Male wild-type (WT) C57BL/6 mice (8weeks old) were purchased from Animal Center of Shanxi Medical University. All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health, and the experiments were approved by the Animal Care and Use Committee of Shanxi Medical University.

Mice were anaesthetized with chloral hydrate (300 mg/kg). Acute Myocardial infarction model was induced by ligation of left anterior descending coronary artery. The same procedure was performed in the sham group without LAD occlusion. Mice were sacrificed 1 week post-surgery for heart tissue collection.

Etv1 overexpressing adeno-associated virus driven by endothelial specific gene Icam2 promoter (AAV2/9-Icam2-mEtv1-Flag-P2A-EGFP, AAV9-Etv1) or control viral vectors (AAV2/9-Icam2-EGFP, AAV9-NC) were constructed by Taitool Bioscience Co., Ltd (Shanghai, China) following standard methods (21). Briefly, the cDNA fragments encoding mouse ETV1 was cloned into inverted terminal repeat (ITR)-containing AAV9 plasmid harboring the human endothelial specific Icam2 promoter. AAV9 vectors, rep2/cap9 packaging plasmids, and helper plasmids were packaged in HEK293T cells (Thermo Scientific). After transfection using polyethylenimine for 72 h, cells were collected and lysed. AAV9 was purified and concentrated by gradient centrifugation. AAV9 titer was determined by qPCR. For the in vivo experiment, 3 days before MI surgery, mice received an intracardiac injection of AAV9-NC or AAV9-Etv1 using an insulin syringe with a 30-gauge needle at a dose of 4 × 10¹¹ viral genome particles per animal as previously described (22). Hearts were collected 1 week after MI.

One week after MI surgery, mice were anaesthetized using 1.5%–2% isoflurane and placed in supine position. Heart function was assessed by echocardiography. Left ventricular ejection fraction (LVEF), left ventricular short-axis shortening rate (LVFS), left ventricular end-systolic diameter (LVIDs) and left ventricular end-diastolic diameter (LVIDd) were measured using the corresponding formulas.

Myocardial tissues from the infarct border zone were harvested at 7 days after MI surgery. Heart tissues were fixed in 4% paraformaldehyde and then dehydrated. After dehydration, the samples were embedded in paraffin and sectioned longitudinally. The sections were stored at −80℃ until further use.

Masson's trichrome staining was performed by using a kit (Solarbio) according to the manufacturer's protocol. The cardiac tissue sections from the infarct border zone were dewaxed and stained with Weigert's hematoxylin. After washing thoroughly with tap water and then rinsed with distilled water, the sections were stained with acid fuchsin solution, differentiated in phosphotungstic‒phosphomolybdic acid, stained with aniline blue, and washed with 1% acetic acid. After dehydration, the slides were mounted and scanned. The fibrotic area was measured with image J software.

Myocardial apoptosis in the infarct border zone was detected using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay kit (Beyotime, China) according to the manufacturer's instructions. The slides were counterstained with DAPI for nuclei labeling. The fraction of apoptotic cells was estimated as ratio of TUNEL-positive cells to total cell nuclei.

The sections from the infarct border zone were fixed with 4% paraformaldehyde. After permeabilization with 0.1% Triton X-100, the slides were blocked with 10% goat serum and then incubated with the primary antibodies against cTnT (Abcam, ab209813, 1:1,000) and CD31 (Abcam, ab222783, 1:1,000). The next day, the sections were washed and stained with the corresponding fluorescent secondary antibodies (Alexa Fluor-488 or Alexa Fluor-555, Abcam, 1:1,000). The nuclei were labeled with DAPI. Images were taken using an inverted fluorescence microscope (Olympus BX51, Japan). To assess the density of capillaries in border zones, the numbers of vessels were counted in 5 random fields on each section per animal and recorded as CD31⁺ vessels/mm².

Cardiac microvascular endothelial cells (CMECs) were isolated as previously described (23). Briefly, the Mice hearts from each group were harvested and minced into small pieces. After digestion with collagenase type II and dispase, the cells were collected and incubated with CD31 magnetic beads (Miltenyi Biotec, Germany) for endothelial cell sorting. The isolated endothelial cells were cultured and used between passages 2 and 4 for subsequent western blot experiments.

