Abstract
Background. Resveratrol (RES) is a polyphenolic compound and natural phytoalexin that plays a potential role in various human diseases. Studies have confirmed that RES has an important function in cardioprotection.
Objectives. To investigate the effect of RES on HO-1 protein expression in rat heart after different duration of hypothermic preservation.
Material and methods. The Langendorff model of isolated rat heart was used. After being stored in 4°C different Celsior solution for 9 h, Sprague–Dawley rats hearts were divided into 6 groups randomly: control group, 9 h group, 3 µM RES group, 10 µM RES group, 30 µM RES group, and 100 µM RES group. The morphological changes of cardiomyocytes were detected with the hematoxylin & eosin (H&E) staining using a light microscope. The mRNA and protein expression of HO-1 were detected using reverse-transcription polymerase chain reaction (RT-PCR) and western blotting.
Results. Compared with the control group, cardiomyocytes were obviously injured in the 9 h group and the protein and mRNA expression of HO-1 were obviously decreased. Compared with the 9 h group, the mRNA and protein expression of HO-1 were increased in dose-dependent manner in the 3 µM RES, 10 µM RES and 30 µM RES group. Compared with the 9 h group, rat myocardial injury was gradually alleviated in 3 µM RES, 10 µM RES and 30 µM RES groups. However, the rat myocardial injury in the 100 µM RES group showed no more obvious improvement than in the 30 µM RES group.
Conclusions. In the isolated rat heart, RES protects cardiomyocytes against hypothermic preservation injury through increasing HO-1 protein expression.
Key words: HO-1, RES, heart preservation
Background
Resveratrol (RES) is a polyphenolic compound and natural phytoalexin that plays a potential role in various human diseases.1 Studies have confirmed that RES has an important role in cardioprotection.2 Heme oxygenase-1 (HO-1) is a cytoprotective gene, also known as a graft survival gene, which has been used as a unique therapeutic target for organ transplantation in recent years.3 In this study, the isolated rat heart cryopreservation model was used to observe the effect of RES on cardiomyocytes in long-term cryopreserved hearts. We found that RES plays a protective role in isolated cryopreserved hearts by upregulating the expression of HO-1, which provides a theoretical basis for the effective improvement of donor heart preservation conditions in clinical practice.
Material and methods
Animals
Healthy male Sprague–Dawley (SD) rats of SPF grade, weighing 200–250 g, were provided by the Experimental Animal Center of Wenzhou Medical University (Wenzhou, China), with the animal use license No. SYXK (Zhejiang) 2010-0150. The animal work took place in Ultrasonic Imaging Department of First Affiliated Hospital of Wenzhou Medical University and was approved by the Ethics Committee of First Affiliated Hospital of Wenzhou Medical University.
Main reagents
Primary antibodies (HO-1 antibody, β-action antibody) were purchased from Abcam (Cambridge, UK). Resveratrol was purchased from Sigma-Aldrich (St. Louis, USA). The RIPA lysate was provided by Shanghai Biyuntian Biotechnology Co., Ltd (Shanghai, China). Polyvinylidene difluoride membrane (PVDF membrane) was purchased from Millipore (Burlington, USA). Both bicinchoninic acid (BCA) protein quantification kit and chemiluminescent substrate (SuperSignal West Pico enhanced substrate, ECL) were obtained from Pierce (Dallas, USA). Resveratrol was dissolved in dimethyl sulfoxide (DMSO) before use, and the final concentration in DMSO was always less than 0.1%.
Celsior solution was used for cardiac arrest and preservation, and its composition and content were as follows (mmol/L): NaOH 100, KCl 15, MgCl2 13, CaCl2 0.25, lactobionate 80, mannitor 60, histidine 30, glutamate 20, with pH 7.4, and osmolarity 320 mOsm/L.
Main methods
The preparation of hypothermic heart
preservation model
Rats were adapted to feeding for 1 week and randomized into the following 6 groups with 6 rats in each group.
1. Control group (blank group): Isolated rat hearts were taken out quickly and the samples were retained for detection;
2. 9 h group (Celsior cryopreservation group): Isolated rat hearts were preserved in Celsior solution at 4°C for 9 h;
3. 3 µM RES group: Isolated rat hearts were preserved in Celsior solution containing 3 µmol/L RES for 9 h;
4. 10 µM RES group: Isolated rat hearts were preserved in Celsior solution containing 10 µmol/L RES for 9 h;
5. 30 µM RES group: Isolated rat hearts were preserved in Celsior solution containing 30 µmol/L RES for 9 h;
6. 100 µM RES group: Isolated rat hearts were preserved in Celsior solution containing 100 µmol/L RES for 9 h.
Langendorff perfusion and cryopreservation of isolated heart was performed according to the following procedure.
