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Deleterious effect in endothelin receptor–mediated coronary artery smooth muscle contractility in high-salt diet rats

  • Author Footnotes
    1 These authors contributed equally to this work.
    Hui Xiao
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    1 These authors contributed equally to this work.
    Affiliations
    Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Haoyang Lu
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    1 These authors contributed equally to this work.
    Affiliations
    Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China
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  • Author Footnotes
    1 These authors contributed equally to this work.
    Yangcheng Xue
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    1 These authors contributed equally to this work.
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    Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China
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  • Zhuoran Jia
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    Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China
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  • Manyu Dai
    Affiliations
    Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China
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  • Ke He
    Correspondence
    Corresponding author. Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, 218 Jixi Road, Hefei, Anhui 230022, China.
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    Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China
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  • Ren Zhao
    Correspondence
    Corresponding author. Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, 218 Jixi Road, Hefei, Anhui 230022, China. Fax: +86 551 65908455.
    Affiliations
    Department of Cardiology, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, China
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  • Author Footnotes
    1 These authors contributed equally to this work.
Open AccessPublished:October 22, 2022DOI:https://doi.org/10.1016/j.numecd.2022.10.010

      Highlights

      • Coronary vasoconstriction was weakened under high-salt diet condition.
      • The ETR-SOCE-mediated coronary contraction was decreased in high-salt diet rats.
      • Coronary fibrosis was observed in high-salt diet rats.
      • Serum ET-1 level was raised but ETR were decreased in Coronary after high-salt diet.

      Abstract

      Background and aims

      High-salt diet has been suggested to increase the risk of heart disease. However, the mechanisms underlying coronary artery tension dysfunction caused by high-salt diet are unclear. Previous studies have shown that coronary artery spasm is often induced by endothelin-1 (ET-1) and thromboxane, leading to myocardial ischemia, while the store-operated Ca2+ entry (SOCE) function of coronary smooth muscle is very important in this process.

      Methods and results

      Tension measurements of endothelium-denuded coronary artery ring segments showed that vasocontraction induced by U46619, ET-1, orSTIM1/Orai1-mediated SOCE was significantly lower in 4% high-salt diet rats than in control rats fed a regular diet. The results of western blotting and immunohistochemistry assays showed lower expression levels of endothelial receptors ETA and ETB, STIM1 and Orai1 in coronary artery of high-salt intake rats compared with control rats. Fibrosis was observed by using Masson's trichrome staining and picrosirius red staining. The plasma ET-1 concentration in high-salt diet rats was significantly higher than that of controls. The interventricular septum and posterior wall of high-salt diet rats were significantly thickened.

      Conclusion

      Our findings indicated that coronary artery tension was significantly decreased in 4% high-salt diet rats and that this decrease may be due to the change of endothelin receptor and its downstream pathway SOCE related protein expression in coronary artery. Coronary fibrosis was observed in rats fed with high-salt diet. This study provides potential mechanistic insights into high-salt intake–induced heart disease.

      Keywords

      Abbreviations

      ET-1
      Endothelin-1
      U46619
      9, 11-dideoxy-11α, 9α-epoxymetha-nopros-taglandin
      IP3R
      1, 4, 5-triphosphate receptor
      GPCRs
      G protein-coupled receptors
      ETA
      Endothelin receptor type A
      ETB
      Endothelin receptor type B
      STIM1
      Stromal interaction molecule 1
      Orai1
      Ca2+-release-activated Ca2+ channel protein 1
      ER
      endoplasmic reticulum
      SOCE
      Store-operated Ca2+ entry
      VSMCs
      Vascular smooth muscle cells
      IVSd
      Interventricular septum thickness in diastole
      IVSs
      Interventricular septum thickness in systole
      LVIDd
      Left ventricle internal diameter in diastole
      LVIDs
      Left ventricle internal diameter in systole
      LVPWd
      Left ventricle posterior wall thickness in diastole
      LVPWs
      Left ventricle posterior wall thickness in systole
      HR
      Heart rate
      LVEF
      Left ventricle ejection fraction
      LVFS
      Left ventricle fractional shortening

