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Introduction

There are different degrees of apoptosis in myocardial ischemia/reperfusion (I/R) injury. Reducing myocardial apoptosis has become a focus of research on myocardial protection. In 2003, Wang et al (1) first reported that calcium-sensing receptor (CaSR) was expressed in myocardial tissue, leading to the hypothesis that CaSRs participated in myocardial I/R injury induced by calcium overload (2); however, the specific mechanism remains unknown.

With I/R, CaSR activation of the phospholipase C (PLC) pathway causes calcium release from the endoplasmic reticulum (ER) (1,3-5), resulting in excessive ER stress (6). Multiple studies have demonstrated that the protein kinase C (PKC) family participates in the protection of cardiomyocytes or damage from myocardial I/R injury (5-7). The promotion or inhibition of apoptosis depends on the PKC subtype (8,9). PKCδ, one of the PKC subtypes, is an important signal in multiple types of cells, including myocardial infarction (10), lung inflammation (11), development of atherosclerosis (12) and the salivary gland (13). Previous studies have confirmed that myocardial I/R injury results in the transfer of PKCδ from the cell cytoplasm to the mitochondria and ER (5,7,14,15). During I/R, CaSR induces phosphorylation of PKCδ and migrates to mitochondria to induce cardiomyocyte apoptosis (5); however, the mechanism by which PKCδ participates in cell apoptosis has not been fully elucidated. The aim of the present study was to confirm that CaSR could activate PKCδ to induce cardiomyocyte apoptosis through ER stress-associated apoptotic pathways during I/R in vivo.

Materials and methods

Animals

Male Wistar rats, weighing 200-250 g, were obtained from the Animal Center of Harbin Medical University. All animals were treated according to the Guidelines for the Care and Use of Laboratory Animals and all protocols were approved by the Ethics Committee of Basic Medical College of Harbin Medical University. The associated permit number is SCXK (Hei) 2013-001.

Materials

NPS-2390, GdCl3, verapamil, rottlerin and amiloride were obtained from Sigma-Aldrich (Merck KGaA). The terminal deoxynucleotidyl transferase (TdT)-mediated-UTP nick end labeling (TUNEL) kit, and the 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocar bocyanine iodide (JC-1) kit were from Roche Diagnostics GmbH. Primary antibodies targeting PKCδ (cat. no. sc-213), glucose-regulated protein 78 (GRP78; cat. no. sc-13968), Caspase-12 (cat. no. sc-5627), c-Jun N-terminal kinase (JNK; cat. no. sc-474), phosphorylated (p-) JNK (cat. no. sc-6254) and Caspase-3 (cat. no. sc-7148) were obtained from Santa Cruz Biotechnology, Inc. The primary antibody targeting CaSR was from Alpha Diagnostic International Inc. (cat. no. ACR-004). β-actin (cat no. M01263-2) and Calnexin (cat. no. A03372) were obtained from Boster Biological Technology.

Experimental protocol

Heparinized rats [2,000 U/kg, by intraperitoneal (i.p.) injection] were anesthetized with 2% pentobarbital sodium (40 mg/kg; i.p.). Following endotracheal intubation, an animal breathing machine was provided with a rodent respirator at a rate of 50 breaths/min and a tidal volume of 20 ml/kg (16). Between the third and fourth ribs, a left parasternal incision was made to expose the heart. The left anterior descending artery (LAD) was ligated using a 6-0 silk suture. Myocardial I/R was induced by the compression of the LAD (17). The changes in the ST-T segment, caused by tightening or loosing of the silk suture, were monitored by ECG. After coronary artery ligation for 30 min, the ligation was removed to restore myocardial blood perfusion. At 2 h after reperfusion, the rats were sacrificed by exsanguination under anesthesia, and aortic inverse irrigation with PBS was performed to the hearts in order to remove residual blood. Subsequently, a portion of the tissue of the anterior wall of the left ventricle near the apex was obtained for further analysis.

