Male Sprague-Dawley rats (SPF grade; age, 6 weeks; weight, 180-220 g; n=30) were fed under standard laboratory conditions with a temperature of 27°C, 40-60% humidity and 12-h light/dark cycle. The rats were acclimated to these conditions for 7 days and provided with free access to water and food. The rats were then anesthetized with 1% pentobarbital sodium (30 mg/kg) by intraperitoneal (i.p.) injection, and the myocardial I/R model was established following the surgical protocol of a previous study (10). A total of 21 rats were successfully induced in the I/R model with a survival rate of 91.3% (21/23), which was similar to previous studies (14,15); ischemia time may be the cause of death of two rats. All rats were randomly divided into 4 groups (n=7 rats/group): i) Control group (Sham operation), in which open heart surgery was performed but the anterior descending branch of the coronary artery was not ligated; ii) I/R group; iii) I/R + lentiviral short hairpin-RNA-NC (Lv-sh-NC) group; and iv) I/R + Lv-sh-GADD45A group. Lentiviral vectors containing shRNA targeting GADD45A (Lv-sh-GADD45A, 5′-CGC AGA GCA GAA GAT CGA AAG-3′) and the Lv-sh-NC (5'-TTC TCC GAA CGT GTC ACG T-3') were constructed by Hanbio Biotechnology Co., Ltd. During ligation, 20 µM lentivirus vectors (4×10⁹ IFU/ml) were injected into the pericardial tissue of rat hearts at four different places around the infarcted area. No obvious side effects were found following I/R surgery and administration of si-GADD45A.

At 7 days post-I/R surgery, the rats were anesthetized with 1% pentobarbital sodium (30 mg/kg; i.p.) and rapid thoracotomy was performed. Blood (10 ml) was collected from the apex of the heart and serum was separated by first letting the collected blood stand at 37°C for 30 min, and then centrifuged at 1,006 × g at 4°C for 15 min to detect the levels of creatine kinase (CK), lactate dehydrogenase (LDH) and NO (described below). Next, the rats were euthanized by the i.p. injection of 1% pentobarbital sodium (150 mg/kg) and pre-cooled saline was injected into the apex of the heart through an intravenous infusion device for irrigation. Cessation of breathing for 3 min was used to verify death. After full irrigation, the quickly separated heart was washed in saline, placed on ice and dried with absorbent paper. The myocardial tissue was collected for 2,3,5-triphenyltetrazolium chloride (TTC) staining and other experiments, as described below.

Animal experiments were approval of the animal care and ethics committee of Yan'an Hospital Affiliated to Kunming Medical University (Kunming, China) and performed following the ARRIVE guidelines (https://www.nc3rs.org.uk/arrive-guidelines). The humane endpoints considered in this experiment were as follows: i) The animals showed mental depression accompanied by hypothermia (<37°C) in the absence of anesthesia; ii) the experiments were terminated before the earliest indicator if an animal experienced severe pain. Any animals reaching these endpoints were to be euthanized with 1% pentobarbital sodium (150 mg/kg; i.p.).

Total RNA was extracted from myocardial tissues or CMECs using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instruction. cDNA was synthesized from the total RNA using the PrimeScript™ RT Reagent kit (cat. no. RR037A; Takara Bio, Inc.) according to the manufacturer's instructions. qPCR was conducted using the SYBR^® Premix EX Taq™ kit (Takara Bio, Inc.) and the following thermocycling conditions: Initial denaturation for 5 min at 94°C; followed by 40 cycles of denaturation for 20 sec at 94°C, annealing for 20 sec at 65°C and elongation for 30 sec at 70°C. The following primer sequences were used: Rat GADD45A forward, 5′-TAA GCA AGA AGC CGG CAA GA-3′ and reverse, 5′-GGG TCT ACG TTG AGC AGC TT-3′; human GADD45A forward, 5′-CGA AAG GAT GGA TAA GGT G-3′ and reverse, 5′-GGA TCA GGG TGA AGT GGA-3′; GAPDH (rat) forward, 5′-TGT GAA CGG ATT TGG CCG TA-3′ and reverse, 5′-GAT GGT GAT GGG TTT CCC GT-3′; and GAPDH (human) forward, 5′-GCA CCG TCA AGG CTG AGA AC-3′ and reverse, 5′-GGA TCT CGC TCC TGG AAG ATG-3′. mRNA expression levels were quantified using the 2^−ΔΔCq method and normalized to the internal reference gene GAPDH (16).

