Syringic acid mitigates isoproterenol‐induced cardiac hypertrophy and fibrosis by downregulating Ereg

Abstract Gallic acid has been reported to mitigate cardiac hypertrophy, fibrosis and arterial hypertension. The effects of syringic acid, a derivative of gallic acid, on cardiac hypertrophy and fibrosis have not been previously investigated. This study aimed to examine the effects of syringic acid on isoproterenol‐treated mice and cells. Syringic acid mitigated the isoproterenol‐induced upregulation of heart weight to bodyweight ratio, pathological cardiac remodelling and fibrosis in mice. Picrosirius red staining, quantitative real‐time polymerase chain reaction (qRT‐PCR) and Western blotting analyses revealed that syringic acid markedly downregulated collagen accumulation and fibrosis‐related factors, including Fn1. The results of RNA sequencing analysis of Ereg expression were verified using qRT‐PCR. Syringic acid or transfection with si‐Ereg mitigated the isoproterenol‐induced upregulation of Ereg, Myc and Ngfr. Ereg knockdown mitigated the isoproterenol‐induced upregulation of Nppb and Fn1 and enhancement of cell size. Mechanistically, syringic acid alleviated cardiac hypertrophy and fibrosis by downregulating Ereg. These results suggest that syringic acid is a potential therapeutic agent for cardiac hypertrophy and fibrosis.

In this study, the therapeutic effects of syringic acid on isoproterenol-induced cardiac hypertrophy and fibrosis and the underlying mechanisms were examined. Syringic acid exerted preventive effects on cardiac hypertrophy and fibrosis by downregulating Ereg.

| Animal model of cardiac hypertrophy
All animal procedures were approved by the Animal Experimental To induce cardiac hypertrophy, male CD-1 mice aged 8 weeks with an average bodyweight of approximately 33 g were infused with isoproterenol (25 mg/kg bodyweight/day) using an osmotic minipump (Alzet). Mice were anaesthetised by intraperitoneally injecting ketamine (120 mg/kg bodyweight) and xylazine (6.2 mg/kg bodyweight). The animals were randomly divided into the following three groups (n = 8/group): vehicle-treated sham, isoproterenoltreated and isoproterenol/syringic acid (100 mg/kg bodyweight/ day)-treated groups. Isoproterenol was solubilized in 0.1% ascorbic acid and 0.9% physiological saline, while syringic acid was dissolved in dimethyl sulfoxide (DMSO) and diluted using 0.9% physiological saline.
To determine the preventive effect of syringic acid, mice were pretreated with vehicle (DMSO) or syringic acid (100 mg/kg bodyweight/day; total volume = 400 μl) 7 days before isoproterenol infusion. Next, the animals were co-treated with isoproterenol and syringic acid for 5 days (n = 8 per group).

| Echocardiography
The echocardiography procedure was performed using the Vivid S5

| Histological analysis and Picrosirius red staining
Mice were sacrificed by exposing them to carbon dioxide for 2-3 min. The cardiac tissues were soaked in 3.7% paraformaldehyde and embedded in paraffin. The paraffin-embedded tissues were sectioned to a thickness of 3 μm.
To measure the cross-sectional area, the tissue sections were stained with haematoxylin and eosin (H&E) as previously described. 12 The cross-sectional area was quantified using NIS Elements software (Nikon Eclipse 80i microscope, Tokyo, Japan). Cardiac fibrosis was determined using Picrosirius red staining (Abcam, Cambridge, UK). The rehydrated cardiac tissue sections were completely covered with Picrosirius red solution for 1 h. The samples were rapidly rinsed twice with 0.5% acetic acid solution, followed by rinsing with absolute alcohol for 1 min. Next, the samples were cleared with xylene and mounted using Canada balsam. Digital images were captured under a microscope (Nikon, Japan).

| Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the cardiac tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The isolated RNA (1 μg) was reverse-transcribed into complementary DNA (cDNA) using TOPscript RT DryMIX (Enzynomics, Daejeon, South Korea). The PCR primers used in this study are shown in Table 1. qRT-PCR analysis was performed using a 7500 Real-Time PCR system with the SYBR Green PCR kit (Enzynomics). The expression levels of the target gene were normalized to those of Gapdh (housekeeping gene).

