Chlorogenic acid attenuates cardiac hypertrophy via up‐regulating Sphingosine‐1‐phosphate receptor1 to inhibit endoplasmic reticulum stress

Abstract

Aims

Cardiac hypertrophy, an adaptive response of the heart to stress overload, is closely associated with heart failure and sudden cardiac death. This study aimed to investigate the therapeutic effects of chlorogenic acid (CGA) on cardiac hypertrophy and elucidate the underlying mechanisms.

Methods and results

To simulate cardiac hypertrophy, myocardial cells were exposed to isoproterenol (ISO, 10 μM). A rat model of ISO‐induced cardiac hypertrophy was also established. The expression levels of cardiac hypertrophy markers, endoplasmic reticulum stress (ERS) markers, and apoptosis markers were measured using quantitative reverse transcription PCR and western blotting. The apoptosis level, size of myocardial cells, and heart tissue pathological changes were determined by terminal deoxynucleotidyl transferase dUTP nick‐end labelling staining, immunofluorescence staining, haematoxylin and eosin staining, and Masson's staining. We found that CGA treatment decreased the size of ISO‐treated H9c2 cells. Moreover, CGA inhibited ISO‐induced up‐regulation of cardiac hypertrophy markers (atrial natriuretic peptide, brain natriuretic peptide, and β‐myosin heavy chain), ERS markers (C/EBP homologous protein, glucose regulatory protein 78, and protein kinase R‐like endoplasmic reticulum kinase), and apoptosis markers (bax and cleaved caspase‐12/9/3) but increased the expression of anti‐apoptosis marker bcl‐2 in a dose‐dependent way (0, 10, 50, and 100 μM). Knockdown of sphingosine‐1‐phosphate receptor 1 (S1pr1) reversed the protective effect of CGA on cardiac hypertrophy, ERS, and apoptosis in vitro (P < 0.05). CGA also restored ISO‐induced inhibition on the AMP‐activated protein kinase (AMPK)/sirtuin 1 (SIRT1) signalling in H9c2 cells, while S1pr1 knockdown abolished these CGA‐induced effects (P < 0.05). CGA (90 mg/kg/day, for six consecutive days) protected rats against cardiac hypertrophy in vivo (P < 0.05).

Conclusions

CGA treatment attenuated ISO‐induced ERS and cardiac hypertrophy by activating the AMPK/SIRT1 pathway via modulation of S1pr1.

Introduction

Cardiac hypertrophy is a cellular response characterized by increased cell volume, up‐regulation of embryonic gene expression, and increased protein synthesis. ¹ , ² , ³ On the one hand, physiological cardiac hypertrophy is generally caused by normal development, pregnancy, and exercise, maintaining the heart's normal physiological function. On the other hand, pathological cardiac hypertrophy occurs upon various pathological stimuli such as cardiomyopathy, hypertension, and myocardial infarction, which will evolve into myocardial fibrosis and cardiac chamber expansion when sustained pathological stimulation exists, eventually leading to heart failure and death. ⁴ Studies have shown that cardiac hypertrophy can cause sustained endoplasmic reticulum (ER) stress (ERS) and eventually lead to apoptosis. ⁵ , ⁶ ERS reactions mainly include ER overload response, unfolded protein response, and steroid regulated cascade reactions. ⁷ Unfolded protein response is the most well‐studied ERS response, which is closely related to the pathogenesis of cardiac hypertrophy. It is mainly manifested in the pause of protein synthesis and the up‐regulation of ER function‐related proteins, which aims to regulate the steady state of ER protein synthesis. ⁸ Persistent and severe ERS triggers ERS‐related apoptosis, resulting in cell damage, which is closely related to neurodegeneration, diabetes, and ischaemia/reperfusion injury. ⁹ The pathogenesis of cardiac hypertrophy is complex and not entirely clear. Therefore, an in‐depth study investigating the underlying mechanism of cardiac hypertrophy is of great significance for its clinical treatment.

Chlorogenic acid (CGA), one of the most abundant polyphenolic compounds in the human diet, is widely found in plants, such as Eucommia ulmoides and honeysuckle. ¹⁰ , ¹¹ CGA can be obtained by eating fruits, vegetables, herbs, and coffee. It has attracted attention due to its antioxidative and anti‐inflammatory properties, as well as its role in the regulation of neuronal apoptosis and autophagy. ¹² , ¹³ A previous study has shown that CGA could prevent isoproterenol (ISO)‐induced hypertrophy in neonatal rat myocytes. ¹⁴ However, the underlying mechanism remains further studied. Sphingosine 1 phosphate (S1p) is a bioactive sphingolipid metabolite that mediates various biological processes by coupling to its membrane protein receptors, the S1p receptors. ¹⁵ S1p receptor 1 (S1pr1) is an S1p receptor that has been shown to protect cardiac function by inhibiting cardiomyocyte autophagy. ¹⁶ However, the effect of S1pr1 on ERS of myocardial hypertrophy remains unclear.

