Interaction of nitric oxide synthase and mitochondrial ATP-sensitive potassium channels in protective impacts of combination therapy with irisin-preconditioning and melatonin-postconditioning in myocardial ischemia/reperfusion in aging

2.1 Animal care

Experimental protocols and animal handling were done following approval of the local ethical committee (ethical code: 7020–21) in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (8th Edition, NRC 2011), according to the ARRIVE guidelines for animal studies (Kilkenny et al., 2010). Male Wistar rats (n = 78, 400–450 g) at 22–24 months of age were provided by the animal center, and maintained in standard cages under a cycle of 12 h lightness and 12 h darkness and a controlled room temperature and humidity and were fed with tap water and standard pellet chow. The procedures were performed according to the eighth edition of “Guide for the Care and Use of Laboratory Animals” (NRC, 2011), and validated by the local Ethics Board (Ethical code: 07020018).

2.2 Rat model of myocardial I/R

After being anesthetized using sodium-pentobarbital (50 mg/kg, intraperitoneally), the animals were ventilated with room air using a small animal ventilator (Cwe, Inc, Ardmore, PA, USA). After the left thoracotomy, the heart was exposed. To induce regional ischemia, the left anterior descending (LAD) coronary artery was occluded using a ligature for 30 min. ST-elevation on the electrocardiograms and pale discoloration of myocardial tissue in the ischemic areas were considered as successful induction of ischemia. Following 30 min of ischemia, the ischemic myocardium was reperfused by removing the ligature for 3 h or 2 weeks. Rats in the sham group experienced a similar surgical procedure, without LAD ligation.

2.3 Experimental protocols

Aged rats were randomly allocated into the five experimental groups in the first protocol, and eight experimental groups in the second protocol (n = 6 per group). In the first protocol, the reperfusion lasted for 2 weeks, and echocardiographical evaluation of cardiac function and infarct size (IS) were performed. Collecting blood samples for measuring lactate dehydrogenase (LDH) and creatine kinase (CK) serum levels were accomplished after 24 h of reperfusion. Echocardiographic changes require more time to occur, so reperfusion time was 2 weeks to evaluate this goal. In the second protocol, the reperfusion lasted for 3 h, and then the animals were sacrificed for biochemical and molecular assessments.

The groups in the first protocol are as follows (with 2 weeks reperfusion):

• (1).

  Sham: without experiencing I/R;

• (2).

  I/R: rats experienced 30 min LAD occlusion and 2 weeks reperfusion;

• (3).

  I/R + Iri: rats were pretreated with an intraperitoneal injection of recombinant irisin (Cloud Clone, Wuhan, China) at 0.5 mg/kg/day for one week, and at the time point of 30 min before I/R surgery (Lu et al., 2020), then the rats experienced 30 min LAD occlusion and 2 weeks reperfusion;

• (4).

  I/R + Mel: rats underwent 30 min LAD occlusion and 2 weeks reperfusion plus intraperitoneal injection of melatonin (Sigma-Aldrich, St Louis, USA) at 20 mg/kg, 10 min before the onset of reperfusion (Zhai et al., 2017); and

• (5).

  I/R + Iri + Mel: the I/R-treated rats received both irisin and melatonin at their corresponding time points.

The groups in the second protocol are as follows (with 3 h reperfusion):

Groups 1–5 are similar to the first protocol, except that the rats in groups 2–5 experienced 30 min LAD occlusion and 3 h reperfusion.

• (6).

  I/R + Iri + Mel + 5HD: the I/R-treated rats received both irisin and melatonin at their corresponding time points, and 5HD (Sigma-Aldrich, St Louis, USA), as a mitoKATP channel blocker, was administrated at 10 mg/kg, five minutes before ischemia;

• (7).

  I/R + Iri + Mel + L-NAME: the I/R-treated rats received both irisin and melatonin at their corresponding time points, and L-NAME (Sigma-Aldrich, St Louis, USA), as an eNOS enzyme inhibitor, was administrated at 16 mg/kg, five minutes before the reperfusion; and

• (8).