      1. Introduction

      It is well established that high-salt diet is an important cause of cardiovascular disease (CVD) [
      • Strazzullo P.
      • D'Elia L.
      • Kandala N.B.
      • Cappuccio F.P.
      Salt intake, stroke, and cardiovascular disease: meta-analysis of prospective studies.
      ,
      • Baldo M.P.
      • Rodrigues S.L.
      • Mill J.G.
      High salt intake as a multifaceted cardiovascular disease: new support from cellular and molecular evidence.
      ]. A recent study show that the risk of cardiovascular disease was increased in people with an average sodium intake exceed 5 g/day [
      • Zhang J.
      • Guo X.
      • Lu Z.
      • Tang J.
      • Li Y.
      • Xu A.
      • et al.
      Cardiovascular diseases deaths attributable to high sodium intake in Shandong Province, China.
      ], but the abnormal changes in coronary artery tension associated with a high-salt diet were still unknown. In previous experimental studies, researchers have found that serotonin-induced coronary artery contraction is enhanced or reduced in different concentrations of extracellular sodium chloride concentration ([NaCl]), due to activation of the sodium/calcium (Na+/Ca2+) exchanger [
      • Coleman D.A.
      • Khalil R.A.
      Physiologic increases in extracellular sodium salt enhance coronary vasoconstriction and Ca2+ entry.
      ]. It was indicated that the distribution of calcium channels may change in coronary artery smooth muscle layer, which was affected by extracellular sodium concentration. Recent experimental studies have shown that more serious coronary microvessel endothelial dysfunction were induced by high salt intake in Goto Kakizaki rats, because of elevated expression of myocardial endothelin, inducible NO synthase (NOS) protein and 3-nitrotyrosine (3-NT) [
      • Pearson James T.
      • Thambyah Hamish P.
      • Waddingham Mark T.
      • Inagaki T.
      • Sukumaran V.
      • Ngo Jennifer P.
      • et al.
      β-blockade prevents coronary macro- and microvascular dysfunction induced by a high salt diet and insulin resistance in the Goto–Kakizaki rat.
      ]. It was known that high-salt intake upregulated a subset of genes encoding for proteins involved in inflammation, extracellular matrix remodeling and regulation of calcium ions [
      • Wang Q.
      • Domenighetti A.A.
      • Schafer S.C.
      • Weber J.
      • Simon A.
      • Maillard M.P.
      • et al.
      Impact of salt on cardiac differential gene expression and coronary lesion in normotensive mineralocorticoid-treated mice.
      ]. Abnormal protein expression causes some changes in vascular function. There were also many studies that have shown that high salt intake has harmful effects on vascular function, such as increasing peripheral arterial stiffness and impairing endothelial function [
      • Liu Z.
      • Peng J.
      • Lu F.
      • Zhao Y.
      • Wang S.
      • Sun S.
      • et al.
      Salt loading and potassium supplementation: effects on ambulatory arterial stiffness index and endothelin-1 levels in normotensive and mild hypertensive patients.
      ,
      • Zhu J.
      • Yu M.
      • Friesema J.
      • Huang T.
      • Roman R.J.
      • Lombard J.H.
      Salt-induced ANG II suppression impairs the response of cerebral artery smooth muscle cells to prostacyclin.
      ], but the specific pathway on coronaryartery tension disfunction is unclear.
      In the recent study researchers has found that endothelial 1 (ET-1) is significantly increased in plasma of high-salt–diet rats [
      • Speed J.S.
      • D'Angelo G.
      • Wach P.A.
      • Sullivan J.C.
      • Pollock J.S.
      • Pollock D.M.
      High salt diet increases the pressor response to stress in female, but not male ETB-receptor-deficient rats.
