Inbred male Lewis rats (8-12 week-old; Charles River Laboratories, Beijing, China) received care in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1996). All procedures and handling of animals during the investigations were reviewed and approved by the Ethical Committee of Laboratory Animal Research Center of Southern Medical University Nanfang Hospital. The animals were housed at constant ambient temperature (22±2 °C) in light-controlled rooms (12-12 hr light-dark cycles), were given food and water access ad libitum, and acclimatized for 1 week.
Rats were randomly assigned to three groups. Donor hearts were arrested and stored for 1 hr in either cold Custodiol (Dr. Franz Köhler, Chemie GmbH, Bensheim, Germany) supplemented with a serum-free medium vehicle (α-MEM) (Vehicle group, 8 rats), or Custodiol supplemented with normoxic CdM-BMSCs (N-CdM group, 8 rats), or Custodiol supplemented with hypoxic CdM-BMSCs (H-CdM group, 8 rats). Then, the donor hearts were heterotopically transplanted.
Isolation and culture of BMSCs
As previously reported(15), BMSCs were harvested from the bone marrow of 8-week-old male Lewis rats. Briefly, rats were euthanized with an overdose of pentobarbital sodium (100 mg/kg, intraperitoneally). Bone marrow was isolated by flushing femurs and tibias with phosphate-buffered saline (PBS, Life Technologies, Grand island, NY, USA). The cells were suspended in MEM Alpha basic(1X) (α-MEM, Life Technologies, Grand island, NY, USA) supplemented with 10% fetal bovine serum (Life Technologies, Grand island, NY, USA), 1% penicillin-streptomycin (Life Technologies, Grand island, NY, USA), and then incubated at 37°C with 5% CO2 on culture flasks. The primary culture was subcultured at a ratio of 1:2 when 80% confluency was reached. Only the third passage was used in subsequent experiments.
Preparation of hypoxic and normoxic CdM-BMSCs
Normoxic and hypoxic CdM-BMSCs were acquired as described previously with slight modifications(19). A simplified schematic of the CdM-BMSCs collection protocol is shown in Figure 1. After BMSCs reached greater than 80% confluency at Passage 3, the medium was aspirated, and BMSCs were rinsed 3 times with PBS. Then, α-MEM was added to culture flasks with BMSCs, and the culture flasks were put into an incubator under the normoxic or hypoxic conditions for 24 hr. For the normoxic group, BMSCs were cultured at 37°C in a humidified atmosphere containing 5% CO2 and 20% O2. The hypoxic group was cultured at 37°C in 1% O2, 5% CO2, and 94% N2 in a hypoxic incubator (Galaxy 48, Eppendorf, Shanghai, China). The primary CdM was collected after incubation for 24 hr, and the cell debris was removed using a syringe filter (Millex‐GP; Millipore, Burlington, MA, USA). The primary CdM-BMSCs was then sequentially concentrated to 15-fold by the centrifugation through Amicon Ultra Centrifugal Filter (5,000g for 2 hr at 4 °C, Amicon Ultra‐15 3K, Millipore, Burlington, MA, USA) according to the manufacturer’s protocol. Finally, the novel donor heart preservation solution was produced by diluting 300 ul α-MEM (Vehicle group), concentrated normoxic (N-CdM group) or hypoxic CdM-BMSCs (H-CdM group) from the filtrate tube of the top unit in 2700 ul Custodiol cardioplegic solution as indicated in Figure 1. α-MEM was regarded as a control (nonconditioned medium).
Screening of Secreted Proteins in CdM-BMSCs
The cytokines/chemokines of isolated CdM-BMSCs were measured by using a G‐series rat cytokine array 67 (Raybiotech, Norcross, GA, USA) according to the manufacturer’s instructions. The Gene Ontology (GO) analysis (R package “org.Hs.eg.db” and “clusterProfiler”) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (R package “clusterProfiler”) were performed using the open-source program R (version 3.5.1) to investigate the biological process categories and pathways of proteins that were highly expressed between hypoxic and normoxic CdM-BMSCs, as previously described(25).
The rat model of heterotopic heart transplantation
Surgical technique of heart transplantation
As described elsewhere(24, 26), heterotopic heart transplantations were performed in an isogenic Lewis-to-Lewis rat strain model to exclude the effects of immune rejections and avoid the application of immunosuppressors such as cyclosporine.
As shown in Figure 2A, heart explantation was performed as follows. Briefly, donor rats were anesthetized with pentobarbital sodium (60 mg/kg, intraperitoneally) and only allowed to be operated on when no palpebral relex of donor rats was observed. After that, both the abdominal artery and inferior vena cava were exposed by careful dissection with the cotton swab after the abdominal cavity was opened. 0.6 ml saline with a high dose of heparin (6250 IU/kg) was slowly injected via inferior vena cava. Abdominal artery cannulation was performed after heparinization. The chest cavity of the donor rat was open, and the inferior vena cava was cut. Then, the donor heart was immersed completely with ice and cold Custodiol cardioplegic solution was slowly and retrogradely perfused into the donor heart via the abdominal artery within 3 minutes. After that, inferior and superior vena cava, pulmonary veins were ligated and cut. Aorta and pulmonary artery were dissected and cut, respectively. Finally, the donor heart was taken out from the chest cavity. 1 ml cold Custodiol solution supplemented with α-MEM or CdM-BMSCs was applied to perfuse slowly and retrogradely the donor heart via the aorta. After that, the donor heart was statically stored in the Custodiol solution supplemented with α-MEM or CdM-BMSCs (2700 ul Custodiol solution with 300 ul α-MEM or CdM-BMSCs) at 0 °C to 4 °C for 60 minutes.