The animals were randomly divided into five groups: i) Control group (n=10), thread only, without ligation; ii) I/R group (I/R; n=10), coronary artery ligation for 30 min, and loosening of the silk suture to restore myocardial blood perfusion for 2 h; iii) I/R+Amiloride+Verapamil+GdCl3 group (I/R+A+V+Gd; n=10), 20 µM amiloride (inhibitor of Na+-Ca2+ exchanger), 1 µM verapamil (L-type calcium channel blocker) and 30 µM GdCl3 (activator of CaSR) were added by injection through the femoral vein 10 min prior to reperfusion; iv) I/R+Amiloride+Verapamil+GdCl3+NPS-2390 group (I/R+A+V+Gd+NPS; n=10), NPS-2390 (inhibitor of CaSR) was added by injection through the femoral vein 10 min prior to reperfusion, while all other procedures were the same as those performed in the I/R+Amiloride+Verapamil+GdCl3 group; and v) I/R+Amiloride+Verapamil+GdCl3+Rottlerin group (I/R+A+V+Gd+PKCδI; n=10), amiloride (20 µM), verapamil (1 µM), GdCl3 (30 µM) and 10 µM rottlerin (specific inhibitor of PKCδ) were added by injection through the femoral vein 10 min before reperfusion, and all other procedures were identical to those performed in the I/R+Amiloride+Verapamil+Gd Cl3+NPS-2390 group.

ER isolation

Frozen heart specimens were minced and ground by pestle on ice and then homogenized using a tissue homogenizer thirty times in 3 ml of cool lysis buffer (250 mM sucrose, 20 mmol/l Tris-HCl pH 7.2, 10 mmol/l KCl, 1.5 mmol/l MgCl2, 1 mmol/l EDTA, 1:300 protease inhibitor cocktail, 1:300 phosphatase inhibitor cocktail). The muscle cells were then collected in a microcentrifuge tube and incubated for 30 min at 4°C. The homogenates were centrifuged at 800 × g for 10 min at 4°C, and the resulting supernatant was centrifuged at 10,000 × g for 20 min at 4°C. The new supernatants were centrifuged at 100,000 × g for 1 h (4°C) to obtain the ER (pellet) and cytosolic (supernatant) extracts. The ER pellet was resuspended in lysis buffer containing 1% Triton X-100 (14). The ER extract was stored at −70°C.

TUNEL staining

Identification of apoptotic cells was performed using an in situ cell death detection kit (Roche Diagnostics GmbH), according to the manufacturer's instructions. After rinsing with PBS, the heart specimens were fixed with 4% formaldehyde solution at room temperature for 24 h. Following routine protocols, the heart specimens were paraffin-embedded, sectioned at 4 µm thickness and dewaxed. After rinsing with PBS, the slides were soaked in a solution containing 0.1% Triton X-100 and 0.1% sodium citrate for 2 min to increase the cell membrane permeability. The slides were incubated with 50 µl of TUNEL reaction mixture for 60 min at 37°C. 3,3′-Diaminobenzidine (DAB; 0.05%) was added to the slide for 30 min and then incubated in the substrate solution (100 µl) for an additional 10-15 min. Labeled myocytes were analyzed using a light microscope (XSZ-D2; Olympus Corporation).

Transmission electron microscopy

Heart samples were fixed with 2.5% glutaraldehyde for 24 h at 4°C, washed with PBS, and fixed with 1% osmium tetroxide for 2 h. Samples were then dehydrated with fractionated acetone, embedded in Epon-Araldite resin, and cut into ultrathin sections of 50-70 nm. Ultrathin sections were placed on copper grids and stained with uranyl acetate and lead citrate. Samples were examined under a JEM-2000EX transmission electron microscope (16,18). The images were captured using a Hamamatsu camera (Hamamatsu Photonics, Ltd.).

