In this study, all experimental procedures involving animals were approved by the Animal Care and Ethics Committee of Kunming Medical University (Kunming, China, Approval number: No. Kmmu2020350). All animal experiments complied with the National Institutes of Health Guide for the Care and Use of Laboratory animals. Significant efforts were made to minimize the sufferings and the number of the rats used.

Male Sprague-Dawley (SD) rats weighing 220–250 g were purchased from Hunan SJA Laboratory Animal Co., Ltd., China. After 1 week acclimatization under laboratory conditions, a natural light cycle at a controlled temperature 25 ± 2°C with food and water available ad libitum, the rats were randomly divided into six groups (eight rats/group) and treated as Figure 1. In brief, the control group received a daily subcutaneous (s.c) injection of normal saline (0.1 ml/100 g) for 28 days. The ISO group received a daily s.c injection of isoproterenol (1.5 mg/kg/day) at day 1 to day 14, followed by a daily s.c injection of normal saline (0.1 ml/100 g) for another 14 days. The ISO+OT postconditioning group received a daily s.c injection of isoproterenol (1.5 mg/kg) for the first 14 days, followed by a daily s.c injection of 0.03 and 3 ug/kg oxytocin acetate (purchased from MedChemExpress) for the second 14 days, respectively. In ISO+OT preconditioning group, oxytocin acetate (3 ug/kg) was administered subcutaneously for 7 days, followed by co-administration of oxytocin and ISO for 7 days, and ISO administration alone for another 7 days. The time interval of administration between oxytocin acetate and ISO was more than 8 h. OT group received a daily s.c injection of oxytocin acetate (3 ug/kg) for the first 14 days, followed by a daily s.c injection of normal saline (0.1 ml/100 g) for the second 14 days. The dose of oxytocin acetate was based on Plante’s study (Plante et al., 2015). After 28-day treatment, echocardiography examinations were performed to assess changes in cardiac morphology and functions. At the end of the study, all animals were anesthetized using isoflurane in a sealed glass jar and hearts excised. Heart weights were measured after the hearts were rinsed with ice-cold saline and dried with filter papers. Some heart samples were immediately stored in liquid nitrogen, while the other samples were fixed in 4% paraformaldehyde.

Transthoracic echocardiograms were performed on rats using a PHILIPS EPIQ7C ultrasound system for cardiology. Rats were isolated in a glass chamber, anesthetized by inhalation of 1.5 to 2% isoflurane mixed with 100% oxygen, and then supinely placed on an operational warming platform. The chest hair was depilated with a hair removal cream. M-mode cine loops were recorded and analyzed by high-frequency ultrasound imaging software to assess myocardial parameters and cardiac functions of left ventricle (LV), including left ventricular posterior wall thickness at end-diastole (LVPWd), left ventricular posterior wall thickness at end-systole (LVPWs), left ventricular internal diameter at end-diastole (LVIDd), left ventricular internal diameter at end-systole (LVIDs), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS).

Heart tissues were fixed in 4% paraformaldehyde for 48 h and then embedded in paraffin. The embedded hearts were cut into 5-μm thickness sections, which were mounted on glass slide and stained with hematoxylin-eosin (H&E) and Masson’s trichrome stainings. The sections were assessed under light microscopic fields by digital image analysis (BH-Z, Olympus Corporation, Tokyo, Japan).

The tissue sections underwent dewaxing and rehydrated, then be boiled in EDTA buffer for 10 min, and phosphate-buffered saline was used to washing the slides three times. Slides were incubated for 30 min with 5 uM wheat germ agglutinin (WGA) dying (sigma) in the dark. The cell nucleus were stained with 4′,6-diamidino-2-phenylindole (DAPI). The slides were sealed with antifade mounting medium and then observed using a fluorescence microscope (Olympus BX53).

Masson’s trichrome staining was used to evaluate the myocardial fibrosis and WGA staining was used to evaluate the cardiomyocyte cross-sectional area. Myocardial fibrosis and cardiomyocyte cross-section areas were quantitatively analyzed with Image J software. Three images for each group and 40 cells for each image were randomly selected to determine the cross-section areas of cardiomyocyte. Five images for each group were randomly selected to measure the collagen content, which was presented by collagen volume fraction (collagen area/field area × 100%).

