Exogenous spermine inhibits hypoxia/ischemia-induced myocardial apoptosis via regulation of mitochondrial permeability transition pore and associated pathways

Isolation of neonatal cardiomyocytes and hypoxia/ischemia (H/I) treatment

Newborn Wistar rats, aged 1–3 days and weighing 5–8 g, were used in this study. Primary cultures of neonatal cardiomyocytes were prepared, as described in a previous study. ¹² All the experiments were approved by the Animal Care Committee for the Use of Experimental Animals at Harbin Medical University, Heilongjiang, China. The cardiomyocytes were plated in 6- or 96-well plates coated with collagen. Thereafter, they were maintained at 37℃ in a 5% CO₂ incubator that was humidified using Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin, and streptomycin. In this experiment, we used cardiomyocytes that were spontaneously beating 72 h after plating. For the hypoxic/ischemic protocol, hypoxic condition was produced using Hank’s buffered saline solution that was saturated with 95% N₂ and 5% CO₂. To mimic ischemic solution, the pH was maintained at 6.8 using lactate. Cells were placed in a hypoxic incubator, which was equilibrated with 1% O₂/5% CO₂/94% N₂ for 24 h.

Experimental protocols

At the end of the initial culture period, cells were randomly divided into the following six groups (n = 8 independent experiments, Figure 1): (1) Control group (Control): cardiomyocytes were cultured with DMEM containing 10% FBS for 24 h; (2) Control + spermine group (Control + Sp): cardiomyocytes were cultured for 24 h in a media using 5 μM/L spermine; (3) H/I group: cardiomyocytes were exposed to H/I condition for 24 h; (4) H/I + spermine group (H/I + Sp): 5 μM/L spermine was added to an ischemic medium showing signs of hypoxia, and the cells were subsequently incubated for 24 h; (5) H/I + PD98059 (or LY294002) group (H/I + PD98059/LY294002): 10 mM of PD98059 (or 10 mM LY294002) was added to the medium 30 min before the development of hypoxia; the resulting medium was incubated for 24 h; (6) H/I + PD98059 (or LY294002) + spermine group (H/I + PD98059/LY294002 + Sp): 10 mM PD98059 (or 10 mM LY294002) was added to the medium 30 min before the commencement of hypoxia and the medium was incubated for 24 h. OPEN IN VIEWER

Determination of PAs by RP-HPLC

Precolumn derivatization was carried out using RP-HPLC, which was equipped with ODS-C18 column (COSMOSIL, Japan).^13–15 In this process, PAs (putrescine, spermidine, and spermine, Sigma, St. Louis, MO) were used as standards, while benzoyl chloride was used as the derivative reagent. Thus, in the control and H/I groups, we compared the content of PAs. Derivatization was carried out at 50℃ for 8 h under the following conditions: methanol–water (45:55) was used as the mobile phase; the flow rate was kept fixed at 1.0 mL/min. The wavelength of detection was also fixed at 234 nm. Thus, each peak of the derivatives was separated clearly in the chromatogram obtained by HPLC instrument. Using the size of peak area, we determined the difference in the content present in the two groups.

Cell viability assay

Cell viability was measured by Cell Counting Kit-8 (CCK-8) (Dojindo, WTS, Japan). Cells were seeded in 96-well plates at a concentration of 3 × 10³ cells/well. After 24 h of each treatment, 10 µL was added to each well of CCK-8 immediately. Subsequently, they were incubated for 2 h at 37℃. Using a microplate spectrophotometer, the plates were read at 570 nm (A570) to determine their optical density.

Assay of lactate dehydrogensase (LDH) and creatine kinase (CK) activity

The extent of cellular injury was determined by measuring the amount of LDH and CK released from cardiomyocytes. The culture media was collected, while LDH and CK activity was measured using commercial kits (Jian Cheng Bioengineering Institute, Nanjing, China); the plates were read using a microplate spectrophotometer.