Western blot analysis

Protein extracts were prepared as aforementioned and subjected to 10% SDS-PAGE. Each lane contained 60 µg of protein. All samples were transferred to nitrocellulose membranes and blocked for 1 h at room temperature with 5% BSA in Tris-buffered saline/Tween-20 (TBST) buffer. The membranes were then exposed to the rabbit monoclonal anti-rat CaSR antibody, the rabbit polyclonal serum against rat total PKCδ, and GRP78, Calnexin, Caspase-12, p-JNK, JNK and Caspase-3 antibodies at a concentration of 1:1,000 overnight at 4°C. The secondary antibody alkaline phosphatase IgG (1:2,000; cat. no. S3731; Promega Corporation) was then added for 2 h at room temperature. The final signals were quantified using a Bio-Rad Chemi Doc EQ densitometer and Bio-Rad Quantity One software (Bio-Rad Laboratories, Inc.). β-actin was used as internal reference for the total cytoplasmic extract. Calnexin was used as internal reference for the ER extract. All band intensities were normalized to β-actin or Calnexin and expressed as a percentage of the control sample.

Isolation of acute myocardial cells

Rats without any treatment were anesthetized with 2% pentobarbital (100 mg/kg, i.p.) and the hearts were quickly removed. Then the hearts were perfused (5 min) through the aorta with standard Tyrode's solution at 37°C until the residue was clear (19). The composition of the standard Tyrode's solution was: 136 mM NaCl, 5.4 mM KCl, 0.35 mM NaH2PO4, 1.0 mM MgCl2, 2.0 mM CaCl2, 10 mM dextrose and 10 mM HEPES (pH adjusted to 7.4 with NaOH). The solution was equilibrated with 95% O2 and 5% CO2 at room temperature prior to use. Then the hearts were sequentially perfused with Ca2+-free Tyrode's solution for 10 min, and a Ca2+-free Tyrode's solution containing 0.015 g/l collagenase for 30 min. The ventricular tissue (2-3 mm in diameter) was excised and placed in KB solution. The composition of the KB solution was: 70 mM glutamic acid, 15 mM taurin, 30 mM KCl, 10 mM KH2PO4, 10 mM HEPES, 0.5 mM MgCl2, 0.5 mM ethylene glycol tetraacetic acid and 10 mM glucose (pH adjusted to 7.3-7.4 with KOH). Myocardial cells were isolated and kept at room temperature (5).

The acutely isolated myocardial cells were then divided into five treatment groups: i) Control group, myocardial cells were incubated in Tyrode's solution without any treatment; ii) I/R group, myocardial cells were incubated with Tyrode's solution for 8 min, then incubated in simulated ischemic solution (NaCl 136 mM, KCl 5.4 mM, NaH2PO4 0.35 mM, MgCl2 1.0 mM and Hepes 10 mM, pH 6.8) at 37°C for 30 min, and finally incubated with Tyrode's solution for another 8 min; iii) I/R+A+V+Gd, myocardial cells were incubated with simulated ischemia solution for 30 min, followed by reperfusion for 8 min in Tyrode solution immediately after addition of 20 µM Amiloride, 1 µM Verapamil and 30 µM GdCl3; iv) I/R+A+V+Gd+NPS, 20 µM Amiloride, 1 µM Verapamil, 30 µM GdCl3 and 10 µM NPS-2390 were added to the myocardial cells before reperfusion with Tyrode's solution for 8 min; and v) I/R+A+V+Gd+PKCδI, 20 µM Amiloride, 1 µM Verapamil and 30 µM GdCl3 and 10 µM Rottlerin were added to the myocardial cells before reperfusion with Tyrode's solution for 8 min. Following treatments, cells were analyzed as indicated.