      ]. ET-1, as a first messenger that activates G protein–coupled receptor (GPCR) pathways, which acts on the 1, 4, 5-triphosphate receptor (IP3R) to increase the release of Ca2+ from the endoplasmic reticulum and activate store-operated Ca2+ entry (SOCE) [
      • Kato K.
      • Okamura K.
      • Hatta M.
      • Morita H.
      • Kajioka S.
      • Naito S.
      • et al.
      Involvement of IP3-receptor activation in endothelin-1-induced Ca(2+) influx in rat pulmonary small artery.
      ]. SOCE, a new calcium channel complex, primarily causes the contraction of VSMCs by rapidly increasing the intracellular concentration of Ca2+ [
      • Avila-Medina J.
      • Mayoral-Gonzalez I.
      • Dominguez-Rodriguez A.
      • Gallardo-Castillo I.
      • Ribas J.
      • Ordonez A.
      • et al.
      The complex role of store operated calcium entry pathways and related proteins in the function of cardiac, skeletal and vascular smooth muscle cells.
      ]. Previous studies have found that the downstream pathway of ET receptor mediated Ca2+ influx, which was known as STIM1/Orai1-mediatedSOCE pathway, regulates the vascular contractile response of pulmonary, cerebral artery, mesenteric and coronary arteries [
      • Avila-Medina J.
      • Mayoral-Gonzalez I.
      • Dominguez-Rodriguez A.
      • Gallardo-Castillo I.
      • Ribas J.
      • Ordonez A.
      • et al.
      The complex role of store operated calcium entry pathways and related proteins in the function of cardiac, skeletal and vascular smooth muscle cells.
      ,
      • Calderon-Sanchez E.M.
      • Avila-Medina J.
      • Callejo-Garcia P.
      • Fernandez-Velasco M.
      • Ordonez A.
      • Smani T.
      Role of Orai1 and L-type CaV1.2 channels in Endothelin-1 mediated coronary contraction under ischemia and reperfusion.
      ].
      ET-1 can also promote vascular fibrosis, such as in the pulmonary artery [
      • Hartopo A.B.
      • Arfian N.
      • Nakayama K.
      • Suzuki Y.
      • Yagi K.
      • Emoto N.
      Endothelial-derived endothelin-1 promotes pulmonary vascular remodeling in bleomycin-induced pulmonary fibrosis.
      ] and in the internal mammary artery [
      • Sutherland A.J.
      • Nataatmadja M.I.
      • Walker P.J.
      • Cuttle L.
      • Garlick R.B.
      • West M.J.
      Vascular remodeling in the internal mammary artery graft and association with in situ endothelin-1 and receptor expression.
      ], which may lead to vascular remodeling. The expression levels of endothelin type A (ETA) receptors and type B (ETB) receptors in the coronary arteries of patients with ischemic heart disease are significantly increased [
      • Wackenfors A.
      • Emilson M.
      • Ingemansson R.
      • Hortobagyi T.
      • Szok D.
      • Tajti J.
      • et al.
      Ischemic heart disease induces upregulation of endothelin receptor mRNA in human coronary arteries.
      ]. Thus, these results indicated that ET-1 and its downstream SOCE protein may also play an indispensable role in the development of high-salt diet induced coronary heart disease.
      In the present study, we fed the rats with a high-salt diet to investigate the effects of vasoconstrictors, namely, a thromboxane A2 analogue (U46619) and ET-1, on the contractile response of isolated coronary artery ring segments. Coronary artery fibrosis, heart function, and plasma ET-1 concentrations were also measured to identify their functional roles in coronary dysfunction in high-salt diet rats. We aimed to give further insights into the downstream signaling pathway that mediates the effect of endothelin receptor in coronary artery with special emphasis on SOCE function regulated by stromal interaction molecule 1 (STIM1) and Orai1.