Apoptosis assay by Annexin V/PI staining

The apoptotic rate was detected by flow cytometry using FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, USA). Cells were washed using cold PBS, and then they were suspended in a binding buffer at a concentration of 1 × 10⁶ cells/mL. The solution (100 µL) was transferred into a 5 mL culture tube; thereafter, we added 5 µL of FITC-Annexin V and 5 µL propidium iodide. The cells were gently vortexed and incubated in the dark for 15 min at room temperature (RT). Thereafter, the fluorescence was analyzed by flow cytometry. The percentage of apoptotic cells was determined using the Mod Fit LT software (Verity Software House Inc., Topsham, ME, USA).

Morphological analysis of apoptotic nuclei

Cells apoptosis was determined by visualizing the morphology of nuclei using the fluorescent DNA-binding dye, Hoechst 33342 (Santa Cruz, Bergheimer, Germany). Cells were rinsed with PBS and incubated for 10 min using 5 mg/mL Hoechst 33342. Nuclei were visualized under a fluorescent microscope at 400 × magnification (Nikon Corporation, Tokyo, Japan); the excitation wavelength was maintained in the range of 330–380 nm. Apoptotic nuclei of cells were assessed by counting the number of cells that displayed morphological changes in the nuclei, such as chromatin condensation and fragmentation.

Measurement of [Ca²⁺]_i

The [Ca²⁺]_i was determined by molecular probes according to manufacturer’s instructions. After treatment, cardiomyocytes (1 × 10⁶ cells/sample) were either cultured on a 12-well plate or slides were washed using a buffer of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid to maintain the pH at 7.4. Then, the washed cells were stained using 5 μM fluo-8 AM (Santa Cruz, Bergheimer, Germany) for 30 min at RT. The fluorescence of Ca²⁺ was measured using a laser confocal scanning microscope (Olympus, LSM, Japan); the excitation wavelength was kept at 485 nm, while the emission wavelength was set at 530 nm. The fluorescent intensity was reflected by [Ca²⁺]_i.

Determination of mitochondrial membrane potential (Δψm)

ΔΨm was estimated using mitochondrial membrane potential assay kit, equipped with JC-1(Santa Cruz, Bergheimer, Germany). Briefly, cells were collected and incubated at 37℃ for 20 min using JC-1 (1×). Then, they were washed twice using phosphate buffered saline. Using a fluorescent microscope, observations were conducted at 400× magnification (Nikon Corporation, Tokyo, Japan). Furthermore, the fluorescence of JC-1 monomer (green) was observed at excitation wavelength of 514 nm; the emission wavelength was maintained at 529 nm. Thereafter, the fluorescence of JC-1 aggregate (red) was observed at an emission wavelength of 585 nm/590 nm. At least 100 areas were randomly selected from each image, and the average intensity of each region was quantified. We calculated the ratio of JC-1 aggregate to monomer (red/green) intensity in each region. A decrease in this ratio was interpreted as a decrease of Δψm, whereas an increase in this ratio was interpreted as a gain of Δψm.

Assay of mPTP opening

The changes of mPTP opening were measured by co-incubating calcein AM (Santa Cruz, Bergheimer, Germany) and cobalt chloride (Sigma Chemical Co., St. Louis, MO, USA). Cardiomyocytes were plated in 24-well plates (5 × 10⁵ cells/well). After stimulating with different treatments, we loaded the cells with 2 μM of calcein AM in the presence of 5 mM of cobalt chloride solution; the reaction mixture was kept in the dark for 15 min, and the temperature of the reaction mixture was maintained at 37℃. Using a laser confocal scanning microscope (Nikon, Japan), we measured fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 505 nm. The fluorescence intensity of individual cells was measured using the Sigma Scan Pro 5 software (SPSS Inc., Chicago). In the control group, the fluorescence intensity was set at 100% viable.

Detection of Cyt c released from mitochondria

Cyt c released from mitochondria was measured by immunoblotting. Mitochondrial and cytosolic protein fractions were prepared using a Cell Mitochondria Isolation Kit (Beyotime, Jiangsu, China). Briefly, cells were collected and suspended in a mitochondrial isolation reagent A, containing phenyl methyl sulfonyl fluoride and other protease inhibitors. Then, the cell culture was vortexed for 5 s and incubated on ice for 15 min. Mitochondrial isolation reagent B was added, vortexed, and centrifuged at 14,000 rpm for 5 min; the temperature was maintained at 4℃ during this process. The supernatant was transferred to a fresh 1.5 mL microcentrifuge tube and was termed as the cytosolic fraction. Lactate dehydrogenase (LDH) served as a cytosolic marker. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was considered as the loading control.