  I/R + Iri + Mel + 5HD + L-NAME: the I/R-treated rats received both irisin and melatonin as well as both inhibitors, 5HD and L-NAME, at their corresponding time points as above.

It should be added that monotherapies had no significant effects on cardioprotection, so, 5HD and/or L-NAME were administered only in rats receiving combined therapy. In addition, according to pilot studies and previous reports (Lu et al., 2020; Zhai et al., 2017), the best cardioprotective effects of irisin and melatonin are achieved when they are administered pre-conditionally and post-conditionally, respectively. Therefore, in this study, these time points were considered for their injection.

2.4 Determination of heart function

Two weeks after reperfusion, echocardiography was carried out by a cardiologist who was blinded to the study design. The animals were reanesthetized, and assessment of the contractile function of the hearts was performed by Vevo 2100 system (VisualSonics, Toronto, ON, Canada). The measurement of ejection fraction (EF), left ventricular end-diastole inner diameter (LVIDd), left ventricular end-systole inner diameter (LVIDs), and fractional shortening (FS) was performed from three consecutive cardiac cycles.

2.5 Measurement of myocardial infarct size

Following completion of the cardiac function assessment, the Evans blue and triphenyl tetrazolium chloride (TTC) double-staining method was applied for the determination of the areas at risk (AAR) and IS. The ligature around the LAD was re-ligated, and the injection of 2 ml Evans blue (2%) was performed through the right jugular vein. The hearts were removed, frozen, sectioned, and stained with 1% TTC. Consequently, transverse sections of the left ventricles (1 mm thickness) were photographed. The AAR and IS were calculated by ImageJ software (Ver. 7.0, CA, USA). A percentage of the left ventricle was reported as an AAR, and a percentage of AAR was reported as IS.

2.6 Measurement of LDH and CK

After 24 h reperfusion, the blood samples were obtained from the rat's tail vein under anesthesia with sodium pentobarbital (40 mg/kg, intraperitoneally). After the samples being centrifuged and the serum is collected, a colorimetric method was applied to determine LDH and CK serum levels, with commercial assay kits (Nanjingjiancheng Bioengineering, China), in accordance with the protocol provided by the manufacturer. The recorded values were reported in IU/l.

2.7 Detection of cardiac mitochondrial function

Cardiac mitochondria from the AAR of LV were isolated after 3 h of reperfusion. Following homogenization of fresh tissue in buffer (pH 7.4) containing 70 mmol/L sucrose, 210 mmol/L mannitol, and 1 mmol/L ethylene-diamine-tetra-acetic acid (EDTA) in 50 mmol/L Tris/HCl, the samples were centrifuged at 1300g for 3 min. To obtain isolated mitochondria, the collected supernatants were then centrifuged at 10000g for 10 min. The pellets were collected and resuspended in a storage buffer (100 µL, pH 7.4) containing 70 mmol/L sucrose, and 210 mmol/L mannitol in 50 mmol/L Tris-HCl. To determine mitochondrial protein concentration, the Bicinchoninic Acid (BCA) assay was performed. Using a ROS assay kit (Beyotime, Shanghai, China), cardiac mitochondrial ROS production was measured by di-chlorohydro-fluorescein-di-acetate (DCFDA) dye (Yarana et al., 2012). By a mitochondrial membrane potential assay kit (Sigma, Germany) with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine-iodide (JC-1), the changes in mitochondrial membrane potential were determined according to the manufacturer’s protocol. Decreased red/green fluorescence intensity ratio was reported as depolarization of mitochondrial membrane (Yarana et al., 2012).

2.8 Determination of pro-inflammatory cytokines

Rat-specific enzyme-linked immunosorbent assay (ELISA) kits (MyBioSource Inc., USA) were applied to measure the levels of cytokines IL-6, IL-1β, and TNF-α in tissue homogenates, based on the manufacturer’s method. The concentrations of these cytokines were quantified by reference to standard curves. The relative absorbance of cytokines was read at 450 nm by a spectrophotometer, and the results were normalized and reported as pg/mg of protein in each sample.

