HUVECs were obtained from ATCC (Manassas, VA, USA) and cultured within complete endothelial medium (F-12K complemented with 10% FBS, 0.1 mg/mL heparin, and 30 μg/mL endothelial cell growth supplement (ECGS), all from Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with 5% CO₂. The HUVECs used in the experiments were between passages 25 and 35. All experiments were performed with mycoplasma-free HUVECs. Additionally, HUVECs were examined for short tandem repeats and were not contaminated with other cells. Subsequently, HUVECs were transfected with different vectors using Lipofectamine 3000 (cat. no. L3000150, Thermo Fisher Scientific).

The AQP1 promoter (1000 bp, pAQP1) was cloned into the pGL3-basic vector and co-transfected with pRL-TK (Promega E2241; Promega, Madison, WI, USA), in combination with ETS1 or MEF2C overexpression vector (pcDNA3.1-ETS1/MEF2C) or shRNA targeting ETS1 or MEF2C (pGPU6/GFP/Neo-shETS1/shMEF2C) into HUVECs via Lipofectamine 3000 (Thermo Fisher Scientific). HUVECs were harvested 48 h post-transfection and washed with 0.1 mol/L phosphate-buffered saline (PBS, PH7.4), and luciferase activity was measured using a dual-luciferase reporter assay system (cat. no. E1910, Promega, Madison, WI, USA).

Total RNA was extracted from HUVECs and cardiac tissues of Sprague-Dawley (SD) rats using a RNeasy Plus Micro Kit (cat. no. 74034-50T, NUO YANG SHENG WU, Hangzhou, China) according to the manufacturer’s instructions. cDNA was prepared by reverse transcription using a TRUEscript One Step RT-PCR kit (PC3401, Aidlab Biotechnology Co., Ltd., Beijing, China) in line with specific instructions, and served as the starting material for qRT-PCR using the Step OnePlus Real-Time PCR system (Life Technologies Corp, Carlsbad, CA, USA). Primer Premier software 5.0 (Sangon Biotech Co., Ltd., Shanghai, China) was used to design all primer sequences. The 2^−ΔΔCT method was normalized to GAPDH.

The following primers were used in the study:

AQP1(rat) forward: 5^′-CTCTTCTGGAGGGCAGTGGTGG-3^′

AQP1(rat) reverse: 5^′-CCTGGACCGTCGTCTGGTTGTT-3^′

MEF2C (rat) forward: 5^′-ATTCGGACTGATGAAGAAG GCT-3^′

MEF2C (rat) reverse: 5^′-TTGTTGGTGCTGTTGAAGATGA-3^′

ETS1 (rat) forward: 5^′-CTCAAGCCGACTCTCACCATC-3^′

ETS1 (rat) reverse: 5^′-AACTCATTCACAGCCCACATC-3^′

GAPDH (rat) forward: 5^′-ACAGCAACAGGGTGGTGGAC-3^′

GAPDH (rat) reverse: 5^′-TTTGAGGGTGCAGCGAACTT-3^′

AQP1(human) forward: 5^′-GGCCTGGGCATCGAGATCAT-3^′

AQP1(human) reverse: 5^′-GGGCTACAGAGAGGCCGATG-3^′

MEF2C (human) forward: 5^′-GCGTGCTGTGTGACTGTGAG-3^′

MEF2C (human) reverse: 5^′-CATGCGGCTCGTTGTACTCC-3^′

ETS1 (human) forward: 5^′-CTGCAGCGTGTTCCCTCCTA-3^′

ETS1 (human) reverse: 5^′-CGGTCCCGCACATAGTCCTT-3^′

GAPDH (human) forward: 5^′-TGGGTGTGAACCATGAGAAGT-3^′

GAPDH (human) reverse: 5^′-TGAGTCCTTCCACGATACCAA-3^′

HUVECs and cardiac tissues of SD rats were lysed via RIPA lysis buffer (cat. no. G2008, Servicebio Biotechnology Co., Ltd., Wuhan, China), and protein content was determined using a bicinchoninic acid (BCA) kit (cat. no. P0011, Beyotime Biotechnology, Shanghai, China). Proteins were separated using 10% SDS-polyacrylamide gels and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk for 1 h at room temperature, and incubated with the following primary antibodies overnight at 4°C: anti-AQP1 (1:5000, 20333-1-AP, Proteintech, Chicago, IL, USA), anti-MEF2C (1:500, 18290-1-AP, Proteintech), anti-ETS1 (1:5000, 66598-1-Ig, Proteintech), and anti-GAPDH (1:500, 13937-1-AP, Proteintech). GAPDH served as the loading control. Horseradish peroxidase (HRP)-labeled secondary antibodies (1:4000, SA00001-15/17, Proteintech) were used as secondary antibodies. Finally, an enhanced chemiluminescence kit (cat. no. WBKlS0100, Millipore, Burlington, MA, USA) was used to observe the protein blots, and ImageJ software version 4.3 (National Institutes of Health [NIH], Bethesda, MD, USA) was used for quantification.

ChIP assay was performed using a ChIP Assay Kit (cat. no. P2078, Beyotime, Shanghai, China) according to the manufacturer’s instructions. Briefly, formaldehyde (37%) was added to the HUVEC culture medium at a final concentration of 1%, and incubated at room temperature for 10 min. The digested chromatin was treated with an ultrasonic crusher (VCX500; Sonics, Newtown, CT, USA) for 3 min, ultrasonicated for 5 s, and stopped for 25 s. Chromatin DNA was fragmented into 100–200 bp fragments. Subsequently, 25 μg DNA was incubated with anti-ETS1 (5 μg, 1:2000, 12118-1-AP, Proteintech), anti-MEF2C (5 μg, 1:500, 18290-1-AP, Proteintech) or mouse IgG (1 mg/mL, ab18413, Abcam, Cambridge, UK) at 4°C for 2 h before adding protein A/G beads (cat. no. P2078, Beyotime, Shanghai, China) to each sample and rotating at 4°C overnight using a rotary instrument (80 rpm, VSZD-IIIAB, Woshin, Wuxi, China). The immunocomplexes were collected using a protein G agarose suspension and washed successively with low-salt, high-salt, and LiCl buffer once each, and TE buffer twice. Protein G was removed using the prepared elution buffer (10%–20% SDS, 20 mm NaHCO₃, and 170 mL of sterilized water per 200 mL of buffer). The complexes were incubated at 65°C for 5 h to denature the cross-linking. The DNA was purified using a PCR purification kit (cat. no. D4013, Zymo Research, Irvine, CA, USA) and amplified by PCR with the following primers: forward (5^′-gaggtctgtgtggtgt-3^′)/reverse (5^′-tcaattccctctgagag-3^′) for the MEF2C binding site and forward (5^′-gggagagagagagaga-3^′)/reverse (5^′-acaccacacagacctc-3^′) for the ETS1 binding site. PCR amplification conditions: 95°C for 5 min, (94°C for 40 s, 53°C for 60 s, 72°C for 60 s) for 30 cycles, and finally 72°C for 10 min.

HUVECs were cleaved into proteins using an IP lysate (cat. no. G2008; Servicebio, Wuhan, China). Proteins were incubated with each primary antibody or mouse IgG at 4°C for 2 h before adding protein A/G beads to each sample and rotating at 4°C overnight. The immunocomplexes were then washed, eluted, and reverse cross-linked. The proteins were extracted using 12% SDS-PAGE and then transferred to PVDF membranes (Millipore, Burlington, MA, USA). The membranes were incubated using 5% skimmed milk, followed by overnight incubation with primary antibodies under 4°C. After rinsing, the membranes were incubated for 1 h with an HRP-labeled secondary antibody (1:4000, SA00004-10, Proteintech, Wuhan, China) at ambient temperature. Finally, an enhanced chemiluminescence kit (cat. no. WBKlS0100; Millipore, Burlington, MA, USA) was used to observe the protein blots, and ImageJ software (NIH, version 4.3) was used for quantification. The primary antibodies used were anti-AQP1 (1:5000, 20333-1-AP, Proteintech), anti-MEF2C (1:500, 10056-1-AP, Proteintech), anti-ETS1 (1:5000, 66598-1-Ig, Proteintech), and anti-GAPDH (1:500, 13937-1-AP, Proteintech), with GAPDH as the loading control.