Immunoblotting

Cells were harvested and lysed. Protein concentration was measured using a BCA Protein Assay Kit (Beyotime, Jiangsu, China). We boiled equal quantities of proteins, and then we separated them by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis. After separating these proteins, we electrophoretically transferred them into a nitrocellulose membrane. Membranes were blocked at RT for 1 h using tris-buffered saline containing 5% non-fat milk. Thereafter, they were incubated overnight at 4℃ along with specific primary antibodies (ornithine decarboxylase [ODC], spermidine/spermine N¹-acetyltransferase (SSAT), anticleaved caspase-3 and -9, Bcl-2, Cyt c, and GAPDH were purchased from Santa Cruz and anti-ERK1/2and PI3K–Akt–GSK-3β antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA). The membrane was washed thrice with 1 × Tris-buffer saline-Tween 20 (TBST) buffer. Subsequently, the membrane was incubated in TBST solution along with alkaline phosphatase-labeled secondary antibody; the incubated mixture was kept in a shaker for 2 h at RT. Finally, the membrane was washed thrice with TBST solution. Antibody–antigen complexes were detected using Western Blue® Stabilized Substrate for Alkaline Phosphatase (Promega, USA). The intensities of protein bands were quantified by a Bio-Rad ChemiDoc™ EQ densitometer and Bio-Rad Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis

The data were expressed as mean ± SE (n = 8 for each group). Each measurement was obtained by performing at least three independent experiments. Statistical comparisons were made using paired or unpaired t-tests or one-way ANOVA followed by Bonferroni post-test. Significance level was set at p < 0.05.

Changes of PAs metabolism under H/I condition

In this experiment, HPLC chromatogram was used to determine the content of PAs. Standard solutions were used as positive control. The peaks of the three derivatives were obtained at about 8, 17, and 37 min, respectively. Compared with the control group, the peak area of putrescine was greater, while that of spermidine and spermine was lower in the H/I group (p < 0.05 for all 3; Figure 2(a)). Furthermore, ODC is an enzyme that performs the first step of PA biosynthesis; SSAT is a key regulatory enzyme catabolizing PA. Compared with the control group, the expression of ODC was lower while that of SSAT was higher in the H/I group (both p < 0.05; Figure 2(b)). These results indicate that H/I inhibited the production of endogenous spermine. OPEN IN VIEWER

The effects of spermine on cell viability and apoptosis

Compared with the control group, cell viability was lower in the H/I group at 54 ± 3% (100%, p < 0.05; Figure 3(a)). In contrast, in the presence of spermine, cell viability increased to 79 ± 4% versus H/I group (p < 0.05). Compared with the control group, there was no significant difference in the control + Sp group. OPEN IN VIEWER

The extent of cellular injury was estimated from the leakage of LDH and CK (Figure 3(b)). With H/I treatment, extracellular LDH increased to 440 ± 13 U/L, while CK increased to 336 ± 16 U/L. In the control group, the corresponding values of LDH and CK were 145 ± 14 and 96 ± 6 U/L, respectively (p < 0.05). With spermine incubation, we significantly reduced the leakage of LDH and CK to 275 ± 15 and 235 ± 13 U/L, respectively. There was no significant difference between control + Sp group and the control group.

Flow cytometry was used to examine cardiomyocyte apoptosis (Figure 3(c)). Thus, we detect whether spermine altered cardiac apoptosis that was induced by H/I. The control group exhibited a low apoptotic rate of 3 ± 1%, while H/I significantly increased the rate to 42 ± 2% (p < 0.05). Compared with the H/I group, the apoptotic rate was reduced to 21 ± 2% in the presence of spermine (p < 0.05). In the absence of H/I, spermine did not influence the apoptotic rate.