      2. Methods

      2.1 Reagents

      ET-1 was obtained from Calbiochem (La Jolla, CA, USA). U46619 and verapamil were obtained from Sigma–Aldrich (St. Louis, MO, USA). The primary rabbit antibodies against the ETA receptor (DF4923) and ETB receptor (DF7104) were purchased from Affinity Biosciences (OH, USA). The primary rabbit antibody against STIM1; GTX113558, and Bosentan were purchased from GeneTex, Inc (St. Antonio, TX, USA). The primary rabbit antibody against Orai1 (sc-74778) was obtained from Santa Cruz Biotechnology (CA, USA). The rat ET-1 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Elabscience Biotechnology Co., Ltd (Wuhan, China).

      2.2 Animal treatment

      Six to eight-week-old male Sprague Dawley rats weighing 150–180 g were randomly placed in a 4% high-salt diet group or a regular-salt diet group and were kept under standard conditions of a 12-h light/dark cycle. Rats had free access to food and tap water. The high-salt diet consisted of 4% (w/w) NaCl, and the regular-salt diet consisted of 0.4% NaCl. The diets were provided for 4 weeks. All animal experiments were performed with approval from the Medical Ethics Committee in Anhui Medical University and followed procedures described in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (publication No. 8523).

      2.3 Coronary artery tension measurement

      Coronary artery tension measurements were performed as described in previous studies [
      • Hedegaard E.R.
      • Nielsen B.D.
      • Kun A.
      • Hughes A.D.
      • Kroigaard C.
      • Mogensen S.
      • et al.
      KV 7 channels are involved in hypoxia-induced vasodilatation of porcine coronary arteries.
      ,
      • Climent B.
      • Santiago E.
      • Sánchez A.
      • Muñoz-Picos M
      • Pérez-Vizcaíno F
      • García-Sacristán A
      • et al.
      Metabolic syndrome inhibits store-operated Ca2+ entry and calcium-induced calcium-release mechanism in coronary artery smooth muscle.
      ]. After the rats were killed with excess carbon dioxide inhalation, the heart was quickly removed from the chest and placed in an oxygenated ice-cold Krebs solution that contained (in mM/L) 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4 (7H2O), 25.2 NaHCO3, and 11.1 glucose. The coronary arteries were dissected from the surrounding heart muscle tissue. The isolated coronary arteries were cut into ring segments 2 mm in length. The endothelial layer was removed by gently rubbing the luminal side of the ring with a stainless-steel wire.
      Each ring segment was mounted in a Multi Myograph system (DMT 620M, Danish Myo Technology, Aarhus, Denmark), and changes in arterial tone were recorded. After the tension was stabilized, the coronary artery rings were stimulated with a high-K+ solution (60 mmol/L KCl solution containing in mM/L: 62.7 NaCl, 60 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4 (7H2O), 25.2 NaHCO3, and 11.1 glucose). When the maximum contraction was reached, the high-K+ solution was replaced with three changes of Krebs solution for 5 min each to achieve a stable baseline tension. After washout, the vessels were stimulated with 100 nM U46619 and 1 μM acetylcholine to examine endothelial integrity. If the effect of the acetylcholine was less than 20% of the maximum contraction caused by U46619, the endothelium was considered successful removed and subsequent experiments were performed. The coronary artery rings were sequentially treated with U46619 and ET-1 to examined concentration-dependent vasocontraction changes. In the SOCE-induced contraction experiments, the coronary artery rings were first pretreated in a Ca2+-free solution (Krebs solution without CaCl2), 100 nM ET-1, and 1 μM verapamil for 10 min, and then the coronary artery contractile response to CaCl2 (from 1 to 10 mM) was recorded.

      2.4 Immunohistochemical staining analysis

      The coronary arteries of the rats were fixed in 4% paraformaldehyde and cut into 10-μm-thick cross sections. These vessel sections were deparaffinized, treated with 0.3% hydrogen peroxide, and then blocked with 10% normal goat serum for 30 min at room temperature. Subsequently, the vessel sections were incubated with a rabbit anti-ETA receptor antibody or a rabbit anti-ETB receptor antibody (all diluted 1:100) overnight at 4 °C. After being washed with phosphate-buffered saline, the sections were incubated with a secondary antibody for 30 min at room temperature. Following a 30-min reaction with the streptavidin-biotin-peroxidase complex, immunoreactivity was visualized using diaminobenzidine, and the sections were counterstained with hematoxylin (Zymed Laboratories). Negative control sections were incubated with the respective secondary antibodies but without primary antibody. All images were obtained using a light microscope, and the integrated optical density of the images was determined using Image-Pro Plus software (version 6.0).

      2.5 Western blotting

      The proteins were extracted from rat coronary arteries with detergent extraction buffer, which contained 150 mM NaCl, 20 mM Tris–HCl (pH 7.5), 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM disodium salt of ethylenediaminetetraacetic acid, and 2.5 mM sodium pyrophosphate plus protease inhibitor cocktail tablets. After separation on 10% sodium dodecyl sulfate–polyacrylamide gels, the proteins were transferred to polyvinylidene difluoride membranes. After blocking, the membrane was incubated with an anti-ETA receptor (1:500), anti-ETB receptor (1:500), anti-STIM1 (1:500), anti-Orai1 (1:500), or anti-β-tubulin (1:1000) primary antibody at 4 °C overnight. Subsequently, the membrane was incubated with horseradish peroxidase–conjugated secondary antibody and stained using an enhanced chemiluminescence detection kit. The optical densities of the protein bands were analyzed using ImageJ software. The optical density of each blot was normalized to β-tubulin in the same lane and is expressed as the relative optical density.

      2.6 Enzyme-linked immunosorbent assay (ELISA)

      Each sample was diluted in phosphate-buffered saline at an appropriate proportion for optimization. The plasma concentration of the ET-1 level in rats was measured using a commercial ELISA kit according to the provided instruction manual and calculated by establishing a standard curve fit with linear regression using OriginPro software (version 8.0).