AMP‐activated protein kinase (AMPK) has been reported to maintain energy homeostasis and attenuate ERS. ¹⁷ Sirtuin 1 (SIRT1) has been shown to protect hearts against ERS‐induced cell death, and impairment of the cardiac SIRT1 signalling contributes to the pathogenesis of cardiovascular diseases. ¹⁸ , ¹⁹ What is more, it has been reported that long non‐coding RNA (lncRNA) NBR2 could attenuate angiotensin II‐induced myocardial hypertrophy by reducing ERS through activating the LKB1/AMPK/SIRT1 pathway. ²⁰ Therefore, we speculate that AMPK/SIRT1 signalling may be implicated in the mediation of cardiac hypertrophy by CGA.

In this study, we explored the effects of CGA on improving cardiac hypertrophy both in vivo and in vitro and the underlying mechanisms involved in S1pr1 up‐regulation and AMPK/SIRT1 activation.

Materials and methods

Cell culture

Rat myocardial cells H9c2 were purchased from the American Type Culture Collection (ATCC) (Manassas, USA). The human cardiomyocyte cell line AC16 was purchased from Procell (Wuhan, China). Cells were cultured in ATCC‐formulated Dulbecco's modified Eagle's medium (DMEM) supplemented with a concentration of 10% foetal bovine serum (FBS) (Hyclone, Hudson, USA), in an incubator with 5% CO₂ at 37°C. The 10th‐ to 12th‐generation cells were used for the follow‐up study. Before the experimental treatment, the cells were cultured with serum‐free medium for 24 h and then pre‐treated with different concentrations of CGA (HY‐N0055, purity: 99.29%, MCE, NJ, USA) for 2 h and next administered with 10 μM ISO (Henry Schein, Melville, NY, USA) until 24 h, in which both CGA and ISO used in the study were dissolved in sterile normal saline. Cells subjected to the combined treatment of CGA and GSK621 (an AMPK agonist) were incubated with 50 μM CGA and 10 μM GSK621 for 2 h.

Cell transfection

The overexpression or down‐regulation of S1pr1 was performed by transfecting lentiviral plasmids carrying S1pr1‐expressing sequence (oe‐S1pr1; GenePharma, Shanghai, China) or short‐hairpin RNA (shRNA) interference (GenePharma), respectively, using a Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, USA). In 24‐well plates, 1 × 10⁵ H9c2 logarithmic‐phase cells were cultured with 0.5 mL of complete medium with 5% CO₂, at 37°C overnight. The next day, 1 μL Lipofectamine 3000 and 30–50 pmol lentiviral plasmids were dissolved in 150 μL Opti‐MEM, respectively, and mixed. After 5 min, the two solutions were mixed evenly. The complete medium in 24‐well plates was replaced by 400 μL of serum‐free medium, and then the transformation solution was added. After being cultured for 6 h, add the complete medium to be transfected for 48 h continuously.

3(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay

The cells were incubated in 96‐well plates (2000 cells per well) for 12 h, with 5% CO₂, at 37°C. After treatment with the corresponding drug, 10 μL of 3(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) with a concentration of 10 mg/mL was added into each well. The plates were incubated continuously for 4 h. Then, 150 μL of dimethylsulfoxide (DMSO) was added to every well for 10 min. Finally, the absorbance value at 490 nm was measured by using an enzyme immunoassay analyser (Thermo Fisher Scientific).

Determination of cell size

At room temperature, H9c2 cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X‐100 (Absin, Shanghai, China) for 15 min, washed with phosphate buffered saline (PBS) three times, and then sealed with 3% bovine serum albumin for 45 min. Cell morphology was visualized by incubating cells with fluorescein isothiocyanate (FITC)‐labelled alpha‐actin (Sigma‐Aldrich, St. Louis, USA) at room temperature for 45 min in the dark and observed under a fluorescent microscope. The cross‐sectional areas were measured by the Image‐Pro Plus 5.1 (Media Cybernetics, Silver Spring, USA).

Quantitative reverse transcription PCR

Total RNA was extracted from H9c2 cells and rat heart tissues by TRIzol reagent (Thermo Fisher Scientific). The TransScript First‐Strand cDNA Synthesis Super Mix Kit (TransGen Biotech, Beijing, China) was used for reverse transcription of first‐strand cDNA. Quantitative reverse transcription PCR (qRT‐PCR) was performed by the Multiplex Quantitative PCR MX3000p System (Stratagene, La Jolla, USA) with SYBR Green Mix (Dongshen Biotech, Guangzhou, China). The primers (Sangon, Shanghai, China) used for qRT‐PCR are listed in Table 1 . The relative expression was determined by 2^−ΔΔCt analysis, with GAPDH as the internal reference.

Table 1.