Rats were fasted overnight with free access to water before surgery. The abdominal cavity of rats was fixed and incised in dorsal position after cervical dislocation. The heart and large vessels were promptly exposed by thoracotomy. We cut the pulmonary veins, and then quickly removed the heart. The blood was removed in Krebs–Henseleit (KH) solution at 4°C, and then transferred and fixed in Langendorff perfusion device rapidly. Conventional retrograde constant pressure perfusion (76 mm Hg) was performed with modified KH solution. The composition of modified KH solution was as below (mmol/L): NaCl 118.9, KCl 4.69, CaCl2 2.2, KH2PO4 1.17, MgSO4 1.17, NaHCO3 17.9, EDTA 0.03, and glucose 9.99, with a pH of 7.4. The temperature was always maintained at 37°C during perfusion, and the perfusate was saturated with 95% O2 and 5% CO2. After a balanced perfusion with KH solution for 30 min, perfusion with Celsior solution was performed at 4°C for less than 3 min. At the same time, the heart surface was cooled. After the cardiac arrest, the hearts in each group were stored in different Celsior preservation solutions at 4°C for 9 h for indicator detection.
Light microscopic observation of H&E staining
We observed the morphological changes of myocardial cells with hematoxylin & eosin (H&E) staining under light microscope. The full thickness myocardium of left ventricular apex was cut and fixed with 4% paraformaldehyde, dehydrated, embedded with paraffin, sectioned, stained with H&E, and then observed under light microscope for the structure and morphological changes of the myocardial tissue and myocardial fibers.
Gene expression assay
Reverse transcription polymerase chain reaction (RT-PCR) was used for the gene expression assay. Total RNA was extracted using Trizol method, and cDNA was obtained with reverse transcription using Thermo Fisher reverse transcription kit (Thermo Fisher Scientific, Waltham, USA) according to the procedure. The PCR products were separated using a 2% agarose gel. Primer sequences can be seen in Table 1. The PCR reaction parameters were as follows: 1 μL of cDNA, 1 μL of each upstream and downstream primer, 12.5 μL of 2 × TaqPCR MasterMix, and deionized water with a final volume of 25 μL. Amplification was performed under the following conditions: initial denaturation – 94°C for 3 min; PCR reaction – 94°C for 1 min; annealing for 1 min 30 s; and extension at 72°C for 5 min. Number of cycles was 33 and annealing temperature was 66°C.
Protein expression assay
We applied western blotting analysis for the protein expression assay. Specimens were lysed in RIPA lysis solution (1% phenylmethylsulfonyl fluoride (PMSF)) on ice, homogenized and centrifuged at 4°C to obtain the supernatant. Protein concentration was determined using the bicinchoninic acid assay (BCA) method. Thirty micrograms of protein were loaded, separated using 10% SDS-PAGE, transferred to PVDF membrane, blocked with 5% nonfat dry milk for 1 h, and then incubated with HO-1 primary antibody (1:500) at 4°C for 16–18 h. After washing on the next day, secondary antibody horseradish enzyme-conjugated goat anti-rabbit immunoglobulin G (IgG; 1:10,000) was added and shaken for 1 h at room temperature. Electrochemiluminescence (ECL) reaction, exposure, development, and fixation were performed in the darkroom after washing. We also employed the digital gel imaging system to take pictures, and then compared the gray values of target protein bands with internal reference β-action for the calculation of the relative gray values.
Statistical processing
All statistical analyses were performed using SPSS v. 18.0 software (SPSS Inc., Chicago, USA). All data was expressed as mean ± standard deviation (SD). Comparisons among multiple groups were conducted with one-way analysis of variance (ANOVA), and those between groups were carried out using the least significant difference (LSD) method.
Results
Myocardial cells in each group after cryopreservation detected with H&E staining
In the control group, the myocardial cells were arranged neatly, the cell morphological structure was basically normal and the nucleus was in the middle (Figure 1A). In the 9 h group, the myocardial cells became shrunk and deformed and were disorganized, with its nucleus bordered. The intercellular space was also widened (Figure 1B). Compared with the 9 h group, the 3 μM RES, 10 μM RES and 30 μM RES groups exhibited significantly improved myocardial cell damage with the increase of RES concentration. Among the groups, the damage improvement was more obvious in the 30 μM RES and 100 μM RES groups in comparison with the 10 μM RES group, with gradually normalized arrangement of myocardial cells, clearer boundaries, natural morphology, and abundant and uniform cytoplasm (Figure 1C–F). Meanwhile, no more significant improvement was observed in the 100 μM RES group compared to the 30 μM RES group (Figure 1E,F).
HO-1 gene expression in each group
HO-1 mRNA expression was remarkable in the cytoplasm of myocardial tissue in the control group and was significantly downregulated in the 9 h group (p < 0.01). Compared with the 9 h group, the expression of HO-1 mRNA in the 3 μM RES, 10 μM RES, 30 μM RES, and 100 μM RES groups gradually went up with the increase of RES concentration (p < 0.01; Figure 2).