HUVECs were cultured at the appropriate density (5 × 10⁵/well), and a wound was scratched with a 10 μL pipette gun when the cells reached 80% confluence and then cultured in serum-free medium in the incubator with 5% CO₂ at 37°C. Photographs were taken at 0 and 48 h, respectively. An inverted microscope (Olympus BX63, Tokyo, Japan) was used to observe the cells at 0 and 48 h (200×). The migration rate of the cells was calculated using the following equation: x−y/x, where x is the width of the scratches at 0 h, and y is the width of the scratches at 48 h.

After Matrigel was liquefied at 4°C overnight, a 24-well plate was placed on ice for pre-cooling. Subsequently, 50 μL of Matrigel was added to each well, and the plate was placed flat and stationary at 4°C for 10 min. The plate with the matrix gel was then placed in the incubator with 5% CO₂ at 37°C for 30 min. The solidified Matrigel was removed, and HUVECs (3 × 10⁵/well) were inoculated into a plate pre-coated with Matrigel and incubated for 24 h. Subsequently, cells were incubated with 20 μm calcein (cat. no. Y271510, Beyotime, Shanghai, China) for 20 min, imaged via a fluorescence microscope (IX71, OLYMPUS, Tokyo, Japan), and the number of lumens was counted using ImageJ software.

30 adult male SD rats (6 weeks, 180~220 g) were purchased from Cavens Laboratory Animal Co. (Changzhou, China) and acclimated for a week. The rats were allowed to access water and food ad libitum, housed under suitable temperature (22 ± 2°C) and humidity (65% ± 5%) with a 12/12-h light/dark cycle. The rats were fed a high-fat diet with 45% energy from lipids and 17% energy from sucrose (D12451 Research Diets, New Brunswick, NJ, USA) for 16 weeks.

Next, ligation of the left anterior descending artery (LAD) was performed. Anesthesia was induced using 5% isoflurane and maintained using 2% isoflurane. The rats were intubated with a 20-G IV catheter through the mouth and connected to a ventilator (KW-10; KEW BASIS, Nanjing, China). The chest was incised in the left lateral intercostal space between the 4th and 5th ribs. Ligation was performed using an 8-0 polypropylene suture on the same segment as the LAD. The suture was removed 30 min later, and the opening was closed. Rats were randomly divided into six groups (n = 5): NC, shETS1, shMEF2C, shETS1+shMEF2C, Model, Model+shETS1+shMEF2C. For shRNA delivery, AAV9-shETS1 or AAV9-MEF2C (Gene Pharma, Suzhou, China) was injected into the tail vein 24 h prior to LAD ligation. Rats were euthanized 7 days after LAD ligation following anesthesia with 5% isoflurane. Heart tissues were harvested for subsequent experiments. This study adhered to the recommendations outlined in ARRIVE Guidelines 2.0, and the experiments were approved by the Institutional Animal Care and Usage Committee of Jilin Medical University (JMU2020-0037).

After fixation with 4% formaldehyde, the cardiac tissues were embedded in paraffin and cut into 5-μm sections. Pathological changes in the sections were assessed using an HE staining kit (G1120, Solarbio, Beijing, China), and cardiac fibrosis was assessed using a Masson staining kit (G1340, Solarbio, Beijing, China). Images were captured using a light microscope (OLYMPUS BX63; Tokyo, Japan).

Briefly, cardiac sections were dehydrated and immersed in a PBS blocking solution containing 1.5% normal goat serum and 0.2% Triton X-100. The sections were then incubated overnight with AQP1 antibody (1:1000, ab300463, Abcam) and CD31 antibody (1:1000, ab9498, Abcam) at 4°C, and then incubated with FITC-labeled goat anti-rabbit IgG (1:100, SA00003-1, Proteintech) or Cy3-labeled secondary antibody (1:200, SA00009-2, Proteintech) at room temperature for 1 h. Then, sections were incubated with 5 μg/mL Hoechst 33258 (C1017, Beyotime) for 5 min in the dark, and washed with 0.1% phosphate buffered saline Tween (PBST) for 4 times. Finally, the samples were incubated in a humid chamber for 15 min under light-protected conditions, and images were captured using a light microscope (OLYMPUS BX63; Tokyo, Japan).