Primer information

Gene name	Sequence (5′–3′)
S1pr1 (rat)	Forward	CCT CGG TGG TGT TCA TTC
	Reverse	GCA GGT TAG CTG TGT AGG
ANP (rat)	Forward	CTC CTT CTC CAT CAC CCT G
	Reverse	GTT GAC CTC CCC AGT CCA
BNP (rat)	Forward	CTG GGA AGT CCT AGC CAG TCT CCA
	Reverse	GCG ACT GAC TGC GCC GAT CCG GTC
β‐MHC (rat)	Forward	GCA GAC AGA GAA TGG GGA GCT GTC C
	Reverse	TCG CAA TCA TGC CGG GCT GAC
CHOP (rat)	Forward	GGG AAA CAG CGC ATG AAG
	Reverse	GCG TGA TGG TGC TGG GTA CA
GRP78 (rat)	Forward	TTC CGA GGA ACA CTG TGG TG
	Reverse	GTC AGG GGT CGT TCA CCT TC
GAPDH (rat)	Forward	CCA TCA ACG ACC CCT TCA TT
	Reverse	GAC CAG CTT CCC ATT CTC AG
ANP (human)	Forward	ACC GTG AGC TTC CTC CTT TT
	Reverse	GGG CAC GAC CTC ATC TTC TA
BNP (human)	Forward	TGG AAA CGT CCG GGT TAC AG
	Reverse	CTG ATC CGG TCC ATC TTC CT
β‐MHC (human)	Forward	GGT GAA AGT GGG CAA TGA GT
	Reverse	TGG TGA AGT TGA TGC AGA GC
S1PR1 (human)	Forward	CCA CAA CGG GAG CAA TAA CT
	Reverse	CAG AAT GAC GAT GGA GAG CA
GAPDH (human)	Forward	AGG TCG GAG TCA ACG GAT TT
	Reverse	TGA CGG TGC CAT GGA ATT TG

ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CHOP, C/EBP homologous protein; GRP78, glucose regulatory protein 78; S1pr1, sphingosine‐1‐phosphate receptor 1; β‐MHC, β‐myosin heavy chain.

Western blotting

H9c2 cells were lysed with cold radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) after being washed twice with PBS. Heart tissues were grinned with liquid nitrogen and then treated with a cold RIPA lysis buffer. The insoluble material was removed by microcentrifugation. The BCA kit (Beyotime) was used to quantify the concentration. The proteins were separated by 10–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific). After blocking with 5% non‐fat milk, the membranes were incubated overnight at 4°C with primary antibodies against atrial natriuretic peptide (ANP; 1:1000; ab225844, Abcam, Cambridge, USA), brain natriuretic peptide (BNP; 1:500; ab19645, Abcam), β‐myosin heavy chain (β‐MHC; 1:500; A7564, ABclonal, Wuhan, China), S1pr1 (1:1000; PA1‐1040, Thermo Fisher Scientific), bax (1:2000; ab32503, Abcam), bcl‐2 (1:1000; ab196495, Abcam), cleaved caspase‐12 (1:1000; ab62484, Abcam), cleaved caspase‐9 (1:1000; 9507, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase‐3 (1:1000; 9661S, Cell Signaling Technology), C/EBP homologous protein (CHOP; 1:1000; 2895, Cell Signaling Technology), glucose regulatory protein 78 (GRP78; 1:2000; ab21685, Abcam), protein kinase R‐like ER kinase (PERK, 1:1000; 3192S, Cell Signaling Technology), phosphorylated PERK (p‐PERK; 1:2000; 3179S, Cell Signaling Technology), AMPK (1:1000; ab80039, Abcam), phosphorylated AMPK (p‐AMPK) (1:1000; ab131357, Abcam), SIRT1 (1:1000; ab110304, Abcam), and GAPDH (1:10 000; ab181602, ab8245, Abcam) separately. After being incubated with the corresponding secondary antibodies, the membranes were detected by an enhanced chemiluminescence detection system. The expression was analysed by Image‐Pro Plus, with GAPDH as the internal reference.

Animals and treatments

Forty male Sprague Dawley rats (aged 5 weeks) were housed in the specific pathogen‐free animal facilities. Two weeks after feeding, the rats were randomized into the following four groups: control, CGA, ISO, and ISO + CGA. Rats in the ISO and ISO + CGA groups were modelled by subcutaneous injection of ISO (5 mg/kg/day) for seven consecutive days to induce cardiac hypertrophy. Rats in the ISO + CGA group were intragastrically administered with CGA (90 mg/kg/day, for six consecutive days) 1 day after the first ISO injection. Adequate clean food and water, meeting the national standard GB 14924.3‐2010, should be provided for rats during feeding. According to the reports by Huang et al., the concentration of CGA in this study was determined. ²¹ Seven days later, the rats were euthanized by cervical dislocation. After euthanasia, the thoracic cavity of rats was opened quickly, the heart was taken out, the surrounding big blood vessels were cut off, the blood stains were washed with pre‐cooled normal saline, the water was absorbed by filter paper, and the whole heart wet weight was measured by electronic balance. The heart samples were divided into the following two groups: half samples were embedded into wax blocks, and the other half samples were stored in liquid nitrogen until use for western blotting and qRT‐PCR detection. Animal assays conducted in this study were reviewed and approved by the Ethics Committee of Xinxiang Medical University (No. XYLL‐2020164).

Measurement of blood pressure in rats

Blood pressure was measured before all rats were killed. The systolic blood pressure (SBP) was measured by tail artery indirect measurement (tail cuff method) under the conditions of being quiet and awake. First, connect the sphygmomanometer pulse compression sleeve, pressure transducer, and blood pressure measurement software system according to the instructions. Then, put the sphygmomanometer pulse compression sleeve and pressure transducer on the third of the root of the rat tail for inflation and deflation training to make it adapt for a moment. At the same time, put the rat in the heater to raise its temperature to expand the tail artery for blood pressure measurement. When the tail feels warm, the skin is pink, and the tail vein dilation is visible, continue to pressurize the balloon until the blood flow of the tail artery is blocked, continue to pressurize until the blood pressure metre reaches 200 mmHg, and then release the gas in the balloon evenly and slowly. At the same time, read the value in the software system. The first peak after the blood flow of the tail artery is read as the systolic pressure of the tail artery; repeat this 10 times, remove the too high and too low values, and take the average value of the next result. Blood pressure was measured by the same person during the whole experiment.