Neonatal rat cardiomyocytes (NRCMs) were prepared from the hearts of 1- to 3-day-old SD rats. In brief, the hearts were excised from neonatal rats and washed in ice-cold phosphate buffered saline (PBS). Then the ventricles were finely separated and minced into 1–2 mm³ pieces, which were digested in 0.25% trypsin at 37°C. The cell suspension was centrifuged and the pellets re-suspended and then transferred into Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) culture medium. Fibroblast growth inhibitor (sciencell) was added to the cell suspension. Immunofluorescence staining was performed to detect the expression of cardiac troponin T (cTnT) and immunofluorescence microscopy to identify primary cardiomyocytes. The cultured cardiomyocytes (CMs) were treated with vehicle alone, ISO (10 uM) alone or OT (10 nM) + ISO (10 uM) respectively and further incubated for 24 h before harvest. In OT + ISO group, the cells were pretreated with OT for 30 min prior to stimulation with ISO.

LncRNA GAS5 and Klf4 gene knock-down were achieved using RNA interference. shRNA-GAS5, shRNA- Klf4 and its corresponding scramble negative control siRNA (siNC) were purchased from TsingKe Biological Technology (Beijing, China). miR-375-3p mimics, miR-375-3p inhibitor and the corresponding negative control were synthesized by RIBOBIO (Guangzhou, China). The Klf4 over-expression plasmid (pcDNA-Klf4) and empty pcDNA3.1 plasmid (control) were purchased from RIBOBIO (Guangzhou, China).

Plasmids were transfected into the cultured CMs using Lipofectamine 3000 (Cat: L3000-015 Invitrogen) according to the manufacturer’s protocol. The transfection efficiency was detected by qRT-PCR and Western blotting. After 24-h transfection, the cells were incubated in DMEM medium supplemented with 10% FBS and 10 µM ISO for 24 h followed by 30 min OT pretreatment.

Total RNA was extracted from the cultured CMs and LV tissues using Trizol reagent (Lifetech 15596026). Reversely transcribed to complementary DNA (cDNA)for miR-375-3p and lncRNA GAS5 and PCR amplification were performed using a Bulge-Loop™ miRNA qRT-PCR Starter Kit, a Bulge-Loop™ miRNA qRT-PCR Primer Set and a lncDETECT™ lncRNA qRT-PCR kit (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. U6 and β-actin served as internal normalized references for miR-375-3p and lncRNA GAS5, respectively. The relative mRNA levels of the target genes were evaluated by qRT-PCR with SYBR Green master mix (KAPA KK4601, Roche, United States) using the LightCycle 96 (Roche, United States). In vitro experiment, all qRT-PCR assays were conducted at least in triplicate. Fold-changes were calculated according to cycle quantitation (Ct) values with 2^−ΔΔCt method. List of the primers used for qRT-PCR is presented in Table 1.

NRCMs or tissue samples were lysed by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China). The total protein concentrations were determined by BCA protein assay kit (Beyotime Biotechnology, China). A total of 30 ug proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. The blots were blocked with 5% non-fat milk and incubated overnight with the primary antibodies against BNP (1:500, rabbit #ab19645 abcam), β-MHC (1:1,000, rabbit #ab172967 abcam), KLF4 (1:1,000, rabbit #bs-1064R BIOSS), PI3K (1:1,000, rabbit #ab182651 abcam), p-PI3K (1:1,000, rabbit #205841-1-AP Proteintech), AKT1 (1:1,000, rabbit # bs-0115M BIOSS), p-AKT1 (1:1,000, rabbit # bs-0876R BIOSS), β-actin (1:1,000, rabbit #abmart P30002) and GAPDH (1:2,000, rabbit #2118S CST). Then, the membranes were washed and treated with horseradish peroxidase-conjugated secondary antibodies (CST 7074) at room temperature for 2 h. ECL System was employed to examine the protein bands and the intensity of protein bands measured by ImageJ software.

LncRNA GAS5 or Klf4 containing the predicted miR-375-3p binding sites were cloned into pGL3-WT-lncRNA Gas5, pGL3-MUT-lncRNA GAS5, pGL3-WT Klf4 or pGL3-MUT Klf4, respectively. The WT or MUT reporter plasmid were co-transfected with the miR-375-3p mimics (50 nM) or the negative control into 293T cells using Lipofectamine 2000 (ThermoFisher), and the luciferase intensity was assessed by the double-luciferase reporter assay kit (Promega, United States) on SpectraMax Gemini EM (Molecular Devices, United States).

After transfection and treatment, cultured cardiomyocytes were washed with cold PBS for three times and subsequently fixed with 4% paraformaldehyde for 30 min. After that, the cells were permeabilized with 3% Triton X-100 for 20 min. Then, washed three times with PBS, the cardiomyocytes were incubated with anti-cTnT antibody (1:300, rabbit bs-10648R) at 4°C overnight and were further incubated with fluorophore-conjugated secondary antibodies at a normal temperature for 1 h, and then stained with 4′,6-diamidino-2-phenylindole (DAPI) solution for 15 min. Immunofluorescence was analyzed under an inverted fluorescence microscope (Olympus BX53).