      2.7 Masson's trichrome staining and Sirius red staining

      To investigate the effect of a high-salt diet on coronary fibrosis and collagen deposition, the coronary artery tissues isolated from the hearts of the two groups of rats were fixed in 4% paraformaldehyde, and paraffin embedded to prepare 5-μm-thick sections. For Masson's trichrome staining, sections were stained with hematoxylin for 5–10 min and Biebrich Scarlet-Acid Fuchsin for 5–10 min. The collagen fibrils stained blue and the cytoplasmic matrix of vascular smooth muscle-stained red. For Sirius red staining, the sections were stained with Picro-Sirius red staining solution for 1 h, and the nucleus were stained with hematoxylin for 8–10 min. Under polarized light microscope, the collagen type I was observed to be red/yellow and the type III was green. Quantitative histological analysis of coronary artery fibrosis assessment was performed using the Image-Pro Plus software (version 6.0).

      2.8 Echocardiography examination

      The rats were anaesthetized with an intraperitoneal injection of pentobarbital sodium (45 mg/kg). The chest hair was shaved, and the rat was placed in the supine position on an operating table. Echocardiograms were recorded using conventional medical ultrasonographic equipment (frequency, 5–11.5 MHz; IE33 S) to evaluate the heart chambers and valves. The following parameters were measured by two-dimensional guided M-mode echocardiography to compare the structure of the heart in 4% high-salt diet rats and control rats: interventricular septum thickness in diastole (IVSd) and systole (IVSs); left ventricle internal diameter in diastole (LVIDd) and systole (LVIDs); left ventricle posterior wall thickness in diastole (LVPWd) and systole (LVPWs); and left ventricle fractional shortening (LVFS). The left ventricle ejection fraction (LVEF) was calculated by Simpson's method [
      • Ribeiro S.
      • Pereira A.R.S.
      • Pinto A.T.
      • Rocha F.
      • Ministro A.
      • Fiuza M.
      • et al.
      Echocardiographic assessment of cardiac anatomy and function in adult rats.
      ]. The mean values of the parameters were obtained from measurements of at least 5 cardiac cycles on M-mode tracings.

      2.9 Statistical analysis

      Data from at least three separate experiments are expressed as the mean ± SEM. Two-way analysis of variance and independent samples t-tests were used to compare differences between the two groups; P < 0.05 was considered statistically significant. All statistical analyses were performed using Sigma Plot software (version 12.5).

      3. Results

      3.1 Agonist-induced coronary artery vasocontraction in high-salt diet rats

      Because coronary blood flow is regulated by vessel tone [
      • Mizuno R.
      • Fujimoto S.
      • Saito Y.
      • Okamoto Y.
      Optimal antihypertensive level for improvement of coronary microvascular dysfunction: the lower, the better?.
      ], the contraction tension was very important in the process, high salt diet is a risk factor, but the change of coronary artery contraction tension under high salt diet is not clear. So we tested for changes in the vasocontraction of coronary arteries between the high-salt diet rats and controls. In order to exam contraction tension, these experiments used the endothelium-denuded vessels. The coronary artery ring segments were stimulated by increasing concentrations of potassium chloride (KCl), the thromboxane A2 receptor–dependent constrictor U46619, or ET-1. Although increasing concentrations of KCl increased vasoconstriction in both groups, no significant changes were observed between the two groups (Fig. 1A-D). The contractile responses to U46619 (0.01–3 μM) in both groups were also concentration dependent; however, the vasoconstriction observed in response to U46619 was significantly reduced in the 4% high-salt diet rats compared with controls (Fig. 2A and B). The vasocontraction induced by ET-1 (1–300 nM) presented a similar result (Fig. 2C and D). We used Bosentan (10 μM), an ET receptor antagonist, to inhibit the ET-1-induced contraction and test the difference of its inhibitory effects between the two groups. The results showed that the contractile response in 4% high-salt diet rats was significantly decreased compared with that in controls (Fig. 2C and D).
      Figure 1
      Figure 1Changes in the contractile response induced by KCl in endothelium-denuded coronary arteries derived from 4% high-salt diet rats vs. controls. A and B, representative traces (A) and summary data (B) of the contractile in response to 60 mM K+. C and D, representative traces (C) and summary data (D) of the concentration-dependent contractile responses to increasing concentrations of KCl. Data are shown as the mean ± SEM; n = 9–12 rats.
      Figure 2
      Figure 2Changes in the contractile response induced by U46619 or endothelin-1 (ET-1) after treatment with bosentan in endothelium-denuded coronary arteries derived from 4% high-salt diet rats vs. controls. A and B, representative traces (A) and summary data (B) of the concentration-dependent contractile responses to increasing concentrations of U46619. C and D, representative traces (C) and summary data (D) of concentration-dependent contractile responses to increasing concentrations of ET-1 and the responses in the presence of bosentan (an inhibitor of ET receptors; 10 μM). Data are shown as the mean ± SEM; n = 7 rats. ∗P < 0.05,∗∗P < 0.01 compared with control.