Haematoxylin and eosin staining

The heart tissues were fixed in 10% formalin, subjected to routine alcohol–xylol processes, and embedded in paraffin. Samples were cut into 5‐mm‐thick sections. Subsequently, they were dehydrated by being treated with xylene three times each for 10 min and then with gradient ethanol one by one. After that, the sections were immersed in a haematoxylin solution for 1 h and washed with distilled water two to three times. Then, they were treated with a hydrochloric acid and alcohol differentiation solution and distilled water to reverse the blue. Further, the sections were treated with eosin solution for 3–5 s, gradient ethanol, and xylene in turn. Phosphomolybdic acid aqueous solution for 5 min, aniline blue dye for 6–8 min, 1% glacial acetic acid for 2–3 min, 95% ethanol for 4–5 s, and xylene in turn. After that, the cover slips were blocked by a cover slip with neutral balsam and the sections were observed under a light microscope (Nikon Eclipse TS100; Nikon).

Masson's staining

The section pre‐treatment was the same as that in haematoxylin and eosin (H&E) staining. The sections were dehydrated by being treated with xylene three times each for 10 min and then with gradient ethanol one by one. Subsequently, they were immersed in potassium dichromate overnight, dyed in a haematoxylin solution for 3 min, and fully washed with a solution composed of ethanol and hydrochloric acid to reverse the blue. Further, the sections were treated with Ponceau and acid fuchsin dye for 10 min, phosphomolybdic acid aqueous solution for 5 min, aniline blue dye for 6–8 min, 1% glacial acetic acid for 2–3 min, 95% ethanol for 4–5 s, and xylene in turn. After that, the cover slips were blocked by a cover slip with neutral balsam and the sections were observed under a light microscope (Nikon Eclipse TS100; Nikon).

Terminal deoxynucleotidyl transferase dUTP nick‐end labelling

Formalin‐fixed paraffin‐embedded (FFPE) sections were made by a slicer (Leica, Germany) and then attached to the slides pre‐treated with poly‐l‐lysine. They were treated with gradient ethanol in turn to transform them into water. Further, a terminal deoxynucleotidyl transferase dUTP nick‐end labelling (TUNEL) experiment was performed by using TUNEL kit purchased from Roche (Switzerland) according to the user guide. Briefly, immerse the dewaxed sections in 0.01 M Tris‐buffered saline (TBS) containing 1:200 of Proteinase K at 37°C for 10 min. After being fully washed with 0.01 M TBS, 20 μL of labelling buffer with 1 μL TdT and DIG‐d‐UTP was added to the slides at 37°C for 2 h. Next, 50 μL of blocking buffer was used to block the slides, which were then incubated with hybridization buffer containing an anti‐digoxin antibody labelled by biotin at 37°C for 30 min. Finally, add 50 μL buffer containing 1:100 of streptavidin–biotin complex (SABC) to the sections to stain them. A fluorescence microscope (Olympus, Tokyo, Japan) was used to identify apoptosis.

Statistical analysis

All results were presented as mean ± SD. All data analyses were performed using the SPSS 19.0 software (IBM Inc., Chicago, USA). The Shapiro–Wilk test was used to test the data normality. The unpaired two‐tailed Student's t‐test was used for statistically significant differences between two groups. A one‐way ANOVA was used for statistically significant differences among several groups. *P < 0.05, ^** P < 0.01, and ^*** P < 0.001 indicated statistical significance. All cell culture experiments were repeated three times, and all animal experiments were repeated five times.

Results

Chlorogenic acid improved hypertrophy of isoproterenol‐treated myocardial cells via activating the AMP‐activated protein kinase pathway