2.9 Western blotting

Western blotting was conducted according to previous work (Palee et al., 2020). The concentrations of proteins from AAR samples were determined by applying an assay kit (Bio-Rad Laboratories, USA). Proteins (50 μg) from AAR samples were loaded for electrophoresis. Thereafter, transferring of proteins to nitrocellulose membranes was performed. The membrane was incubated for an hour at 23 °C and blocked with 5% skimmed milk, followed by overnight exposure to primary antibodies as follows: anti B cell lymphoma-2 (Bcl-2), anti-Bcl-2-associated × protein (Bax), anti cleaved caspase 3, anti endothelial nitric oxide synthase (eNOS), and anti-GAPDH (Cell Signaling Technology, USA). Then the membrane was exposed to a secondary antibody (Cell Signaling Technology, USA) for an hour at room temperature. Enhanced chemiluminescence substrate was applied to visualize bands. Alpha Ease ® FC Imaging System was used for band density analysis per their optical densities. Finally, the intensity of bands was normalized with GAPDH.

2.10 Measurement of NO

Griess colorimetric method was applied for measuring NO levels following the NO assay kit protocol (Thermo-Fisher Scientific, Germany) (Castillo et al., 2000). Color development with Griess reagent (N-naphthyl ethylene diamine and sulfanilamide) in an acidic medium following nitrate conversion into nitrite by copperized cadmium granules is the principle of this method. The calibration of this assay was performed by standard solutions of sodium nitrite. Employing a 96-well microplate reader, the optical densities of NO metabolites were measured at 540 nm following 3 h incubation time, and the results were reported as µmol/mg protein.

2.11 Statistical analysis

The values were reported in means ± standard deviation (SD). Data analysis was performed with SPSS statistical software (Version 15.0, SPSS Inc., Chicago, IL). One-way analysis of variance (ANOVA) followed by Tukey post hoc test was employed to determine multiple comparisons between groups. P-value <0.05 was considered statistically different.

3.1 Effect of combined conditioning on cardiac function of the aged rats

Our data revealed that induction of 30 min ischemia and 2 weeks reperfusion significantly decreased EF and FS in comparison with the Sham group (P < 0.01). Irisin preconditioning or melatonin postconditioning could not significantly increase EF and FS when compared with the I/R group. Nevertheless, combined therapy with irisin and melatonin significantly increased these parameters as compared to I/R (P < 0.01), I/R + Iri, and I/R + Mel (P < 0.05) groups (Fig. 1A and 1B).

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Fig. 1

The changes of EF (A); FS (B); LVIDs (C); and LVIDd (D); and representative echocardiogram recordings (E) in experimental groups (n = 6 per group). The data were expressed as mean ± SEM. (** P < 0.01 vs. Sham group, ## P < 0.01 vs. I/R group, $ P < 0.05 vs. I/R + Iri group, @ P < 0.05 vs. I/R + Mel group). I/R: Ischemia/reperfusion; Iri: Irisin; Mel: Melatonin; EF: Ejection fraction; FS: Fractional shortening; LVIDs: Left ventricular inner diameter in end systole; LVIDd: Left ventricular inner diameter in end diastole.

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Fig. 1C and 1D show that LVIDs and LVIDd in the I/R group were significantly higher than the Sham group (P < 0.01 for both). In addition, individual treatments could not significantly decrease LVIDs and LVIDd in comparison with the I/R group. Combined therapy with irisin and melatonin caused a significant reduction in LVIDs and LVIDd when compared to I/R (P < 0.01 for both), I/R + Iri, and I/R + Mel (P < 0.05 for both) groups.

3.2 Effect of combined conditioning on AAR and IS in the aged heart

As shown in Fig. 2A, the AAR was similar among all groups. The expansion of IS to 53.67 ± 5.03% was detected in the I/R group as compared to the zero IS in the Sham group (P < 0.001). Alone treatments could not significantly reduce the IS in comparison to the I/R group. The IS was significantly reduced to 20.33 ± 2.50% in the group receiving combined therapy as compared to I/R, I/R + Iri, and I/R + Mel groups (P < 0.01 for all) (Fig. 2B).