SPSS 20.0 (IBM Corp. Armonk, NY, United States) and Prism 8 (GraphPad Software, CA, United States) were used for statistical analysis and plotting. Data were expressed as mean ± standard deviation (sd). Unpaired Student’s t-test was used to determine the significance between two groups.

First, to investigate the effects of OT pretreatment on cardiac hypertrophy, NRCMs were incubated and pretreated with OT for 30 min and then stimulated with 10 uM ISO for 24 h. Cardiomyocyte hypertrophy was evaluated by cell surface area and expressions of BNP and β-MHC, which have been well-established as cardiac hypertrophy markers. As shown in Figure 2, ISO triggered significant hypertrophic responses in NRCMs, as indicated by the increased cell surface area (Figures 2A,B) and elevated protein levels of BNP and β-MHC (Figures 2C,D,K), which were reduced by pretreatment of OT. At the same time, we observed the expressions of lncRNA GAS5 (Figure 2E), Klf4 mRNA (Figure 2G) and KLF4 protein (Figures 2H,K) were down-regulated in ISO group when compared with control group, while up-regulated significantly after OT treatment. Inversely, the expression of miR-375-3p (Figure 2F) was increased significantly in ISO group when compared with control group and decreased after OT treatment. We also observed that OT modulated PI3K/AKT signaling pathway in a cardiomyocyte hypertrophy model. As shown in Figures 2I−K, the ratios of phosphorylated-PI3K/PI3K (p-PI3K/PI3K) and phosphorylated-AKT1/AKT1 (p-AKT1/AKT1) were significantly increased in ISO-induced hypertrophic cardiomyocytes and significantly decreased after OT treatment.

These results indicated that oxytocin could attenuate cardiomyocyte hypertrophic responses, promote the expressions of lncRNA GAS5 and KLF4 protein, repress miR-375-3p expression, and modulate PI3K/AKT signaling pathway.

To evaluate the effects of OT on cardiac hypertrophy in vivo, we established a rat cardiac hypertrophy model by ISO subcutaneous infusion for 14 days and treated rats with different dosages of OT before (preconditioning) and after (postconditioning) ISO infusions. Heart weight/body weight (HW/BW) ratio served as a measurement of cardiac hypertrophy. HW/BW ratio was significantly increased in ISO group compared to control group, but significant lower in ISO+OT (3) post group than that in ISO group (Figure 3A). HW/BW ratio was not significantly different in ISO+OT (0.03) post group and in ISO+OT (3) pre group when compared with ISO group (Figure 3A). Furthermore, no significant difference in HW/BW ratio was found between control group and OT (3) group (Figure 3A). Besides, ISO markedly increased the expressions of pathological cardiac hypertrophy markers (BNP, β-MHC) and fibrosis marker (Alpha-smooth muscle actin, α-SMA) compared with control group, whereas different concentrations of OT preconditioning or postconditioning inhibited ISO-induced increases in BNP, β-MHC and α-SMA (Figures 3B–D). Likewise, only high dosage (3 ug/kg) of OT postconditioning could significantly reduce expressions of hypertrophic and fibrosis markers (Figures 3B–D). From HE staining (Figure 3G), we observed a large number of inflammatory cells and fibroblasts gathered around endocardium region in ISO group and OT treatment alleviated it. Masson’s trichrome staining (Figure 3G) showed a significant increase in collagen deposition in ISO-infusion group and reduced in OT (3 ug/kg) postconditioning group (Figures 3E,G). To elucidate the mechanisms underlying the anti-hypertrophic effects of OT, lncRNA GAS5 expressions were detected in left ventricular tissues of different groups. As shown in Figure 3F, lncRNA GAS5 expressions in hypertrophic heart tissues were decreased, while up-regulated significantly in rat hearts treated with OT.

The results of WGA staining (Figure 4A) showed that the cardiomyocytes were not orderly arranged and the cross-sectional areas of cardiomyocytes were significantly enlarged after ISO insulted, while OT (3 ug/kg) postconditioning could prevent these pathomorphological changes (Figure 4B).

We performed transthoracic echocardiography to evaluate cardiac function and left ventricular remodeling (Figure 5A). ISO infusion induced cardiac hypertrophy of rats, as evidenced by increased LVPWd (Figure 5B) and LVPWs (Figure 5C) in ISO group compared with control group. OT (3 ug/kg) postconditioning treatment effectively prevented left ventricular wall from thickening (Figures 5B,C). There were no significant differences in LVIDd (Figure 5D), LVIDs (Figure 5E), LVEF (Figure 5F), and LVFS (Figure 5G) between ISO group and control group, suggesting that cardiac hypertrophy induced in our study was not typical concentric hypertrophy and left ventricular function was not worsened by ISO and not affected by OT.