To investigate the optimal concentration of CGA, we set up different concentration gradients of CGA to treat H9c2 cells. After induction for 24 h, the MTT assay was used to detect H9c2 cell viability. Different concentrations of CGA had no obvious effect on the activity of H9c2 cells (Figure 1 A ). Because the effect of CGA on cell viability is <10% when its concentration is lower than 150 μM, we selected 10, 50, and 100 μM concentrations of CGA for the following experiments. As shown in Figure 1 B,C , CGA alleviated ISO‐induced cell hypertrophy. What is more, ISO treatment significantly increased the expression of both mRNA and protein of the cardiac hypertrophy markers (ANP, BNP, and β‐MHC), which was reversed by CGA treatment in a concentration‐dependent manner (Figure 1 D–F,H ). According to previous reports, the expression of S1pr1 was significantly down‐regulated in cardiac hypertrophy induced by ISO treatment. ¹⁶ , ²² Consistent with previous reports, the result showed that S1pr1 expression decreased by ISO treatment, while CGA treatment could alleviate this decline in a dose‐dependent manner (Figure 1 G,H ). We also examined the effects of CGA treatment on AC16 human cardiomyocytes. The results showed that ISO significantly increased the mRNA levels of ANP (Supporting Information, Figure S1 A), BNP (Supporting Information, Figure S1 B), and β‐MHC (Supporting Information, Figure S1 C) but down‐regulated the mRNA expression of S1pr1 (Supporting Information, Figure S1 D) in AC16 cells, while CGA treatment effectively reversed the effects of ISO. Consistently, western blotting analysis showed that CGA treatment inhibited ISO‐induced up‐regulation of the hypertrophy markers' protein levels but restored ISO‐inhibited expression of S1pr1 in AC16 cells (Supporting Information, Figure S1 E). To examine whether AMPK could enhance the effect of CGA on improving cardiac hypertrophy, we selected a medium dose (50 μM) of CGA combined with an AMPK agonist (GSK621) to treat cells. The effects of CGA and GSK621 on ISO‐induced cardiomyocyte hypertrophy were investigated. ISO significantly up‐regulated the mRNA levels of cardiac hypertrophy markers (Supporting Information, Figure S2 A–C) in H9c2 cells, while the effects of ISO were reversed by both CGA and GSK621 alone. The combined treatment with CGA and GSK621 further suppressed ISO‐induced up‐regulation of cardiac hypertrophy markers in H9c2 cells. In line with the qRT‐PCR results, western blotting analysis revealed that CGA or GSK621 reversed the effects of ISO on hypertrophy markers, and the combination of CGA and GSK621 further enhanced the inhibitory effect of CGA on these proteins (Supporting Information, Figure S2 D). These data suggested that an AMPK agonist (GSK621) significantly enhanced the effect of CGA in ameliorating cardiomyocyte hypertrophy. Collectively, the above results indicated that CGA might play an important role in cardiac hypertrophy via activating the AMPK pathway and regulating S1pr1.

Figure 1.

CGA improved hypertrophy of H9c2 cells treated with ISO. (A) Effects of different CGA concentrations on H9c2 cell viability detected by MTT. (B) H9c2 cell morphology observed under fluorescence microscope (scale, 50 μm). (C) Area values of H9c2 cells in each group with different drug treatment. (D) ANP expression level measured by qRT‐PCR. (E) BNP expression level tested by qRT‐PCR. (F) β‐MHC expression level detected by qRT‐PCR. (G) S1pr1 expression level measured by qRT‐PCR. (H) The expression of ANP, BNP, β‐MHC, and S1pr1 proteins detected by western blotting. n = 3 per group, *P < 0.05, ^** P < 0.01, and ^*** P < 0.001.

Chlorogenic acid inhibited endoplasmic reticulum stress and apoptosis of H9c2 cells treated with isoproterenol

It is known that the up‐regulation of ERS levels during myocardial hypertrophy can result in myocardial cell apoptosis. To confirm the regulatory mechanism of CGA on ISO‐induced cardiac hypertrophy, the ERS and apoptosis levels in H9c2 cells after ISO induction were determined. Both mRNA and protein levels of the ERS markers, CHOP and GRP78, in the cardiac hypertrophy cell model induced by ISO increased significantly, which was reversed by CGA treatment in a concentration‐dependent manner (Figure 2 A–C ). The ERS signal factor PERK also exhibited a similar trend (Figure 2 C ). In addition, ISO promoted the apoptosis level of H9c2 cells, which was reversed by adding CGA (Figure 3 A ). The expression levels of apoptosis‐related factors, bax, cleaved caspase‐12, cleaved caspase‐9, and cleaved caspase‐3, significantly increased, but the expression of bcl‐2 significantly decreased in the cardiac hypertrophy model induced by ISO. However, the expression changes of these factors could be reversed by CGA treatment (Figure 3 B ). These data indicated that CGA treatment reduced ERS and apoptosis in ISO‐treated H9c2 cells.

Figure 2.

CGA inhibited ERS of H9c2 cells induced by ISO. (A) CHOP expression level detected by qRT‐PCR after the combination treatment of CGA and ISO in H9c2 cells. (B) GRP78 expression level detected by qRT‐PCR after the combination treatment of CGA and ISO in H9c2 cells. (C) The expression of CHOP, GRP78, and ERS‐related proteins belonging to ERS signal pathway (p‐PERK and PERK) by western blotting. n = 3 per group, *P < 0.05, ^** P < 0.01, and ^*** P < 0.001.

Figure 3.

CGA reversed cell apoptosis of ISO‐stimulated H9c2 cells. (A) The apoptosis in H9c2 cells at different concentrations of ISO and/or CGA. (B) The expressions of bax, bcl‐2, and cleaved caspase‐12/9/3 detected by western blotting. n = 3 per group, *P < 0.05, ^** P < 0.01, and ^*** P < 0.001.