| Western blotting
Total protein was extracted from the cardiac tissues using radioimmunoprecipitation assay lysis buffer (150 mM NaCl, 1% Triton

| Cell culture and cell size measurement
H9c2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS at 5% CO 2 and 37°C in an incubator.
To determine cell size, cells were seeded on coverslips at a density of 1 × 10 4 cells/well, serum-starved overnight, and pretreated with vehicle or syringic acid (10 μM) 3 h before isoproterenol stimulation. Next, the cells were co-treated with isoproterenol (10 μM) and syringic acid for 24 h. Cells were fixed with 3.7% paraformaldehyde for 30 min, washed twice with phosphate-buffered saline, permeabilized with 0.2% Triton X-100 and incubated with Alexa

| RNA sequencing
Total RNA concentration was determined using Quant-IT RiboGreen (#R11490; Invitrogen). The integrity of the total RNA was examined using the TapeStation RNA Screentape (#5067-5576; Agilent, Santa Clara, CA, USA). Only high-quality RNA preparations (RNA integrity number > 7.0) were used for library construction.

| Transfection
To knockdown Ereg, H9c2 cells were transfected with control shortinterfering RNA (siRNA) or siRNA against Ereg (si-Ereg; 100 nM, Bioneer, Daejeon, South Korea) using RNAiMAX reagent for 24 h, following the manufacturer's instructions. The transfected cells were serum-starved overnight and treated with isoproterenol for 9 h.

| Statistical analysis
All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, La Jolla, CA, USA) and IBM-SPSS Statistics for Windows software (ver. 25.0; SPSS Inc., Chicago, IL, USA). The data are expressed as mean ± standard error of mean. The means between two groups were compared using the Student's t-test, whereas those between three or more groups were compared using one-way analysis of variance, followed by Bonferroni post hoc test.

| Syringic acid pretreatment alleviates cardiomyocyte hypertrophy and downregulates the expression of cardiac hypertrophy and fibrosisrelated genes
Syringic acid (4-hydroxy-2,3-dimethoxybenzoic acid), a phenolic compound, is found in fruits and vegetables ( Figure 1A). In this study, the effects of syringic acid on cardiomyocyte hypertrophy were examined.
Syringic acid was not cytotoxic to H9c2 cells up to a concentration of The results of Western blotting analysis were consistent with those of qRT-PCR analysis ( Figure 1H-J).  Figure 3D-E). The ejection fraction in the isoproterenol group was higher than that in the sham group ( Figure 3F).

| Syringic acid alleviates cardiac fibrosis in isoproterenol-treated mice
To evaluate the preventive effect of syringic acid on cardiac fibrosis, Picrosirius red staining, qRT-PCR and Western blotting analyses were performed. Pretreatment with syringic acid markedly mitigated the isoproterenol-induced upregulation of collagen deposition (pink) in the interstitial cardiomyocytes ( Figure 4A,B). The mRNA and protein levels of Col1a1 and Fn1 in the isoproterenoltreated group were higher than those in the sham group. However, syringic acid mitigated the isoproterenol-induced upregulation of Col1a1 and Fn1 mRNA and protein levels ( Figure 4C,D). Western blotting analysis revealed that syringic acid pretreatment mitigated the isoproterenol-induced upregulation of cardiac Col1a1 and Fn1 levels ( Figure 4E-G).

| Syringic acid mitigates isoproterenol-induced upregulation of cardiac Ereg levels
To investigate the syringic acid-regulated genes, RNA-seq was and Myc mRNA levels ( Figure 5A). The upregulation of these three genes was confirmed using qRT-PCR ( Figure 5B-D).

| Ereg knockdown mitigated isoproterenolinduced cardiomyocyte hypertrophy and downregulated fibronectin expression
To examine the effect of Ereg on cardiomyocyte size, cell area was measured after F-actin staining. Cell size was similar between si-Eregtransfected and si-control-transfected cells in the absence of isoproterenol stimulation ( Figure 6A,B). In the si-control-transfected cells, the cell area in the isoproterenol-treated group was significantly higher than that in the vehicle-treated group. The mitigation of isoproterenolinduced increased cell size in the si-Ereg-transfected group was higher than that in the si-control-transfected group ( Figure 6A,B). Western

| DISCUSS ION
This study demonstrated that syringic acid mitigates isoproterenolinduced cardiac hypertrophy and fibrosis by downregulating Ereg

CO N FLI C T O F I NTE R E S T
None of the authors have any conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this research are available from the corresponding author upon request.