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Fig. 2

The percentages of area at risk (AAR) (A), and infarct size (IS) (B) in experimental groups (n = 6 per group). The data were expressed as mean ± SEM. (*** P < 0.001 vs. Sham group, ## P < 0.01 vs. I/R group, $$ P < 0.01 vs. I/R + Iri group, @@ P < 0.01 vs. I/R + Mel group). I/R: Ischemia/reperfusion; Iri: Irisin; Mel: Melatonin.

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3.3 Effect of combined conditioning on CK and LDH serum levels in the aged heart

The CK and LDH serum levels were increased after 24 h of reperfusion when compared to the Sham group (P < 0.001). Only combined therapy with irisin and melatonin caused a significant reduction in CK and LDH serum levels when compared to I/R, I/R + Iri, and I/R + Mel groups (P < 0.01 for both) (Fig. 3A and 3B).

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Fig. 3

Serum levels of CK (A), and LDH (B) in experimental groups (n = 6 per group). The data were expressed as mean ± SEM. (*** P < 0.001 vs. Sham group, ## P < 0.01 vs. I/R group, $$ P < 0.01 vs. I/R + Iri group, @@ P < 0.01 vs. I/R + Mel group). I/R: Ischemia/reperfusion; Iri: Irisin; Mel: Melatonin; CK: Creatine kinase; LDH: lactate dehydrogenase.

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3.4 Effect of combined conditioning on mitochondrial function in the aged heart and its reversal by 5HD and L-NAME

The production of mitochondrial ROS and the alterations of mitochondrial membrane potential were evaluated as indicators of mitochondrial function following 3 h reperfusion (Fig. 4A and 4B). Induction of I/R led to increased production of mitochondrial ROS, as well as the enhanced ratio of red to green fluorescent intensity when compared with those of the Sham rats (P < 0.01 for both). The levels of mitochondrial ROS and membrane potential did not differ between monotherapy groups and those of non-treated rats. However, these mitochondrial functional indices were significantly declined in rats receiving combined therapy in comparison to the I/R group (P < 0.05 for both). Since monotherapies had no significant effects on cardioprotection, 5HD and/or L-NAME were administered only in rats receiving combined therapy. The results demonstrated that the beneficial effects of combination therapy on mitochondrial ROS production and membrane potential were eliminated following 5HD administration (P < 0.05). Interestingly, administration of L-NAME was able to partially prevent the beneficial effects of combination therapy on mitochondrial indices (P < 0.05). The concomitant administration of these two inhibitors had a complete inhibitory effect on cardioprotection induced by the combined therapy (P < 0.001).

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Fig. 4

Mitochondrial ROS production in the heart assessed by DCFH-DA dye (A), and the alterations of mitochondrial membrane potential in the heart assessed by JC-1 dye (B) (n = 6 for each group). The data were expressed as mean ± SEM. (** P < 0.01 vs. Sham group, # P < 0.05 vs. I/R group, & P < 0.05, && P < 0.01, and &&& P < 0.001 vs. I/R + Iri + Mel group). I/R: Ischemia/reperfusion; Iri: Irisin; Mel: Melatonin; 5HD: 5-hydroxydecanoate; L-NAME: NG-nitro-L-arginine methyl ester; ROS: Reactive oxygen species.

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3.5 Effect of combined conditioning on inflammatory response in the aged heart and its reversal by 5HD and L-NAME

As shown in Fig. 5A-C, the myocardial levels of IL-1β, IL-6 and TNF-α did not differ between monotherapy and I/R groups. Nevertheless, the levels of these cytokines were significantly decreased in the group receiving combined therapy as compared to the I/R group (P < 0.05 for all). Co-administration of 5HD or L-NAME significantly attenuated the favorable effects of combination therapy on the levels of inflammatory cytokines (P < 0.05), however, the effect of 5HD in eliminating the inhibitory effects of combined therapy on these inflammatory cytokines was more potent as compared to L-NAME (P < 0.05). In addition, the administration of both 5HD and L-NAME at the same time completely neutralized the inhibitory effects of combined therapy on inflammatory cytokines (P < 0.001, and P < 0.01).