JC-1 fluorescence staining

The decrease of mitochondrial membrane potential is a landmark event in the early stages of apoptosis. This method can identify apoptotic cells earlier than other methods. Mitochondrial membrane potential was measured by JC-1 fluorescence staining. JC-1 is an ideal fluorescent probe for detecting mitochondrial membrane potential. When the mitochondrial membrane potential is high, JC-1 aggregates in the matrix of the mitochondria to form a polymer, which can produce red fluorescence. When the mitochondrial membrane potential is low, JC-1 cannot accumulate in the matrix of mitochondria, and therefore JC-1 exists as a monomer which can produce green fluorescence. Therefore, changes in mitochondrial membrane potential can be easily detected through the change of fluorescence color. The relative ratio of green and red fluorescence is commonly used to measure the proportion of mitochondrial depolarization. The appearance of green fluorescence indicates a decrease in mitochondrial membrane potential, and the cell is likely to be in the early stages of apoptosis. Red fluorescence indicates that the mitochondrial membrane potential is normal and the state of the cells is normal.

In the present study, isolated cardiomyocytes were stained with JC-1 (1 µg/ml) for 15 min at 37°C and washed with Tyrode's solution three times. The changes of mitochondrial membrane potential were observed using confocal laser microscopy. The excitation light was set to 488 nm when the JC-1 monomer (green) was detected, and the emission light was set to 530 nm. When the JC-1 polymer (red) was detected, the excitation light was set to 525 nm, and the emission light was set to 590 nm. Multiple fields of view (>10) were captured from each sample and the average intensity of each field was quantified. The intensity ratio of JC-1 polymer to JC-1 monomer in each field was calculated. A decrease of the ratio indicated a decrease in the membrane potential, and an increase of the ratio indicated an increase in the membrane potential.

Statistical analysis

Data were expressed as the mean ± SD from three independent experiments. Data were analyzed using one-way ANOVA followed by Bonferroni post hoc tests. All statistical analyses were performed using Prism 5 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results

CaSR involved in cardiomyocyte apoptosis during I/R

Previous studies have confirmed that myocardial I/R results in the translocation of PKCδ from the cell cytoplasm to the ER (7,14,15), and activation of CaSR by myocardial I/R can induce cardiomyocyte apoptosis through the ER stress-related apoptosis pathway (6). However, the interaction of the CaSR with PKCδ to induce cardiomyocyte apoptosis in vivo has not been fully elucidated. Thus, the following experimental methods were used to confirm the relationship between CaSR and PKCδ in cardiomyocyte apoptosis during I/R in vivo.

Cardiomyocyte apoptosis was detected by TUNEL staining and JC-1 fluorescence staining. In the TUNEL experiment, normal cardiomyocyte nuclei were stained blue, while apoptotic cell nuclei were stained brown. The ratio of TUNEL-positive cells was increased significantly in the I/R and I/R+A+V+Gd groups compared with the Control group (P<0.05; Fig. 1). In the I/R+A+V+Gd+NPS group and I/R+A+V+Gd+PKCδI group, the ratio of apoptotic cells was lower compared with the I/R+A+V+Gd group (P<0.05; Fig. 1). The results of cardiomyocyte JC-1 staining revealed that the intensity ratio of JC-1 polymer/monomer decreased significantly in the I/R and I/R+A+V+Gd groups compared with the Control group (P<0.05; Fig. 2). However, compared with the I/R+A+V+Gd group, the ratio of JC-1 polymer/monomer in the I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups remained at a high level (P<0.05; Fig. 2).

Figure 1 Representative images from TUNEL staining of cardiomyocytes following I/R. Nuclei with brown staining indicate TUNEL-positive cells. (A) Control group, (B) I/R group, (C) I/R+Amiloride+Verapamil+GdCl3+NPS-2390 (I/R+A+V+Gd+NPS) group, (D) I/R+Amiloride+Verapamil+GdCl3 (I/R+A+V+Gd) group and (E) I/R+Amiloride+Verapamil+GdCl3+Rottlerin (I/R+A+V+Gd+PKCδI) group (magnification, ×200). Quantitative results of TUNEL staining for different groups is shown in the lower panel. Values represent means ± SEM (n=7). #P<0.05 vs. Control group; *P<0.05 vs. I/R+A+V+Gd group. I/R, ischemia/reperfusion.