Total proteins were isolated from rat myocardial tissue and cell samples using RIPA (Beyotime Institute of Biotechnology) and semi-quantified using a BCA kit (Beyotime Institute of Biotechnology). Proteins (20 µg/lane) were separated on 12% gels by SDS-PAGE and transferred to PVDF membranes (cat. no. FFP24; Beyotime Institute of Biotechnology). PVDF membranes were blocked with 5% skimmed milk at room temperature for 2 h and then incubated at 4°C overnight with primary antibodies against: GADD45A [1:1,000; cat. no. 4632; Cell Signaling Technology, Inc.(CST)], CD31 (1:1,000; cat. no. ab281583; Abcam), phosphorylated (p)-eNOS (1:1,000; cat. no. bs-3589R; BIOSS), eNOS (1:1,000; cat. no. ab300071; Abcam), entothelin-1 (ET-1; 1:1,000; cat. no. ab2786; Abcam), p-p38 MAPK (1:1,000; cat. no. bs-5476R; BIOSS), p38 MAPK (1:1,000; cat. no. bs-0637R; BIOSS), p-JNK (1:1,000; cat. no. 4668; CST), JNK (1:1,000; cat. no. 9252; CST), early growth response 1 (EGR1; 1:1,000; cat. no. 4154; CST), p-STAT3 (1:1,000; cat. no. ab32143; Abcam), STAT3 (1:1,000; cat. no. ab68153; Abcam), VEGF (1:1,000; cat. no. 19003-1-AP; Proteintech Group, Inc.), BCL2 (1:1,000; cat. no. ab196495; Abcam), Bax (1:1,000; cat. no. ab32503; Abcam), cleaved caspase 3 (1:1,000; cat. no. 9661; CST) and GAPDH (1:10,000; cat. no. ab181602; Abcam). Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit (1:2,000; cat. no. ab6721; Abcam) or goat anti-mouse (1:2,000; cat. no. ab6789; Abcam) secondary antibodies at 4°C for 1 h. BeyoECL Plus (Beyotime Institute of Biotechnology) was used to visualized the protein bands, and densitometric analysis was conducted using ImageJ 1.8.0 software (National Institutes of Health).

The heart slices (1.5 mm) were firstly placed in TTC solution (Sigma-Aldrich; Merck KGaA) with pH of 7.4 at 37°C for 15 min, and then fixed in 4% paraformaldehyde for 24 h at room temperature. The color of the normal myocardium became red, and that of the ischemic myocardium was gray. The MI area was calculated using an EOS 90D digital camera (Canon, Inc.) to capture images and the staining was quantified by ImageJ2x software (National Institutes of Health).

Serum (100 µl) was used to determine the contents of LDH (Beyotime Institute of Biotechnology) and CK (Sigma-Aldrich; Merck KGaA) in rats according the manufacturer's protocols. NO level in the serum was detected using an NO kit (cat. no. S0021S; Beyotime Institute of Biotechnology) in line with the kit instructions. CK, LDH and NO levels were examined using a CHEMIX-180 automatic biochemistry analyzer (Sysmex Corporation).

The myocardial tissues were fixed in 4% paraformaldehyde at room temperature. After 12 h, the tissues were dehydrated in an ascending gradient of ethanol and then made transparent with xylene. Next the tissues were embedded in paraffin wax and sliced into 4-µm-thick sections. Finally, the sections were stained with hematoxylin for 5 min at 4°C and then with eosin for 3 min at 4°C. H&E staining was observed under a BX43 light microscope (Olympus Corporation).

Cardiac tissues and CMECs were fixed with 4% paraformaldehyde at room temperature for 12 h and for 20 min, respectively, and then permeabilized with 0.5% Triton X-100 at room temperature for 5 min. The 5-µm-thick sections and CMECs were blocked with 5% normal goat serum (Beijing Solarbio Science & Technology Co., Ltd.) for 1 h at room temperature, incubated at 4°C overnight with primary antibodies against CD31 (1:1,000; cat no. 28083-1-AP; Proteintech Group, Inc.) and subsequently incubated with goat anti-rabbit Alexa Fluor^® 488 IgG secondary antibody (1:100; cat. no. ab150077; Abcam) or goat anti-rabbit Alexa Fluor^® 555 IgG secondary antibody (1:200; cat. no. ab150078; Abcam) at 37°C for 1.5 h in the dark. At least five randomly selected fields were examined using an IX73 inverted fluorescence microscope (Olympus Corporation).