Sphingosine‐1‐phosphate receptor 1 knockdown inhibited the protective effect of chlorogenic acid on cardiac hypertrophy in H9c2 cells

To examine the role of S1pr1 in ISO‐induced cardiac hypertrophy in vitro, we first overexpressed S1pr1 in H9c2 cells. Transfection efficiency was evaluated using qRT‐PCR and western blotting assays. The results showed that oe‐S1pr1 markedly up‐regulated the mRNA and protein expressions of S1pr1 (Supporting Information, Figure S3 A,B). What is more, S1pr1 was also up‐regulated by oe‐S1pr1 in ISO‐treated H9c2 cells (Supporting Information, Figure S3 C). Subsequently, we examined how S1pr1‐overexpressing cells responded to ISO treatment. The results demonstrated that overexpression of S1pr1 suppressed ISO‐induced up‐regulation of cardiac hypertrophy markers, including ANP (Supporting Information, Figure S3 D), BNP (Supporting Information, Figure S3 E), and β‐MHC (Supporting Information, Figure S3 F). Consistently, western blotting analysis showed that overexpression of S1pr1 inhibited ISO‐induced up‐regulation of the ANP, BNP, and β‐MHC proteins (Supporting Information, Figure S3 G). These findings indicated that S1pr1 overexpression diminished the effects of ISO on cardiomyocyte hypertrophy. To further examine the role of S1pr1 in the protective effect of CGA on cardiac hypertrophy, we knocked down S1pr1 in H9c2 cells (Figure 4 A,B ). We found that CGA significantly increased the mRNA (Figure 4 C ) and protein (Figure 4 G ) levels of S1pr1 in ISO‐treated cells, which was abolished by S1pr1 knockdown. Additionally, CGA significantly inhibited the expression of ANP, BNP, and β‐MHC in ISO‐induced H9c2 cells, while the role of CGA was relieved after knockdown of S1pr1 (Figure 4 D–G ).

Figure 4.

Knockdown of S1pr1 inhibited the protective effect of CGA on cardiac hypertrophy in vitro. (A) S1pr1 expression was down‐regulated by gene silencing, which was determined by qRT‐PCR. (B) Protein levels of S1pr1 after S1pr1 knockdown detected by western blotting. (C) S1pr1 expression measured by qRT‐PCR. (D) ANP expression tested by qRT‐PCR. (E) BNP expression assessed by qRT‐PCR. (F) β‐MHC expression detected by qRT‐PCR. (G) The expression of ANP, BNP, β‐MHC, and S1pr1 proteins detected by western blotting. n = 3 per group, *P < 0.05, ^** P < 0.01, and ^*** P < 0.001.

Sphingosine‐1‐phosphate receptor 1 knockdown abolished the inhibition effect of chlorogenic acid on endoplasmic reticulum stress and cell apoptosis in vitro

Then we further confirmed the role of S1pr1 in the inhibitory effect of CGA on ERS in vitro. The down‐regulation of mRNA and protein levels of CHOP and GRP78, and the protein level of p‐PERK, caused by CGA was reversed by S1pr1 inhibition in cardiac hypertrophy cells (Figure 5 A–C ). CGA reversed ISO‐induced the inactivating effects on AMPK/SIRT1 signalling in H9c2 cells, while S1pr1 knockdown abolished the effects of CGA (Figure 5 D ). Moreover, the inhibitory effect of CGA on cell apoptosis disappeared when S1pr1 expression was down‐regulated (Figure 6 A,B ). These experimental results indicated that CGA inhibited ERS, activated the AMPK/SIRT1 pathway, and repressed cell apoptosis through increasing S1pr1 in cardiac hypertrophy cells.

Figure 5.

S1pr1 knockdown blocked the improvement of CGA on ERS level in H9c2 cells. (A) CHOP expression evaluated by qRT‐PCR. (B) GRP78 expression measured by qRT‐PCR. (C) Protein expression levels of CHOP, GRP78, p‐PERK, and PERK detected by western blotting. (D) The expression of p‐AMPK, AMPK, and SIRT1 detected by western blotting. n = 3 per group, *P < 0.05, ^** P < 0.01, and ^*** P < 0.001.

Figure 6.

S1pr1 knockdown blocked the inhibition of CGA on cell apoptosis of H9c2 cells. (A) The apoptosis of H9c2 cells at different concentrations of ISO and/or CGA in which S1pr1 expression was down‐regulated. (B) The expression of bax, bcl‐2, and cleaved caspase‐12/9/3 detected by western blotting. n = 3 per group, *P < 0.05, ^** P < 0.01, and ^*** P < 0.001.

Chlorogenic acid alleviated cardiac hypertrophy in vivo

To investigate whether CGA plays a role in treating cardiac hypertrophy in vivo, we constructed a cardiac hypertrophy rat model and treated it with CGA. Firstly, the myocardial hypertrophy of rats induced by ISO was measured by the ratios of heart weight to body weight. The results showed that CGA had a therapeutic effect on cardiac hypertrophy (Figure 7 A ). In addition, we monitored the changes in blood pressure in rats. The blood pressure of the CGA group was similar to that of the control group. ISO treatment increased the blood pressure more than that of the control group. When CGA and ISO were used together, the effect of elevated blood pressure by ISO was restored (Figure 7 A ). The size of the heart was significantly decreased in rats treated with CGA in ISO‐induced rats (Figure 7 B ). The H&E staining revealed that myocardial cells were irregularly arranged and the cell size was significantly increased by ISO treatment, which was alleviated after being treated with CGA (Figure 7 C ). Masson's staining and TUNEL staining found more collagen fibres (Figure 7 C ) and less apoptosis (Figure 7 D ), respectively, in rats treated with CGA compared with the ISO group. Moreover, the up‐regulated effects of cardiac hypertrophic markers caused by ISO treatment were reversed by CGA (Figure 7 E,F ).

Figure 7.