Figure 2 Relative changes in JC-1 fluorescence intensity of cardiomyocytes following I/R. The fluorescence intensity ratio of JC-1 in its aggregate and monomer forms was calculated in the different groups. Values represent means ± SEM (n=6). #P<0.05 vs. Control group; *P<0.05 vs. I/R+A+V+Gd group. JC-1, 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide; I/R, ischemia/reperfusion.

Morphological characterization of cardiomyocyte apoptosis

In the present study, ultrastructural changes in cardiomyocytes were examined by transmission electron microscopy. Morphological characteristics indicating apoptosis include condensation, aggregation and margination of nuclear chromatin, ER expansion, capsule bubble and mitochondrial swelling, and mitochondrial crest fracture. These apoptotic characteristics were observed in the I/R+A+V+Gd and I/R groups, but not in the Control group (Fig. 3). By contrast, in the I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups, only a slight margination of the nuclear chromatin, expansion of the ER and swelling of the mitochondrion were observed (Fig. 3).

Figure 3 Transmission electron micrographs of cardiac tissue ultrastructure changes. (A) Control group, (B) I/R group, (C) I/R+A+V+Gd+NPS group, (D) I/R+A+V+Gd group and (E) I/R+A+V+Gd+PKCδI group (magnification, ×8,000). I/R, ischemia/reperfusion.

CaSR protein expression in myocardial cells

Since CaSR serves a key role in the elevation of [Ca2+]i in cardiomyocytes (5), the expression levels of the CaSR protein, with a relative molecular mass of 130 kDa, were determined by western blot analysis. Compared with the Control group, the protein expression levels of CaSR in the I/R and I/R+A+V+Gd groups were increased (P<0.05; Fig. 4). Additionally, the CaSR protein expression levels in the I/R+A+V+Gd+NPS group was decreased compared with the I/R+A+V+Gd group (P<0.05; Fig. 4). These results demonstrated that GdCl3 could upregulate the expression of CaSR (Fig. 4).

Figure 4 Western blot analysis for CaSR in rat cardiomyocytes of different groups. Representative blots and quantification of CaSR expression levels relative to the Control group are shown. Values represent means ± SEM (n=3-4). #P< 0.05 vs. Control group; *P<0.05 vs. I/R+A+V+Gd group. CaSR, calcium-sensing receptor; I/R, ischemia/reperfusion.

Effect of CaSR in myocardial I/R on the expression of ER stress-related proteins in vivo

The ER environment can be stimulated by various stimuli, such as a perturbation of Ca2+ homeostasis, which leads to initiation of the unfolded protein response (UPR). The UPR is a protective mechanism against ER stress, which is conducive to the stability of the cellular environment. If excessive ER stress is not resolved, it will induce cell apoptosis (20). To detect the occurrence of ER stress, protein expression levels of ER stress-related proteins, such as GRP78, can be detected, which is resistant to the UPR and has been extensively used as a marker for ER stress and UPR (21). In the present study, the protein expression levels of GRP78 were detected by western blot analysis. The results demonstrated that GRP78 expression in the I/R and I/R+A+V+Gd groups was significantly higher compared with the Control group, but in the I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups, GRP78 expression was significantly lower compared with the I/R+A+V+Gd group (P<0.05; Fig. 5). The present results demonstrated that activated CaSR in I/R induced ER stress in vivo. These results also determined that inhibition of PKCδ may protect the myocardium from ER stress in an in vivo model of I/R myocardial injury.

Figure 5 Western blot analysis for GRP78 in rat cardiomyocytes by activating CaSR. Representative blots and quantification of GRP78 expression levels relative to the Control group are shown. Values represent means ± SEM (n=3-4). #P<0.05 vs. Control group; *P<0.05 vs. I/R+A+V+Gd group. GRP78, glucose-regulated protein 78; I/R, ischemia/reperfusion.