We further confirmed the morphological changes in the nuclei by Hoechst 33342 staining (Figure 3(d)). In the H/I group, the number of apoptotic nuclei with typical features of fragmentation and condensation was significantly increased to (60 ± 2%) as compared to that in the control group (10 ± 1%, p < 0.05). In the H/I + Sp group, the percentage of apoptotic nuclei was lower as compared to that in the H/I group (36 ± 2%, p < 0.05). Compared to the control, there was no significant difference in the number of apoptotic nuclei of control + Sp group.

Spermine decreases calcium overload

Calcium overload occurred in the early stage of H/I, and we detected [Ca²⁺]_i by fluo-8 AM staining 24 h after H/I (Figure 4). Since the fluorescence intensity in the H/I group was 3.8 ± 0.2 a.u., it was significantly higher than that of control group (1.0 ± 0.1 a.u., p < 0.05). After administering spermine, the [Ca²⁺]_i was much lower (2.2 ± 0.1 a.u.) than H/I (p < 0.05). Spermine had no effect in the absence of H/I. OPEN IN VIEWER

Spermine increased Bcl-2 expression, decreased the expressions of cleaved caspase-3 and -9 expressions, and suppressed the release of Cyt c

The expression of Bcl-2 and cleaved caspase-3 and -9 decreased, while Cyt c release increased in the H/I group (p < 0.05 versus control; Figure 5). By spermine treatment, the effect of H/I was reversed (p < 0.05); however, the use of spermine had no effect in the absence of H/I. OPEN IN VIEWER

Spermine activates ERK1/2 and PI3K–Akt–GSK-3β pathway

To determine whether ERK1/2 and PI3K–Akt–GSK-3β pathways were activated by spermine in the presence of H/I, we cultured cardiomyocytes using spermine and a media conditioned with hypoxia (Figure 6). Compared with the control group, the levels of phosphorylated ERK1/2, PI3K, Akt, and GSK-3β were lower in the H/I group (p < 0.05). Spermine prevented H/I-induced changes of ERK1/2 and PI3K–Akt–GSK-3β pathways (p < 0.05); however, spermine had no effect in the absence of H/I. The total amount of ERK1/2, PI3K, Akt, and GSK-3β protein remained unchanged in different conditions. OPEN IN VIEWER

To determine whether ERK1/2 and PI3K–Akt–GSK-3β pathways were involved in spermine-inhibited apoptosis induced by H/I, we used PD98059 (an ERK1/2 inhibitor) and LY294002 (a PI3K inhibitor) to inhibit the signaling pathways (Figure 6). We found that the inhibitors of ERK1/2 and PI3K significantly suppressed phosphorylation of ERK1/2, PI3K, Akt, and GSK-3β, which was previously induced by spermine treatment.

Effects of spermine on ΔΨm and mPTP opening

As shown in Figure 7(a), cardiomyocytes exposed to H/I exhibited disruption of ΔΨm, a phenomenon that was not observed in the control group; this effect was detected in the cardiomyocytes with an increase in green fluorescence. However, a co-treatment with spermine served as a protection against this effect. The ratio of red and green fluorescence was used to determine the ΔΨm change induced by H/I treatment and the protective effect offered by Sp. In the control group, JC-1 aggregated in mitochondria at a ratio of 1.5 ± 0.1, while the H/I group was 0.7 ± 0.1 (p < 0.05 versus control group). The ratio was increased to 1.0 ± 0.1 following co-treatment with spermine (p < 0.05 versus H/I group), while PD98059 and LY294002 reversed the effect of spermine. These results indicate that the mitochondria-mediated pathway was involved in the cardioprotective effect of spermine. OPEN IN VIEWER

In different groups, there were also changes in the rates of mPTP opening in response to calcein AM. The fluorescence intensity in the control group was set at 100%. The level of fluorescence intensity was inversely proportional to the degree of mPTP opening. As shown in Figure 7(b), fluorescence intensity was reduced from 100 ± 3 to 32 ± 2% in the H/I group (p < 0.05 versus control). With the use of spermine, the fluorescence intensity increased to 77 ± 5% versus the H/I group (p < 0.05); however, spermine did not have any effect in the absence of H/I. Both PD98059 and LY294002 individually reversed an increase in fluorescence intensity that was observed with spermine treatment.