All experiments were repeated three times or more. Data were analyzed using GraphPad Prism 5.0 (GraphPad Software, Dotmatics, Waltham, MA, USA) and are presented as mean ± SD. Differences between the groups were compared by ANOVA and Tukey’s POC host analysis. Statistical significance was set at p < 0.05.

ETS1 activates MEF2C in the early stage of vasculogenesis and thereafter vascular development. Furthermore, MEF2C is a critical regulator of endothelial AQP1 expression [19,28]. Therefore, we examined whether ETS1 regulated AQP1 expression in endothelial cells. As shown in Fig. 1A, AQP1 and MEF2C protein levels were correlated with ETS1 levels. Notably, ETS1 overexpression significantly increased AQP1 and MEF2C protein levels, whereas silencing ETS1 markedly reduced their protein levels (Fig. 1A–D). Similarly, ectopic ETS1 promoted, whereas silencing ETS1 inhibited the transcriptional activity of the AQP1 promoter (Fig. 1E). Interestingly, silencing MEF2C dampened the ETS1-induced increase in AQP1 promoter activity (Fig. 1E).

Next, we investigated how ETS1 and MEF2C regulated AQP1 transcription. SP1 was a widely existing transcription factor that bound to the GC-rich sequences in the promoter region of genes, thereby regulating gene expression. It has been reported that SP1 regulates AQP1 expression as a transcription factor [29]. As expected, the protein complex containing ETS1, MEF2C, and SP1 was precipitated using antibodies against ETS1 or MEF2C (Fig. 2A). Moreover, both ETS1 (Fig. 2C) and MEF2C (Fig. 2B) antibodies enriched AQP1 promoter sequences containing either ETS1- or MEF2C-binding site.

AQP1 has been shown to be critical for the biological functions of endothelial cells, including migration and angiogenesis [30]. Therefore, we next examined whether the regulation of AQP1 expression by ETS1 affected these processes. ETS1 overexpression significantly increased the motility of HUVECs, which was similar to AQP1 and MEF2C overexpression (Fig. 3A,B,D,E). Silencing MEF2C partially inhibited ETS1-induced HUVEC migration, whereas MEF2C synergized with ETS1 to promote cell migration (Fig. 3A,B,D,E).

Both ETS1 and MEF2C strongly promoted angiogenesis in HUVECs, and the increase in tube formation was more prominent when ETS1 and MEF2C were overexpressed simultaneously (Fig. 3C,F). AQP1 knockdown strongly inhibited HUVEC lumen formation (Fig. 3C,F).

AQP1 is an intrinsic membrane protein located on the cell membrane that mediates water flux between blood arteries and the interstitium and plays a significant role in myocardial I/R injury [31]. The impact of ETS1 and MEF2C regulation of AQP1 on I/R-induced cardiac damage was the subject of our further research. Transient LAD ligation induced the disarrangement of a large number of cardiomyocytes with shrinkage or disappearance of some nuclei, and the interstitial space was significantly widened with fibrous tissue filling, which was relieved by silencing of ETS1 and MEF2C (Fig. 4A,B). Moreover, ETS1 and/or MEF2C silencing alone induced the shrinkage and disarrangement of a small number of myocardial cells (Fig. 4A) and a small amount of fibrous tissue filling the interstitial space of the heart (Fig. 4B) in control rats. Studies on cardiac tissue have mainly focused on the myocardial vascular endothelium. In the immunofluorescence experiments shown in Fig. 4C, the part that expressed CD31 was the endothelium (Red fluorescence), and the part that expressed both CD31 (Red fluorescence) and AQP1 (Green fluorescence) was AQP1 expressed by endothelial cells. Transient LAD ligation (ischemia/reperfusion) induced the upregulation of AQP1 and increased the number of CD31-positive microvessels, which was inhibited by the knockdown of ETS1 and MEF2C (Fig. 4D,E).

ETS1, MEF2C, and AQP1 mRNA (Fig. 5A–C) and protein levels (Fig. 5D–G) were substantially elevated in the rat hearts after transient LAD ligation. Silencing of ETS1 and MEF2C abrogated the upregulation of AQP1 induced by I/R injury (Fig. 5).