CaSR activates PKCδ induction of the ER stress-induced apoptosis pathway in vivo

The translocation of PKCδ to the ER, to participate in ER stress-induced apoptosis, has been previously documented (14). Next, the present study investigated whether CaSR activating PKCδ participated in the apoptotic pathway in vivo. Previous studies have shown that several molecules, including JNK, CCAAT/enhancer-binding protein-homologous protein (CHOP) and Caspase-12, are involved in the ER stress-induced apoptosis pathway (22). In the current study, the protein expression of Caspase-12, p-JNK and Caspase-3 in cardiomyocytes was examined by western blot analysis. Cleaved Caspase-12 (the 42 kDa proteolytic fragment of Caspase-12) was significantly increased in the I/R and I/R+A+V+Gd groups compared with the Control group (P<0.05; Fig. 6). The expression levels of cleaved Caspase 12 were decreased in the I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups compared with the I/R+A+V+Gd group (P<0.05, Fig. 6). As presented in Fig. 7, the protein expression levels of Caspase-3 and the ratio of p-JNK/JNK in the I/R and I/R+A+V+Gd groups were higher compared with the Control group. The levels of Caspase-3 and the p-JNK/JNK ration in the I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups were decreased compared wih the I/R+A+V+Gd group (P<0.05; Fig. 7). The protein expression levels of Caspase-12, p-JNK and Caspase-3 are related to the rate of apoptosis of cardiomyocytes. Therefore, the present results revealed that CaSR activated PKCδ to participate in apoptosis through the ER stress-induced apoptosis pathway.

Figure 6 Western blot analysis for activated Caspase-12 in rat cardiomyocytes. Representative blots and quantification of cleaved Caspace-12 expression levels relative to the Control group are shown. Values are expressed as mean ± SEM (n=3-4). #P<0.05 vs. sham Control group; *P<0.05 vs. I/R+A+V+Gd group. I/R, ischemia/reperfusion.

Figure 7 Western blot analysis for JNK activation and Caspase-3 expression in rat cardiomyocytes following I/R. (A) Representative blots and quantification of p-JNK and total JNK. (B) Representative blots and quantification of Caspase-3 protein expression levels. All values are plotted as relative to the Control group and as means ± SEM (n=3-4). #P<0.05 vs. Control group; *P<0.05 vs. I/R+A+V+Gd group. I/R, ischemia/reperfusion; p-, phosphorylated.

CaSR induces PKCδ translocation to the ER following I/R and participates in the ER stress-induced apoptosis pathway

In the myocardium, inhibition of PKCδ activation could significantly inhibit the ER stress response at the beginning of reperfusion (14). In the present study, expression of total-PKCδ in the cytoplasm of myocardial cells was detected by western blotting in each group (Fig. 8A). In the I/R, I/R+A+V+Gd, I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups, total-PKCδ protein expression levels were significantly higher compared with the Control group (P<0.05; Fig. 8A). The present study also quantitatively analyzed the expression of PKCδ in the ER fractions (Fig. 8B). The PKCδ expression levels of the I/R and I/R+A+V+Gd groups were significantly higher compared with the Control group (P<0.05; Fig. 8B), while the levels of PKCδ from the ER fractions were significantly inhibited in the I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups compared with the I/R+A+V+Gd group (P<0.05; Fig. 8B). These results demonstrated that CaSR induced PKCδ translocation to the ER following I/R and participated in the ER stress-induced apoptosis pathway.

Figure 8 Protein expression of PKCδ in the total cytoplasm and in ER fractions of rat cardiac tissue following I/R. (A) Levels of total-PKCδ in the cytoplasm and (B) in ER-specific fractions in cardiomyocytes from the experimental groups. Values are plotted as relative to the Control group and as means ± SEM (n=3-4). #P<0.05 vs. Control group; *P<0.05. vs. I/R+A+V+Gd group. PKCδ, protein kinase C δ; ER, endoplasmic reticulum; I/R, ischemia/reperfusion.

Discussion

The results of the present study confirmed that the activation of CaSR induced PKCδ translocation to the ER following I/R and participated in cardiomyocyte apoptosis via the ER stress-associated apoptotic pathway in vivo.