HO-1 protein expression in each group
The condition of HO-1 protein expression was similar to the HO-1 mRNA expression in both control group and 9 h group (p < 0.01). In comparison with the 9 h group, the protein expression of HO-1 in the 4 different concentration RES groups was also gradually increasing with increasing RES concentration (p < 0.01; Figure 3).
Discussion
The key to a successful heart transplantation is to alleviate the hypothermic preservation injury of donor heart.4 Researchers have continued to adjust various components of heart preservation solution, such as the addition of oxygen free radical scavengers, calcium ion antagonists, plasma active ingredients, nutrient matrix, etc., all of which improved the damage of heart during cryopreservation to varying degrees.5, 6 Cell injury during long-term cryopreservation is a critical factor affecting organ recovery after transplantation.7 During simple hypothermic preservation, the intracellular and extracellular ion imbalance due to hypoxia leads to cellular edema.8 At the same time, intracellular anaerobic glycolysis is increased, intracellular acidosis occurs, and swelling and degeneration come up in cells due to ischemia and hypoxia. If effective drugs can be added to the preservation solution to reduce the cell damage caused by long-term cryopreservation, it may provide a new drug target for organ cryopreservation and improve the preservation effect of donor heart during heart transplantation.9 In this study, the purpose of adding RES in the preservation solution was to improve the damage of cells in the environment of hypothermic ischemia and hypoxia, reduce the damage caused by intracellular ion imbalance, acidosis and other changes, and try to maintain the state of cells in this condition.
Resveratrol has quite a wide range of biological activities, including anti-cancer, anti-apoptotic, anti-aging, anti-insulin resistance, anti-oxidant, anti-arteriosclerosis, anti-inflammatory, anti-platelet, and anti-estrogen bioactivities.10 It has positive significance for the prevention and treatment of heart disease, atherosclerosis, diabetes, and neurodegenerative diseases.11 Many studies have confirmed that RES plays an important role in cardioprotection.2, 12 The results showed that RES could protect suckling mice cardiomyocytes and human umbilical vein endothelial cells from injury.13 However, high concentration of RES (100 mM) produce toxicity on human umbilical vein endothelial cells.14 During cryopreservation, the morphological changes of myocardial cells can directly reflect the damage of myocardial tissue structure. The damage reflected by light microscopy results is manifested in the arrangement and boundary of myocardial cells, cell contour, cell edema, or cell shrinkage. As measured using light microscopy and H&E staining, in this experiment, myocardial cell injury was more obvious in the 9 h group compared with the control group. These results indicated that long-term hypothermic preservation could lead to severe cardiac injury in rats. The long-term hypothermic preservation model has been successfully constructed. However, in 3 μM RES, 10 μM RES, 30 μM RES, and 100 μM RES groups, myocardial cell injury was gradually alleviated compared with the 9 h group, which affirmed the protective role of RES during long-term hypothermic preservation of the heart. In the meantime, no significant improvement was observed in cardiac myocytes in the 30 µM RES and 100 µM RES groups. There are 2 possibilities for this phenomenon: 100 µM of RES exerted a protective effect on cardiac myocytes at a plateau close to 30 µM RES, or 100 µM of RES produced some toxicity to cardiac myocytes, partially counteracting its protective effect. More experiments need to be conducted to confirm whether higher concentrations are toxic to cardiomyocytes.
Heme oxygenase-1 has been found to be closely associated with many diseases.15, 16, 17 Its upregulation reduces graft injury and can prevent chronic graft function loss after transplantation, hence it is known as a graft survival gene. The HO-1 overexpression is cytoprotective in transplantation models such as rat heart.18 It has been found that RES can protect human hepatocytes from alcohol-induced injury to some extent, and its protective effect is associated with the induction of increased HO-1 enzyme activity.19 Inducing the upregulation of HO-1 expression can significantly reduce the injury of the transplanted liver and decrease the level of serum transaminases in the recipients,20 as well as dramatically prolong the duration of liver cryopreservation.21, 22 Therefore, the protective effect of HO-1 on liver transplantation was basically confirmed. However, there are only few studies on the effects of HO-1 on heart transplantation. Recombinant adeno-associated virus (rAAV)-mediated HO-1 gene transfer has been found to be a novel therapeutic approach for chronic allograft injury in clinical heart transplantation. In this experiment, the expression of HO-1 in the 9 h group compared with the control group was remarkably decreased, and H&E staining showed that the myocardial cell injury was obvious. This result proves the protective role of HO-1 and the regulation of HO-1 by RES is involved in the protective effect of long-term hypothermic preservation of the heart.
Conclusions
We believe that RES plays a pivotal role in protecting the isolated cryopreserved hearts by upregulating HO-1.