Based on bioinformatic prediction (Figures 6A,C), we performed dual-luciferase reporter gene assays to validate the interactions between lncRNA GAS5 and miR-375-3p as well as miR-375-3p and Klf4. The results revealed that luciferase values were decreased in WT GAS5+miR-375-3p mimics group, but there was no significant alteration in MUT-GAS5+miR-375-3p mimics group or in mimics-NC+WT-GAS5/MUT-GAS5 groups (Figure 6B). Similarly, after co-transfection of WT Klf4 and miR-375-3p mimics, the luciferase value was decreased significantly when compared with co-transfection of WT Klf4 and miR-375-3p mimics NC group. There was no significant difference in luciferase value between MUT Klf4+mimics NC and MUT Klf4+miR-375-3p mimics groups (Figure 6D). These results confirmed that lncRNA GAS5 could sponge miR-375-3p and Klf4 was a direct target gene of miR-375-3p.

To validate the regulatory relationships among lncRNA GAS5, miR-375-3p and KLF4 in the in vitro model under OT action, NRCMs were transfected with shRNA GAS5 followed by OT pretreatment and ISO stimulation, and expressions of lncRNA GAS5, miR-375-3p and Klf4 mRNA, as well as the protein levels of KLF4, BNP and β-MHC were evaluated. At the beginning, we assessed the transfection efficiency of shRNA GAS5#1, shRNA GAS5#2, shRNA GAS5#3 in cardiomyocytes and selected the shRNA GAS5#3 (shRNA-GAS5) for the subsequent experiments due to the highest efficiency (Figure 7A). The expression of lncRNA GAS5 was significantly decreased in ISO+OT+shRNA-GAS5 group compared with ISO+OT+shRNA-scramble group (Figure 7B), suggesting that knock-down of lncRNA GAS5 was effective and sustained. Compared to ISO+OT+shRNA-scramble group, the expressions of BNP, β-MHC were significantly increased in ISO+OT+shRNA-GAS5 group (Figures 7C,D,J). Meanwhile, we also found that after lncRNA GAS5 knock-down, the expression of miR-375-3p was increased (Figure 7E), while Klf4 mRNA (Figure 7F) and protein (Figures 7G,J) levels were decreased. These results indicated that knock-down of lncRNA GAS5 inhibited anti-hypertrophic effects of oxytocin and that lncRNA GAS5 negatively regulated miR-375-3p and positively regulated Klf4 mRNA and protein.

Subsequently, we co-transfected shRNA GAS5 and miR-375-3p inhibitor to NRCMs followed by OT and ISO treatment. The expression of miRNA-375-3p was markedly down-regulated in ISO+OT+shRNA-GAS5+miR-375-3p inhibitor group compared with ISO+OT+shRNA-GAS5+miR-375-3p inhibitor NC group (Figure 7E). The inhibiting effects of shRNA-GAS5 on Klf4 mRNA and protein expressions were reversed by transfection of miR-375-3p inhibitor (Figures 7F,G,J). Meanwhile, the expressions of hypertrophic markers (BNP and β-MHC) (Figures 7C,D,J) as well as the size of the cardiomyocytes evaluated by immunofluorescent staining (Figures 7H,I) were significantly decreased in ISO+OT+shRNA-GAS5+miR-375-3p inhibitor group compared with ISO+OT+shRNA-GAS5+miR-375-3p inhibitor NC group. These results indicated that miR-375-3p inhibitor rescued the pro-hypertrophic effects of shRNA-GAS5.

Taken together, these data demonstrated that lncRNA GAS5 mediated anti-hypertrophic effects of OT through negative regulation of miR-375-3p expression.