CGA alleviates cardiac hypertrophy in vivo. (A) Comparison of heart weights and blood pressure among experimental groups. (B) Pictures of heart size and morphology from different experimental groups. (C) H&E and Masson's staining results of rat cardiac tissues. (D) TUNEL staining was employed to determine apoptosis level of rat cardiac tissues. (E) qRT‐PCR was used to detect mRNA levels of ANP, BNP, and β‐MHC in rat heart tissues. (F) Western blotting was used to detect protein levels of ANP, BNP, and β‐MHC in rat heart tissues. (G) The mRNA levels of S1pr1, CHOP, and GRP78 detected by qRT‐PCR in rat heart tissues. (H) The protein levels of S1pr1, CHOP, GRP78, p‐AMPK, AMPK, and SIRT1 detected by western blotting in rat heart tissues. (I) The protein levels of bax, bcl‐2, and cleaved caspase‐12/9/3 detected by western blotting in rat heart tissues. n = 10 per group, *P < 0.05, ^** P < 0.01, and ^*** P < 0.001.

To determine the underlying mechanism of CGA in improving cardiac hypertrophy, the level of markers related to ERS, AMPK/SIRT1 signalling, and apoptosis in the hearts of ISO‐treated rats was examined. It could be seen that ISO treatment significantly down‐regulated S1pr1 and up‐regulated CHOP and GRP78 in rat cardiac tissues, while CGA treatment restored S1pr1 expression and decreased the levels of CHOP and GRP78 (Figure 7 G,H ). CGA treatment also abrogated the inhibitory effect of ISO on phosphorylation of AMPK and SIRT1 expression (Figure 7 H ). In addition, CGA significantly impeded ISO‐induced apoptosis in rat hearts (Figure 7 I ). Taken together, these findings supported the statement that CGA played a protective role in cardiac hypertrophy in vivo.

Discussion

Cardiac hypertrophy is one of the risk factors for cardiovascular diseases, which often leads to increased morbidity and mortality. ²³ However, the pathogenic mechanisms remain elusive. Currently, there are many drugs to treat cardiac hypertrophy, including angiotensin‐converting enzyme inhibitors, beta‐blockers, calcium channel blockers, diuretics, and angiotensin receptor inhibitors. ⁴ However, the current treatment still has some disadvantages and new therapeutic drugs need to be developed. In the current study, we showed that CGA protected against cardiac hypertrophy and reduced ERS by up‐regulating the S1pr1/AMPK/SIRT1 axis.

Previous studies have found that CGA improved ventricular remodelling in mice after myocardial infarction. ²⁴ CGA also protected cardiomyocytes from oxidative emergency damage caused by doxorubicin. ²⁵ In addition, CGA was found to prevent ISO‐induced hypertrophy in neonatal rat myocytes by reducing the expression level of cardiac hypertrophic markers. ¹⁴ The present study elucidated the protective mechanism of CGA in cardiac hypertrophy. We simulated the hypertrophic response of H9c2 cells by ISO and found that CGA inhibited ERS and apoptosis in ISO‐induced H9c2. The protective effect of CGA on cardiac hypertrophy was also observed in the rat model.

S1pr1 plays a critical role in modulating cardiac function under both normal and pathological conditions. Clay et al. found that S1pr1 was required for normal cardiac development. ²⁶ Kuang et al. showed that S1pr1 ameliorated adverse cardiac remodelling after myocardial infarction by inducing reparative macrophage proliferation. ²⁷ Liu et al. reported that endothelial‐specific deletion of S1pr1 significantly aggravated cardiac dysfunction and deteriorated cardiac hypertrophy and fibrosis. ²⁸ In addition, transgenic mice with cardiac fibroblasts overexpressing S1PR1 developed bi‐ventricular hypertrophy and diffuse interstitial fibrosis without haemodynamic stress in the heart. ²⁹ A recent study reported that CGA alleviated fructose‐induced non‐alcoholic fatty liver disease in rats through mediating the level of S1pr1. ³⁰ In this study, we found that CGA recovered decreased S1pr1 expression in cardiac hypertrophy model. In addition, S1pr1 knockdown significantly inhibited CGA's therapeutic effects and increased the levels of apoptosis and ERS in cardiomyocytes, confirming the role of S1pr1 in the improvement of cardiac hypertrophy by CGA. Our results also demonstrated that up‐regulation of S1pr1 expression was the main way for CGA to improve cardiomyocyte apoptosis and ERS. Moreover, we performed animal experiments to further verify the above conclusion. Herein, we found that CGA treatment significantly reversed the pathological phenomenon of cardiac hypertrophy, the expression of cardiac hypertrophy markers, and ERS and apoptosis levels induced by ISO in rats. It demonstrated that CGA played an important role in improving cardiac hypertrophy via regulating S1pr1 in vivo and in vitro.