It is well-established that I/R injury can lead to activation of PKC. PKC, as a kind of Ca2+ and phospholipid-dependent protein kinase, is involved in the apoptosis of cardiac muscle cells by regulating [Ca2+]i homeostasis. The PKC family contains 13 subtypes, and the PKCε and PKCδ subtypes have been well-studied. The former mainly serevs a role in the inhibition of apoptosis, while the latter mainly acts in the promotion of apoptosis. CaSR acts as a protein-coupled receptor, causing the accumulation of inositol phosphate (IP), which leads to an increase in intracellular calcium. However, the PKCδ inhibitor, rottlerin, can reverse the occurrence of this phenomenon by inhibiting CaSR (5,23). The links between PKCδ, CaSR and ER stress during I/R remain unclear. To investigate the effect of activation of PKCδ by CaSR on myocardial I/R, GdCl3 (an activator of CaSR), NPS-2390 (an inhibitor of CaSR), amiloride (an inhibitor of the Na+-Ca2+ exchanger), verapamil (an L-type calcium channel blocker) and rottlerin (a PKCδ inhibitor) were used in the present study. The experimental animals were divided into five groups: Control, I/R, I/R+A+V+Gd, I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups.

First, the expression of CaSR in myocardial I/R was detected by western blot analysis. CaSR, also known as the parathyroid cell CaSR, is an integral membrane protein belonging to the G-protein coupled receptor 3 family. The molecular weights of CaSR are 110, 130 and 150 kDa, of which the 150 kDa form is the mature form of CaSR. CaSR is present on the cell membrane. In the present study, the 130 kDa CaSR protein was detected in the cardiac tissues. Compared with the Control group, the expression of CaSR in the I/R and I/R+A+V+Gd groups increased. However, in the I/R+A+V+Gd+NPS group, the CaSR protein expression levels was lower compared with the I/R+A+V+Gd group. Thus, I/R and GdCl3 were demonstrated to increase CaSR expression.

Multiple studies have indicated that PKC can regulate the intracellular Ca2+ concentration (24,25) and participate in the protection or damage of cardiac muscle cells by CaSR-mediated intracellular calcium release. In the present study, cardiomyocyte apoptosis was examined by JC-1 fluorescence staining and TUNEL staining. The decrease of mitochondrial membrane potential is a landmark event in the early stages of apoptosis. JC-1 is an ideal fluorescent probe for detecting mitochondrial membrane potential. Therefore, the JC-1 method was used to detect early apoptosis following myocardial I/R. The sensitivity of the TUNEL method for detecting apoptosis is very high, and it is also possible to quantitatively analyze apoptotic cells of both the middle or late stages. The results of cardiomyocyte JC-1 straining analysis revealed that the intensity ratio of JC-1 polymer/monomer decreased significantly in the I/R and I/R+A+V+Gd groups compared with the Control group. Similarly, the ratio of TUNEL-positive cells was significantly higher in the I/R and I/R+A+V+Gd groups compared with the Control group. In addition, the ultrastructural changes of the myocardial cells were observed by transmission electron microscopy. In the I/R and I/R+A+V+Gd groups, the myocardial cell structure was disordered, and the nuclear chromatin was condensed and concentrated. By contrast, in the I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups, only a slight margination of the nuclear chromatin, expansion of the ER and swelling of the mitochondrion were observed. Based on the aforementioned results, the experiments revealed that GdCl3 significantly increased the occurrence of cardiomyocyte apoptosis following I/R. The data also demonstrated that inhibition of PKCδ or CaSR significantly reduced the rate of cardiomyocyte apoptosis and protected cardiomyocytes from myocardial I/R injury.