Rat hearts were fixed at room temperature for 24 h in 4% paraformaldehyde and dehydrated with 30% sucrose at room temperature for 24 h. Subsequently, the hearts were embedded in paraffin and then sliced into 4-5 µm sections. Apoptotic cells in the heart tissues were stained using a One-step TUNEL Apoptosis Detection kit (cat. no. C1086; Beyotime Institute of Biotechnology) in accordance with manufacturer's protocol and observed under an IX73 inverted fluorescence microscope (Olympus Corporation).

Human CMECs were purchased from BLUEFBIO, and HUVECs were from American Type Culture Collection; both were incubated in Dulbecco's Modified Eagle Medium supplemented with 10% FBS (both from HyClone; Cytiva) in an incubator containing 5% CO₂ at 37°C.

Plasmids inducing GADD45A silencing (siRNA-GADD45A-1 and -2; siB08722144103-1-5 and siB08722144126-1-5, respectively; 80 nM; Guangzhou RiboBio Co., Ltd.) or an siRNA-NC (siN0000002-1-5; 80 nM) were transfected into human CMECs using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h at 37°C following the standard procedures of the manufacturer. After 48 h, the transfected cells were incubated in a hypoxic incubator (95% N₂ and 5% CO₂) at 37°C for 45 min and then under normoxic conditions (21% O₂, 5% CO₂ and 74% N₂) at 37°C for 90 min, then used for subsequent experimentation.

In rescue experiments, prior to H/R induction, CMECs were treated with the JNK and p38 MAPK activator, anisomycin (5 µM), or with an inhibitor of STAT3, AG490 (50 µmol/l), for 2 h at 37°C.

CMEC viability in each group was detected using the CCK-8 kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. A total of 2×10⁴ cells/well were seeded into 96-well plates for 24 h at 37°C. Then, 10 µl CCK-8 solution was added to each well and the cells were incubated for further 2 h at 37°C. Finally, a microplate reader was used to assess the absorbance at λ=450 nm.

CMECs were cultured in 6-well plates (1×10⁶ cells/well) for 24 h at 37°C. The apoptotic level of CMECs in each group was assessed by Annexin V/FITC-PI Apoptosis Detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Total apoptotic rates (equal to early-plus late-stage apoptosis) were analyzed using a CytoFLEX flow cytometer with FlowJo v10 software (FlowJo; BD Biosciences).

Matrigel (50 µl) was spread into 96-well plates at 37°C for 30 min. After H/R treatment for 30 min at 37°C, 100 µl HUVECs (2×10⁵ cell/ml) were added to each well. To confirm the role of JAK2-STAT3 pathway in angiogenesis, JAK2-STAT3 pathway inhibitor AG490 was added. Angiogenesis was observed after 6 h incubation at 37°C under a BX43 light microscope (Olympus Corporation).

All experiments were repeated at least three times. Data are presented as the mean ± SD and analyzed with GraphPad Prism v7.0 (GraphPad Software; Dotmatics). One-way ANOVA was used for comparisons among groups, followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results obtained from RT-qPCR and western blotting revealed that GADD45A mRNA and protein expression levels, respectively, of the I/R group were significantly elevated compared with those in the Control group (Fig. 1A and B). TTC staining results showed that I/R induction significantly increased the size of the ischemic area in rats compared with the Control group (Fig. 1C and D). In addition, the levels of CK and LDH were significantly higher in the I/R group compared with those in the Control group (Fig. 1E and F).

To assess the role of GADD45A in I/R induction, GADD45A silencing was induced by intramyocardial injection of Lv-si-GADD45A. GADD45A level was successfully decreased following Lv-si-GADD45A induction in I/R model rats compared with that in I/R + Lv-si-NC group (Fig. 2A and B). The sizes of the infarcted area were significantly decreased by GADD45A silencing compared with those in the I/R + Lv-si-NC group (Fig. 2C and D). In the Control group rats, no changes in myocardial tissue were observed, whereas myocardial fibers were disordered with nuclear splitting, edema and enlarged myocardial spaces after I/R induction (Fig. 2E). Myocardial fiber arrangement disorder and interstitial edema were improved in I/R + Lv-si-GADD45A group rats compared with those in I/R + Lv-si-NC group rats (Fig. 2E). In addition, the levels of CK and LDH in the serum were significantly decreased in I/R + Lv-si-GADD45A rats compared with those in the I/R + Lv-si-NC group (Fig. 2F and G).