AMPK, as a key mediator of cardiomyocyte energy homeostasis, regulates various processes that are essential for cardiomyocyte survival and health. ³¹ AMPK activation has been shown to prevent excess nutrient‐induced hepatic lipid accumulation by inhibiting ERS response. ³² SIRT1 is a key factor in the AMPK signalling. ³³ The potential of SIRT1 as a therapeutic target for ERS‐induced cardiac injury has been highlighted. ¹⁸ S1pr1 may be an important regulator of AMPK/SIRT1 signalling. It is known that S1pr1 is a receptor for S1p, and the diverse biological effects of S1p primarily depend on the subtypes of coupled protein receptors and the expression of G‐protein receptors in cells. There have been five identified subtypes of S1p receptors, with S1pr1, S1pr2, and S1pr3 being ubiquitously expressed in various tissues in vivo. ³⁴ Previous studies have shown that promoting S1pr1 activity leads to the phosphorylation of AMPK. ³⁵ , ³⁶ Another study reported that S1p activated AMPK through the stimulation of AMPK phosphorylation via its receptor S1pr1/S1pr3. ³⁷ Here, our data showed that the S1pr1 silencing abolished the activating effects of CGA on the AMPK/SIRT1 pathway in cardiac hypertrophy model rats. Therefore, S1pr1/AMPK/SIRT1 was essential for the protection of CGA against cardiac hypertrophy. However, whether CGA directly regulates S1pr1 expression or through which pathway it regulates S1pr1 expression still needs further study.

According to the above studies, CGA may be an effective drug for the therapy of cardiac hypertrophy and has broad clinical prospects. Honeysuckle, Eucommia, and Capillaris are commonly used Chinese herbal medicines for the treatment of cardiovascular system diseases, ¹⁰ , ³⁸ of which CGA is the main active component; thereby, they are effective drugs for the treatment of cardiac hypertrophy. At present, CGA has been used as a food or health product for weight loss. ³⁹ In China, CGA was also used as an antitumor drug for phase II clinical research on advanced recurrent glioblastoma. ⁴⁰ However, there were no studies on the clinical treatment of cardiac hypertrophy with purified CGA. As CGA is an abundant polyphenol in the diet, it is possible to prevent or treat cardiac hypertrophy by oral administration of CGA or the use of foods rich in CGA. In order to further verify the efficacy and safety of purified CGA for the treatment of cardiac hypertrophy, more in‐depth study and identification are needed, especially in clinical trials.

The limitations of this study should also be acknowledged. First, the clinical efficacy of CGA was not evaluated. Secondly, the direct impact of CGA on the S1p (the S1pr1 ligand) levels was not explored. Lastly, the upstream and downstream molecular mechanisms of how CGA regulates S1pr1 were not further investigated.

In summary, our results show that CGA can treat cardiac hypertrophy by inhibiting ERS‐induced apoptosis through up‐regulation of S1pr1 expression. The study provides a new theoretical basis for the future clinical use of CGA as a drug to alleviate cardiac hypertrophy.

Conflict of interest

None declared.

Funding

This work was supported by grants from the Excellent Youth Incubation Program of Chinese People's Liberation Army General Hospital (2020‐YQPY‐007), the Natural Science Foundation of Hainan Province (821QN389, 820QN383, 820MS126, 822MS198, 821MS112, 820MS124, 823MS161, and 821MS117), the 2021 Military Training Injury Prevention Treatment Research Task (21xls37), the Major Science and Technology Programme of Hainan Province (ZDKJ2019012), the National Key R&D Program of China (2018YFC2000400), the National S&T Resource Sharing Service Platform Project of China (YCZYPT[2018]07), the Specific Research Fund of Innovation Platform for Academicians of Hainan Province (YSPTZX202216), the Sanya Medical and Health Science and Technology Innovation Project (2019YW12, 2016YW21, 2018YW10, and 2018YW11), the Heatstroke Treatment and Research Center of PLA (413EGZ1D10), and the Simulation Training for Treatment of Heat Stroke. The sponsors had no role in the design, conduct, interpretation, review, approval, or control of this article.

Supporting information

Figure S1. CGA improved hypertrophy of AC16 cells treated with ISO. A‐D. ANP, BNP, β‐MHC and S1pr1 mRNA levels measured by qRT‐PCR. E. The expression of ANP, BNP, β‐MHC, and S1pr1 proteins detected by Western blotting. n = 3 per group, *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure S2. AMPK agonist (GSK621) enhanced the effect of CGA in ameliorating cardiomyocyte hypertrophy. A‐C. ANP, BNP, β‐MHC and S1pr1 mRNA levels measured by qRT‐PCR. D. The expression of ANP, BNP, and β‐MHC proteins detected by Western blotting. n = 3 per group, *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure S3. S1pr1 overexpression diminished the effects of ISO on cardiomyocyte hypertrophy. A‐B. Overexpression transfection efficiency of the S1pr1 measured by qRT‐PCR and Western blotting. C. S1pr1 mRNA level evaluated by qRT‐PCR. D‐F. ANP, BNP, β‐MHC and S1pr1 mRNA levels measured by qRT‐PCR. G. The expression of ANP, BNP, and β‐MHC proteins detected by Western blotting. n = 3 per group, *P < 0.05, **P < 0.01 and ***P < 0.001.

Ping, P. , Yang, T. , Ning, C. , Zhao, Q. , Zhao, Y. , Yang, T. , Gao, Z. , and Fu, S. (2024) Chlorogenic acid attenuates cardiac hypertrophy via up‐regulating Sphingosine‐1‐phosphate receptor1 to inhibit endoplasmic reticulum stress. ESC Heart Failure, 11: 1580–1593. 10.1002/ehf2.14707.