The ER environment can be impaired by various stimuli, such as a perturbation of Ca2+ homeostasis, which leads to the UPR. Excessive UPR can stimulate ER stress (20). Previous studies have demonstrated that CaSR-induced Ca2+ release from internal stores is a PLC-mediated/IP3-dependent process. The enhancement of [Ca2+]i may be related to the release of Ca2+ by the ER (6,26). To examine the occurrence of ER stress, the protein levels of GRP78 were detected. GRP78 is an ER molecular chaperone protein that promotes protein folding and the internal environment of calcium ions, protecting cells against oxidative stress and apoptosis, and has been extensively used as a marker for ER stress and the onset of the UPR (21,27). Protein expression of GRP78 can thus be used as a marker of ER stress (28-30). In the present study, the expression of GRP78 was detected by western blot analysis. The results demonstrated that GRP78 protein expression levels in the I/R and I/R+A+V+Gd groups were significantly higher compared with the Control group; however, the levels of the I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups were decreased compared with the I/R+A+V+Gd group. Based on the present results, activated CaSR in I/R was demonstrated to induce ER stress in vivo. These data also determined that inhibition of PKCδ may protect the myocardium from ER stress in an in vivo model of I/R injury.

Next, the current study investigated whether activation of PKCδ by CaSR participated in ER stress-initiated apoptotic signaling. Studies have confirmed that several molecules, including Caspase-12, CHOP and JNK, are involved in the ER stress-induced apoptosis pathway (22). Caspase-12, an ER membrane-resident apoptotic molecule, can lead to cardiomyocyte apoptosis and cardiac dysfunction (31,32). The JNK signaling pathway is one of the important mitogen-activated protein kinase pathways, and serves an important regulatory role in a variety of physiological and pathological programmed cell death processes (33). Both in vitro and in vivo experiments have demonstrated that high-intensity ER overstress activates inositol dependent enzyme 1 (IRE1) and its downstream tumor necrosis factor receptor-associated factor 2 (TRAF2)/apoptosis signal-regulated kinase (ASK1)/JNK apoptotic signaling pathway (34,35). JNK exerts its pro-apoptotic effect by activating the downstream caspase-9 and caspase-3 (36). In the present study, the protein expression levels of Caspase-12, p-JNK and Caspase-3 in cardiomyocytes were measured by western blot analysis. The results demonstrated that the activation of Caspase-12, p-JNK and Caspase-3 were significantly higher following I/R injury. Therefore, CaSR-activated PKCδ induced endoplasmic reticulum Ca2+ depletion and activated the ER stress-associated pathway to participate in the process of cardiomyocyte apoptosis during I/R injury.

Several studies have indicated that inhibition of PKCδ activation could significantly inhibit the ER stress response at the beginning of reperfusion in the myocardium (14). In the present study, the expression of total-PKCδ in the cytoplasm of myocardial cells was detected by western blotting. Compared with the Control group, the expression levels of total-PKCδ protein in the I/R, I/R+A+V+Gd, I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups were significantly increased. Expression of PKCδ was also quantitatively analyzed in the ER fractions. The levels of PKCδ in the I/R and I/R+A+V+Gd groups were significantly higher compared with the Control group, while the levels of PKCδ in the ER fractions was significantly lower in the I/R+A+V+Gd+NPS and I/R+A+V+Gd+PKCδI groups compared with the I/R+A+V+Gd group. These results revealed that CaSR induced PKCδ translocation to the ER, which then participated in cardiomyocyte apoptosis via ER stress-associated pathways.

In summary, the present results confirmed that activation of CaSR induced the translocation of PKCδ to the ER following I/R and participated in cardiomyocyte apoptosis via ER stress-associated apoptotic pathways in vivo.

Acknowledgments

Not applicable.

Funding

The present study was funded by the National Natural Science Foundation of China (grant nos. 81670344, 81370421 and 81370330), the Natural Science Foundation of Heilongjiang (grant no. D201070) and the High-Priority Health Projects of Tianjin (grant no. 16KG146).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

WZ conceived and designed the experiments. CL, MZ, HZ, TF, FL, HL and SD performed the experiments and analyzed the data. CL and HL wrote the paper. HZ, MZ and HL contributed to the manuscript revisions. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of Basic Medical College of Harbin Medical University [permit no. SCXK (Hei) 2013-001].

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References