To assess the effect of GADD45A on the loss of vascular endothelial cells, the cells were stained with CD31 and TUNEL. The data showed that I/R induction contributed to increased apoptotic level and decreased CD31 staining compared with the Control group (Fig. 3A); these effects were reversed by GADD45A silencing. The protein expression levels of CD31 and p-eNOS were significantly downregulated, whereas that of ET-1 was upregulated in the I/R group compared with those in the Control group (Fig. 3B); however, GADD45A silencing significantly reversed this effect. In addition, the significantly decreased expression of NO following I/R was partially reversed in I/R model rats treated with si-GADD45A (Fig. 3C). These results demonstrated that GADD45A silencing may ameliorate the injury induced by myocardial I/R.

To assess the regulatory role of GADD45A in I/R, the protein expression levels of p-p38 MAPK, p38 MAPK, p-JNK, JNK, EGR1, p-STAT3, STAT3 and VEGF were determined. The levels of p-p38 MAPK, p-JNK and EGR1 were significantly increased by I/R induction compared with those in the Control group, and these were subsequently decreased following GADD45A silencing (Fig. 4A). Furthermore, the expression levels of p-STAT3 and VEGF were significantly decreased in the I/R group compared with the Control group (Fig. 4B); these effects were significantly reversed in the I/R + Lv-si-GADD45A group compared with rats in the I/R + Lv-si-NC group.

To examine the mechanism by which GADD45A is involved in H/R-induced CMEC injury in vitro, the cells were subjected to H/R induction. H/R treatment significantly increased GADD45A mRNA and protein expression levels compared with those in the untreated Control group (Fig. 5A and B, respectively). Next, the effects of GADD45A silencing on CMECs were examined. RT-qPCR and western blotting analysis confirmed that GADD45A mRNA and protein expression levels, respectively, were successfully reduced following transfection with siRNA-GADD45A-1 and siRNA-GADD45A-2 compared with siRNA-NC transfection (Fig. 5C and D). Based on the transfection efficiency, siRNA-GADD45A-1 was selected for use in subsequent experiments. H/R stimulation activated p38 MAPK/JNK signaling and suppressed STAT3/VEGF signaling, as shown by the increased levels of p-p38 MAPK and p-JNK (Fig. 5E), and the decreased levels of p-STAT3 and VEGF in H/R group (Fig. 5F). In addition, EGR1 expression was increased compared with the Control group (Fig. 5E). GADD45A silencing reversed the effects of H/R induction on these protein expression levels (Fig. 5E and F).

The mechanisms by which GADD45A may affect the injury and apoptosis of CMECs induced by H/R were investigated. H/R induction diminished cell viability and promoted apoptosis (Fig. 6A-C). The knockdown of GADD45A subsequently increased the viability and decreased the apoptosis of H/R-induced CMECs. To verify that GADD45A modulated viability and apoptosis in H/R-treated CMECs via J JNK/p38 MAPK signaling, NK and p38 activator anisomycin was used here. Treatment with anisomycin significantly reduced these effects. The declined BCL2 expression and the elevated Bax and Cleaved caspase 3/caspase 3 expressions in the H/R group were all reversed by GADD45A silencing (Fig. 6D). Furthermore, anisomycin treatment also reduced BCL2 protein expression but raised the expression levels of Bax and cleaved caspase 3 in CMECs subjected to H/R + siRNA-GADD45A.

AG490 is an inhibitor of STAT3 phosphorylation, which may enhance myocardial cell apoptosis and eliminate the protective effect of ischemic pre-conditioning and ischemic post-conditioning in the heart (17). The decreased CD31 expression caused by H/R induction was elevated following GADD45A silencing (Fig. 7A and B); however, AG490 treatment partially reversed this effect. The decreased protein expression levels of CD31 and p-eNOS (Fig. 7B), as well as the reduction in NO (Fig. 7C), in H/R-induced CMECs were significantly increased in H/R + siRNA-GADD45A cells, which were then reduced by AG490 treatment; ET-1 expression exhibited the opposite trend. In the angiogenesis assay, fewer tubes were observed in the H/R group compared with the Control group, whereas the number of tubes was significantly increased in H/R cells transfected with siRNA-GADD45A (Fig. 7D and E); H/R + siRNA-GADD45A cells treated with AG490 formed fewer tubes compared with the untreated H/R + siRNA-GADD45A group.