To further verify whether down-regulation of miR-375-3p mediated the anti-hypertrophic effects of OT, we upregulated miR-375-3p by transfection of miR-375-3p mimics into NRCMs followed by OT pretreatment and ISO insult. The transfection efficiency of miR-375-3p mimics in cardiomyocytes was detected and demonstrated to be effective (Figure 8A). Forced expression of miR-375-3p was observed in ISO+OT+miR-375-3p mimics group, but not in ISO+OT+miR-375-3p mimics NC group (Figure 8B). As compared with ISO+OT+miR-375-3p mimics NC group, ISO+OT+miR-375-3p mimics group presented notable increases in BNP and β-MHC expressions (Figures 8C,D,J), along with remarkable decreases in Klf4 gene (Figure 8E) and protein (Figures 8F,J), suggesting that upregulation of miR-375-3p dampened anti-hypertrophic effects of OT and miR-375-3p could negatively regulate expressions of KLF4. Rescue assays were carried out to test whether upregulation of Klf4 could abolish the effects conferred by miR-375-3p mimics. We co-transfected miR-375-3p mimics and pcDNA-Klf4 vector or empty pcDNA vector into NRCMs followed by OT and ISO treatments. The results showed that the expression of Klf4 mRNA was significantly increased in ISO+OT+miR-375-3p mimics+pcDNA-Klf4 group compared to ISO+OT+miR-375-3p mimics+pcDNA-NC group (Figure 8E), whereas the expressions of KLF4 protein of two groups were not statistically significant (Figures 8F,J). β-MHC protein was reduced observably in ISO+OT+miR-375-3p mimics+pcDNA-Klf4 group compared with ISO+OT+miR-375-3p mimics+pcDNA-NC group (Figures 8D,J), while BNP protein was not significantly reversed (Figures 8C,J). Immunofluorescent staining was also used to detect the changes in cardiomyocyte sizes (Figure 8K). Cardiomyocytes were markedly enlarged in ISO+OT+miR-375-3p mimics group compared with ISO+OT+miR-375-3p mimics NC group. Compared with ISO+OT+miR-375-3p mimics+pcDNA NC group, the sizes of cardiomyocytes were diminished notably in ISO+OT+miR-375-3p mimics+pcDNA-Klf4 group (Figures 8I,K).

Furthermore, we also evaluated the protein expressions of p-PI3K, PI3K, p-AKT1, AKT1 after transfection of miR-375-3p mimics and co-transfection of miR-375-3p mimics and pcDNA-Klf4, respectively. The ratios of p-PI3K/PI3K (Figures 8G,J) and p-AKT1/AKT1 (Figures 8H,J) were significantly increased in ISO+OT+miR-375-3p mimics group compared with ISO+OT+miR-375-3p mimics NC group, suggesting that PI3K/AKT signaling pathway was also regulated by miR-375-3p. When the cells were simultaneously transfected with miR-375-3p mimics and pcDNA-Klf4, the ratio of p-AKT1/AKT1 (Figures 8H,J) was significantly reduced, whereas p-PI3K/PI3K (Figures 8G,J) was reduced but not statistically significant.

We first assessed the interference efficiency of shRNA Klf4#1, shRNA Klf4#2, and shRNA Klf4#3 in cardiomyocytes and then selected the shRNA Klf4#3 for the subsequent experiments based on its highest efficiency of interference (Figures 9A–C). We transfected shRNA-Klf4 #3 (shRNA-Klf4) and its negative scramble into NRCMs followed by OT pretreatment and ISO stimulation. The expressions of Klf4 mRNA and protein were significantly lower in ISO+OT+shRNA-Klf4 group than those in ISO+OT+shRNA-NC group (Figures 9D,E,L). The anti-hypertrophic effects of OT was dampened in ISO+OT+shRNA-Klf4 group compared with ISO+OT+shRNA-NC group, as shown by higher protein expressions of BNP and β-MHC (Figures 9F,G,L), and larger surface areas of cardiomyocytes observed in ISO+OT+shRNA-Klf4 group (Figures 9J,K).

The ratios of p-PI3K/PI3K (Figures 9H,L) and p-AKT1/AKT1 (Figures 9I,L) in ISO+OT+shRNA-Klf4 group were significantly higher than those in ISO+OT+shRNA-NC group, suggesting PI3K/AKT pathway was modulated by KLF4. After transfecting shRNA-Klf4 into NRCMs, we pretreated NRCMs with 10 uM LY194002 (a PI3K inhibitor that prevents PI3K phosphorylation) and then treated with OT and ISO. The results revealed that the ratios of p-PI3K/PI3K (Figures 9H,L) and p-AKT1/AKT1 (Figures 9I,L) were notably lower in ISO+OT+shRNA-Klf4+LY194002 group than those in ISO+OT+shRNA-Klf4 group. The expressions of BNP and β-MHC (Figures 9F,G,L) as well as the sizes of cardiomyocytes were remarkably reduced in ISO+OT+shRNA-Klf4+LY194002 group compared with ISO+OT+shRNA-Klf4 group (Figures 9J,K). These findings suggested that blockade of PI3K/AKT signaling pathway rescued the pro-hypertrophic effects of shRNA- Klf4.

The above results indicated that KLF4 mediated the anti-hypertrophic effects of OT, and PI3K/AKT is the downstream signaling pathway of miR-375-3p/KLF4 axis.