The degree of rat cardiac allografts injury correlates with increased cold ischemia time(27), therefore, to minimize the variability among experiments, the duration between harvest, cold storage, and reperfusion was standardized to 110 minutes (10 minutes (harvest of donor hearts) + 60 minutes (cold storage of donor hearts) + 40 minutes (heart transplantation)) as previously described (28).
As shown in Figure 2B, heterotopic abdominal heart transplantation was performed as follows. Recipient rats were anesthetized with isoflurane (5% for induction and 2% for maintenance of anesthesia). Firstly, left external jugular vein canulation was performed in the recipient rat so that 1 ml saline with a low dose of heparin (250 IU/kg) could be administered. Both the abdominal artery and inferior vena cava were exposed by careful dissection with the cotton swab after the abdominal cavity was opened. 1.5 cm abdominal artery and inferior vena cava below the level of the renal artery were carefully clamped by two delicate vessel clamps. All the branches of both the abdominal artery and inferior vena cava were tightly ligated. At the end of 60-minute cold storage of donor hearts, 3 mm incisions were made in the abdominal artery and inferior vena cava. Then, the donor heart was wrapped by the moist gauze with cold Custodiol solution supplemented with α-MEM or CdM-BMSCs and placed next to the abdominal artery. Finally, end-to-side anastomoses were performed between the aorta and the abdominal aorta and between the pulmonary artery and the inferior vena cava with 8-0 prolene suture under the microscope. The vessel clamps were carefully released to reperfuse the donor heart after the completion of anastomoses. In-vivo reperfusion lasted for 1.5 hr after the donor heart was heterotopically transplanted into the recipient rat. The time to return of spontaneous contraction of donor hearts immediately after successful heart transplantation was also recorded. Measurement of LV graft function and sample collection for histologic and molecular analyses were performed after 1.5 hr reperfusion. The experimental protocol is shown in Figure 2C.
Functional measurement in the graft
As previously reported (29), at 1.5 hr after transplantation, transabdominal echocardiographic imaging was performed by a blinded observer using a dedicated Vevo® 2100 System (21 MHz broadband sector transducer, Visualsonics Inc., Toronto, Canada). Ejection fraction (EF), left ventricular internal diameter at end-diastole (LVIDd), and at end-systole (LVIDs), stroke volume (SV), heart rate (HR), and cardiac output (CO) were measured between the anterior wall and the posterior wall of the left ventricle from the short-axis view at the level of papillary muscles from M-mode recordings. For each measurement, three consecutive cardiac cycles were traced and averaged. Fractional shortening (FS) was calculated as [(LVIDd - LVIDs)/ LVIDd] x 100 (%).
Histology and immunohistochemistry in the graft
The donor hearts were explanted after the functional measurements. Pieces of myocardial tissue were fixed immediately in paraformaldehyde solution (4%) and embedded in paraffin. Five-micron-thick slices of myocardium were stained with hematoxylin and eosin. The pathology changes of each heart slice were evaluated by scoring according to the grades 0-4(30): 0: Nil, 1: Minimum (Focal myocytes damage), 2: Mild (Occasionally disordered myocardial fibers with multifocal myofibrillar degeneration and inflammatory process), 3: Moderate (Diffuse inflammation and/or comprehensive myofibrillar degeneration with wave-shaped myocardial fibers and shed nuclei), 4: Severe (Diffuse inflammatory process with myocardial necrosis: The nuclei shrink and the cells are severely damaged). We selected four random and nonoverlapping visual fields for each heart slice under a light microscope, and the average histopathological score of four different fields was calculated for each sample in a blinded way.
Detection of DNA strand breaks in the graft
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to detect DNA-strand breaks of donor hearts after functional measurements was performed as previously described(23). The number of TUNEL-positive cells was counted under a fluorescence microscope and the final results were expressed as the ratio of 4′,6-diamidino-2-phenylindole (DAPI)-TUNEL double-labeled nuclei to the total number of nuclei stained with DAPI.
Proinflammatory cytokines and markers of myocardial injury
Blood samples from the recipient rats were obtained at 1.5 hr after reperfusion just before the sacrifice of rats. Blood samples were drawn from the abdominal aorta. Then, plasma samples were obtained after centrifugation (3000 rpm, 15 min, 4°C). The levels of proinflammatory cytokines (Tumor necrosis factor (TNF)-α, Interleukin (IL)-1β, IL-6), and cardiac troponin I (cTnI) were measured via Rat ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA).
Myocardial protein expression was assessed by western blot as previously described(16). The ratio of phosphorylated-Akt to Akt, phosphorylated-Smad2 to Smad2, phosphorylated-Smad3 to Smad3, and phosphatidylinositol 3-kinase (PI3K) (1:1000 dilution, Cell Signaling Technology (Shanghai) Biological Reagents Company Limited, Shanghai, China) were calculated.
The results were expressed as mean ± standard error of the mean (SEM). GraphPad Prism 7.02 software (GraphPad Sofware, Inc., San Diego, CA, USA) was used to perform statistical analysis. Shapiro-Wilk test was performed to test the normality of data before statistical tests were applied. For data with normal distribution, a two-sample Student’s t-test was applied to analyze the difference between N-CdM and H-CdM groups. If the normality test failed, a nonparametric Mann-Whitney-test was used. One-way ANOVA followed by Tukey’s post-hoc-test was performed for multiple comparisons between three experimental groups. If the data failed the normality test, the nonparametric Kruskal-Wallis test followed by Dunn’s post-hoc-test was used. A value of p<0.05 was considered statistically significant.