Myocardial tissues from the infarct border zone and HUVECs were harvested and homogenized. The samples were lysed with RIPA buffer containing protease inhibitors. The concentration of protein was then quantified by BCA method. Equal amounts of total protein was separated by SDS-PAGE gels and then transferred to PVDF membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies overnight against ETV1 (Abcam, ab314874, 1:1,000), VEGF Receptor 2 (CST, #2,479, 1:1,000), VEGFA (Abcam, ab214424, 1:1,000), eNOS (CST, #32,027, 1:1,000), and GAPDH (Abcam, ab9485, 1:1,000). The next day, the membranes were incubated with secondary antibodies, and protein signals were visualized using an Odyssey infrared imaging system. The gray value of protein bands was quantitatively analyzed by Image J software.

Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC (American Type Culture Collection, USA). The cells were cultured in Endothelial Cell Growth Medium supplemented with 5% FBS, penicillin, and streptomycin. HUVECs between passages 3 and 5 were used for subsequent experiments.

Lentivirus to overexpress ETV1 (Lenti-esEF1a-hETV1-Flag-IRES-MataGFP, Lenti-ETV1) and blank lentivirus (Lenti-esEF1A-3xFlag-IRES-MataGFP, Lenti-NC) were designed and constructed by Taitool Bioscience Co., Ltd (Shanghai, China). Briefly, human ETV1 cDNA was cloned into lentiviral vector at the MCS locus. The HEK293T cells were transfected with the lentiviral vector plasmid and packaging plasmids. The transfected cells were cultivated, and the supernatants were collected 72 h after transfection. Viral supernatants were concentrated using ultracentrifugation. Lentivirus titers were measured using FACS analysis and determined by infection of 293 T cells (24). Lentiviruses expressing GFP gene were used as the control. For the in vitro experiment, HUVECs were infected with lentiviruses at a multiplicity of infection of 3, and the transfection was performed according to the manufacturer's instructions. Briefly, cells were seeded at 1 × 10⁵/ml per well in 12-well plates. 2 × 10⁸ TU/ml ETV1 overexpression lentivirus or 2 × 10⁸ TU/ml control lentivirus were added to HUVECs. The medium was changed 24 h post-transfection.

Parallel lines were drawn on the back of 6-well plate. 5 × 10⁵ HUVECs were inoculated in 6-well plate and incubated overnight with 5% CO2 at 37℃. The next day, a scratch wound perpendicular to the parallel lines was made in the middle of the well using a 200 μl pipette tip. The cells were washed with PBS to remove the floating cells and then infected with lenti-ETV1 or lenti-NC for 24 h. The area of scratch wound was photographed at 0 and 24 h following infection, and the distance was measured using ImageJ.

Before the experiment, Matrigel was thawed at 4°C overnight. Firstly, 50 μl Matrigel matrix was added to each well of a 96-well plate and incubated at 37°C for 30 min. After polymerization, 1 × 10⁴ HUVECs per well were seeded into the 96-well plate coated by Matrigel. Next, the cells were infected with lenti-ETV1 or lenti-NC for 24 h. Tubule formation was then observed and photographed under an inverted microscope. The length of tubes and number of branch points were quantified using ImageJ software.

No statistical methods were used to predetermine sample sizes; however, our sample sizes were similar to those reported in a previous publication (25, 26). The results were presented as mean ± standard deviation (SD). Comparisons between groups were performed using Student's t test or one-way ANOVA followed by LSD post hoc test. P < 0.05 was considered to be statistically significant.

Our previous study demonstrated that ETV1 is a potential transcription factor involved in ventricular remodeling after AMI (20). To explore the role of ETV1 in the process of pathological ventricular remodeling after AMI, the expression level of Etv1 in CMECs isolated from sham and MI mouse hearts were examined. Our data indicated that the ETV1 protein level was significantly downregulated in CMECs isolated from the infarct border zone compared with that in the sham group (Figure 1A). To further determine whether Etv1 improves cardiac function after MI, Etv1 was overexpressed in endothelium through intramyocardial injection of adeno-associated virus serotype 9 (AAV9) carrying the endothelial-specific promoter Icam2 (AAV9- Etv1) 3 days before LAD ligation. Western blot showed that AAV9-Etv1 injection resulted in significantly increased level of ETV1 in endothelial cells (Figure 1A). Cardiac functions were then monitored by echocardiography 1 week after MI surgery.