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Fig. 5

The levels of IL-1β (A), IL-6 (B), and TNF-α (C) in experimental groups (n = 6 per group). The data were expressed as mean ± SEM. (** P < 0.01 and *** P < 0.001 vs. Sham group, # P < 0.05 vs. I/R group, & P < 0.05, && P < 0.01, and &&& P < 0.001 vs. I/R + Iri + Mel group). I/R: Ischemia/reperfusion; Iri: Irisin; Mel: Melatonin; 5HD: 5-hydroxydecanoate; L-NAME: NG-nitro-L-arginine methyl ester; IL: Interleukin; TNF-α: Tumor necrosis factor α.

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3.6 Effect of combined conditioning on apoptosis response in the aged heart and its reversal by 5HD and L-NAME

As shown in Fig. 6A-C, upregulation of Bax and cleaved caspase-3, and downregulation of Bcl-2 were observed following 30 min ischemia and 3 h reperfusion (P < 0.01 for all). Monotherapies had no significant influences. Simultaneous application of irisin preconditioning and melatonin postconditioning significantly suppressed Bax and cleaved caspase-3 expressions, and enhanced Bcl-2 expression, in comparison with the I/R group (P < 0.05 for all). The effect of combination therapy on the expressions of Bax, Bcl-2, and cleaved caspase-3 was reduced following 5HD or L-NAME administration (P < 0.05), however, L-NAME was not as potent as 5HD in inhibiting the combination effect on apoptosis response (P < 0.05). The concomitant administration of these two inhibitors had a complete inhibitory effect on the antiapoptotic response induced by the combined therapy (P < 0.01, and P < 0.001).

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Fig. 6

The expressions of Bax (A), Bcl-2 (B), and cleaved caspase 3 (C), and their representative bands correlated to the GAPDH band in experimental groups (n = 6 per group). The data were expressed as mean ± SEM. (** P < 0.01 vs. Sham group, # P < 0.05 vs. I/R group, & P < 0.05, && P < 0.01, and &&& P < 0.001 vs. I/R + Iri + Mel group). I/R: Ischemia/reperfusion; Iri: Irisin; Mel: Melatonin; 5HD: 5-hydroxydecanoate; L-NAME: NG-nitro-L-arginine methyl ester; Bax: Bcl-2 associated × protein; Bcl-2: B-cell lymphoma 2.

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3.7 Effect of combined conditioning on eNOS/NO pathway in the aged heart and its reversal by 5HD and L-NAME

In comparison to the Sham group, significant reductions in the expression of eNOS (P < 0.01) and the levels of NO (P < 0.05) were detected in the I/R group. Monotherapies had no significant effects on eNOS expression and NO levels in I/R rats. However, combined therapy with irisin and melatonin significantly increased eNOS expression and NO level when compared to the I/R group (P < 0.05 for both). These positive effects of combination therapy on eNOS expression and NO levels were eliminated following 5HD administration (P < 0.05 for both). Administration of L-NAME partially prevented the positive effects of combination therapy on eNOS expression and NO levels (P < 0.05). The concomitant administration of these two inhibitors had a complete inhibitory effect of combined therapy on these parameters (P < 0.01 for both) (Fig. 7A and B).

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Fig. 7

The expression of eNOS correlated to the GAPDH band (A), and the levels of NO (B) in experimental groups (n = 6 per group). The data were expressed as mean ± SEM. (* P < 0.05 and ** P < 0.01 vs. Sham group, # P < 0.05 vs. I/R group, & P < 0.05 and && P < 0.01 vs. I/R + Iri + Mel group). I/R: Ischemia/reperfusion; Iri: Irisin; Mel: Melatonin; 5HD: 5-hydroxydecanoate; L-NAME: NG-nitro-L-arginine methyl ester; eNOS: Endothelial nitric oxide synthase; NO: Nitric oxide.

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