure.

Although the main mechanism of excitation-contraction coupling in cardiac muscle is calcium-induced calcium release via RyR, previous studies have revealed that IP₃Rs also play a role in IP₃-induced calcium release. IP₃Rs are ligand-gated Ca²⁺ channels that function to release Ca²⁺ in response to IP₃ [28,29]. Moreover, some evidence has demonstrated that IP₃R expression in the ventricles of patients with end-stage chronic heart failure is upregulated when compared to that in normal patients, in contrast to RyR, which is down-regulated [30]. However, our results have not found that type 3 IP₃R expression is up-regulated in the failing hearts, which may be due to our heart failure model not in the end stage.

Abnormal intracellular Ca²⁺ cycling and SR function are the main reasons leading to failing cardiomyocytes [31,32]. Because the function of SR-resident chaperones is Ca²⁺-dependent, the accumulation of unfolded proteins and aberrant Ca²⁺ regulation in the SR lead to the activation of ER stress [33]. Although some studies have implicated that ER stress induces heart failure, how ER stress is initiated in failing hearts in still unknown. Our present study suggests that CaR activation may play an important role in ER stress in cardiac hypertrophy and failure. Therefore, we analyzed the expression of ER stress-related proteins in our Iso and calindol groups as well as in our TAC model. While the initial responses to ER stress may be adaptive, leading to an elevation in the levels of classic ER chaperones, such as GRP78 and p60ATF6, prolonged ER stress could initiate the apoptotic signaling pathway, inducing CHOP, JNK and caspase-12 activation [34,35]. Some studies have shown that cytosolic Ca²⁺ were released from ER and lead to the activation of several transcription factors, such as CHOP, PERK and elf2, essential components ER stress-mediated apoptosis [36,37]. Caspase-12 has been shown to be located at the cytosolic side of the ER membrane and to be actively cleaved in response to ER stress, caspase-12 is the only known substrate to caspa-se-12 [38].We found that the expression of GRP78 and p60ATF6 is up-regulated in cardiac hypertrophy and failure. CHOP and cleavage of caspase-12 expression levels were increased only in the heart failure groups and with the treatment of calindol, whereas theses expression levels were markedly lower in the heart failure groups treated with the CaR inhibitor. A strong ER stress response was also found in isoproterenol-stimulated neonatal ventricular cardiomyocytes (NVCMs), and the expression of GRP78 and caspase-12 was significantly higher in animals treated with Iso and with Iso plus calindol than in animals treated with Iso plus calhex231p. Taken together, these results suggest that the activation of CaR induces ER stress and associated apoptosis in heart failure. To further demonstrate the mechanism of activation of CaR that is involved in ER stress-mediated apoptosis, [Ca²⁺]_SR was examined with Fluo-5N in NVCMs. Our data showed that [Ca²⁺]_SR was significantly reduced upon CaR activation with calindol. [Ca²⁺]_SR was maintained with the administration of calhex231, 2-APB and U73122, respectively. These results demonstrate that CaR-induced Ca²⁺ release from the SR is a PLC-IP₃-dependent process.

It has been demonstrated that the mitochondria and SR form an interconnected network with a restricted number of close contacts, which is called the mitochondria-associated membrane (MAM) [8]. Ca²⁺ accumulates in the local MAM, where high concentration Ca²⁺ triggers the mitochondrial calcium uniporter (MCU) in mitochondria to induce an increase of Ca²⁺ uptake in mitochondria [39]. IP₃Rs play an important role in the macromolecular complexes on the surface of the SR membranes and promote Ca²⁺ release from the SR. The transport of Ca²⁺ through MCU can be stimulated with Gq-coupled receptor agonists, which induce IP₃ production and the subsequent release of Ca²⁺ from the SR, which causes a rapid rise in [Ca²⁺]_m [40,41]. Thus, we postulate that CaR may be involved in promoting Ca²⁺ release from the SR and subsequently in the increase in [Ca²⁺]_m. Therefore, we analyzed [Ca²⁺]_m using x-rhod-1 AM and found that [Ca²⁺]m was elevated in the Iso and Iso plus calindol groups, whereas [Ca²⁺]_m was maintained in the Iso plus calhex231 group. The rapid mitochondrial Ca²⁺ uptake is related to the low affinity of the Ca²⁺ transport system. Ruthenium red, an inhibitor of the MCU, was also used in this experiment. The results revealed that [Ca²⁺] was maintained in the Iso-RuR group.

SR-mitochondrial Ca²⁺ cross-talk may play a critical role in heart failure as an inducer of programmed cardiomyocyte death. Ca²⁺ transportation to the mitochondria plays a key role in initiating apoptosis by opening the mitochondrial permeability transition pore (mPTP) [42,43]. These results suggest that both the SR and the mitochondria orchestrate the regulation of Ca²⁺ signaling between these two organelles. It has been reported that [Ca²⁺]_SR depletion causes bax- and bak-mediated permeability of the outer mitochondrial membrane, thereby releasing pro-apoptotic factors, particularly cytochrome c [44,45]. Stimulation of these pathways triggers the translocation of death-promoting proteins (e.g., cytochrome c) from the mitochondria to the cytoplasm [46]. Our present data show that CaR activation induced cytochrome c release from the mitochondria during heart failure. Morphological analysis of apoptosis in cardiomyocytes was also performed using EM in the present study, and the results showed that CaR activation caused aggregated mitochondrial derangements, such as swelling, disorganized cristae and loss of normal striations. TUNEL assay also showed that the activation of CaR increased the number of apoptotic cardiomyocytes. Thus, CaR causes the cardiomyocyte apoptosis related to the interplay between the SR and mitochondria in the progression of heart failure.

In conclusion, our results are the first to report that CaR plays an important role in SR-mitochondrial inter-organelle Ca²⁺ signaling through IP₃Rs, which are also involved in apoptosis during heart failure. We propose that CaR could be a valuable pharmacological target for heart failure therapy.

The authors declare that they have no conflict of interest.

This study was supported by the National Natural Science Foundation of China (81170289, 81170218, 81170178), the Yu Weihan grant for excellent younger scientists of Harbin Medical University (to Weihua Zhang) and a post-doctoral fellowship (2012M520766).