Our results showed that MI challenge significantly decreased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), while left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were significantly increased in AAV9-NC MI mice compared with sham mice. However, after transfection with AAV9-Etv1, the values for LVEF and LVFS were markedly higher, and the values for LVEDD and LVESD were markedly lower compared with AAV9-GFP mice (Figure 1B), indicating that Etv1 ameliorates cardiac dysfunction after MI.

Myocardial fibrosis and apoptosis are important pathological mechanisms responsible for the development of pathological ventricular remodeling (PVR) and cardiac dysfunction after MI. We then investigated whether overexpression of Etv1 could attenuate pathological ventricular remodeling. Masson staining showed that increased expression of ETV1 by AAV9-Etv1 transfection significantly alleviated cardiac interstitial fibrosis compared with AAV9-NC MI mice in the border regions, as evidenced by reduced collagen deposition (Figure 1C). Moreover, Etv1 overexpression significantly reduced cardiac apoptosis compared with the AAV9-NC MI mice (Figure 1D). These results suggest that ETV1 inhibited deterioration of cardiac function and at least partially ameliorated adverse cardiac remodeling in MI mice through its anti-fibrotic and anti-apoptotic effects.

Angiogenesis is essential for cardiac repair after MI. The insufficient neovascularization and subsequent shortage of oxygen supply in ischemic myocardium are major reasons leading to pathological ventricular remodeling and heart failure after MI. As mentioned previously, overexpression of Etv1 alleviated adverse ventricular remodeling. To further explore the mechanisms of the protective role of Etv1, angiogenesis was assessed by CD31 immunofluorescence staining in the myocardium at 1 week after MI. Our results showed that overexpression of Etv1 significantly increased the density of capillaries in the peri-infarct areas in comparison with AAV9-NC MI mice, as evidenced by increased CD31-positive vessels (Figure 2A), indicating that ETV1 enhances angiogenesis after MI.

As mentioned above, overexpression of ETV1 promotes angiogenesis in mice after MI. In order to further clarify the effect of ETV1 on HUVECs cultured in vitro and validate the role of ETV1 in regulating angiogenesis, we transfected HUVECs with ETV1 overexpression lentivirus (Lenti-ETV1) and negative control lentivirus (Lenti-NC) for 24 h under normoxia. The wound healing test and tube formation assay were then performed. The scratch experiment showed that overexpression of ETV1 promoted the migration of HUVECs (Figure 2B). At the same time, transfection of HUVECs with Lenti-ETV1 significantly increased the length and amount of tubes compared to Lenti-NC HUVECs (Figure 2C). Together, these results demonstrate that ETV1 played an important role in regulating cardiac angiogenesis.

VEGF is a key angiogenetic factor and VEGF-VEGFR2 is considered as one of the most important pathways regulating angiogenesis. To gain insights into mechanisms underlying the proangiogenic role of ETV1, the levels of VEGFA and VEGFR2 were measured by western blot. Our data showed that the ETV1 protein level was significantly downregulated, and the levels of VEGF and VEGFR2 was significantly upregulated under MI conditions. More importantly, transfection with AAV9-Etv1 further elevated the levels of VEGFA and VEGFR2 in comparison with AAV9-NC MI mice. eNOS is a critical modulator implicated in angiogenesis. Consistent with the expression of VEGFA and VEGFR2, overexpression of Etv1 significantly increased the expression of eNOS (Figure 3). Collectively, our results suggest that ETV1 played an important role in upregulating the expression of pro-angiogenic factors, which further promoted endothelial cell proliferation, migration, and angiogenesis.

Our in vitro experiment demonstrated that overexpression of ETV1 promoted endothelial migration and tube formation in HUVECs. As described above, overexpression of ETV1 increased the levels of angiogenetic factors (eNOS, VEGFR2, VEGFA) after MI. To investigate the molecular mechanisms of proangiogenic effects of ETV1 on HUVECs, we then also examined the effect of ETV1 on expression of eNOS, VEGFR2, and VEGFA in HUVECs after transfection with Lenti-ETV1. Consistent with the expression pattern in vivo, transfection of HUVECs with Lenti-ETV1 significantly elevated the levels of VEGF, VEGFR2, and eNOS compared with Lenti-NC HUVECs (Figure 4). Taken together, these data demonstrate that ETV1 promoted angiogenesis via VEGF/VEGFR2/eNOS signaling pathway.