      3.2 Role of SOCE in coronary artery vasocontraction in high-salt diet rats

      SOCE plays an important role in vasocontraction and is involved in coronary artery vasocontraction [
      • Groschner K.
      • Shrestha N.
      • Fameli N.
      Cardiovascular and hemostatic disorders: SOCE in cardiovascular cells: emerging targets for therapeutic intervention.
      ]. Human urotensin-II (UII) promotes concentration-dependent vasocontraction in the coronary artery and elicits Ca2+ influx, with both processes sensitive to classical SOCE inhibitors [
      • Dominguez-Rodriguez A.
      • Diaz I.
      • Rodriguez-Moyano M.
      • Calderon-Sanchez E.
      • Rosado J.A.
      • Ordonez A.
      • et al.
      Urotensin-II signaling mechanism in rat coronary artery: role of STIM1 and Orai1-dependent store operated calcium influx in vasoconstriction.
      ]. In the present study, we measured coronary artery tension to explore the differences in SOCE-induced contractile responses between high-salt diet rats and control rats. Endothelium-denuded coronary artery ring segments were incubated in Krebs solution without Ca2+ for 15 min and then incubated with the L-type Ca2+ channel blocker verapamil (1 μM) and with ET-1 (100 nM) for 10 min to deplete intracellular Ca2+ stores. After the Ca2+released from the ER induced the transient contraction of the coronary artery, we added CaCl2 (1–10 mM) to the bath to induce coronary artery contractions. Our results showed that the SOCE-induced contraction of the coronary artery in both groups but was significantly decreased in high-salt diet rats compared with controls (Fig. 3A and B).
      Figure 3
      Figure 3Changes in the contractile response induced by store-operated calcium entry (SOCE) in endothelium-denuded coronary arteries derived from 4% high-salt diet rats vs. controls. Representative traces (A) and summary data (B) of SOCE-induced contraction in coronary artery rings. The rings were pretreated with 100 nM ET-1 and 1 μM verapamil in a Ca2+-free solution for 10 min. The values are shown as the mean ± SEM; n = 6 rats. ∗P < 0.05 compared with controls.

      3.3 Changes in protein expression levels in coronary arteries derived from high-salt diet rats

      As presented earlier (Fig. 2C and D), we found that Bosentan (10 μM) inhibited ET-1–induced coronary artery contractions in both high-salt diet rats and controls. However, the degree of ET-1–induced vasocontraction inhibited by Bosentan was 11.4% in the high-salt diet rats and 30.9% in the control group, suggesting that inhibition effection was reduced in the group of high-salt diet rats compared with controls. This reduced inhibition of ET-1–induced coronary artery contraction by Bosentan might be associated with decreased endothelin receptors expression in VSMCs in these high-salt diet rats.
      Our western blotting and immunohistochemical analysis showed that compared with those in the control group, the expression levels of the ETA receptor and the ETB receptor in coronary artery were significantly decreased in high-salt diet rats (Figs. 4A, B, E, F, I).
      Figure 4
      Figure 4Expression levels of ETA, ETB, STIM1, Orai1 in coronary arteries derived from 4% high-salt diet rats vs. controls. Western blot images (A–D) and summary data (E–H) of endothelial receptors ETA and ETB as well as of STIM1 and Orai1 in fresh isolated coronary arteries derived from rats fed a regular diet (control) and rats fed a high-salt diet. Protein expression levels were normalized to β-tubulin. Immunostaining results of ETA, ETB in segments of coronary arteries derived from control (I (a–b)) and high-salt diet (I (d–e)) rats (three rats in each group). The values are shown as mean ± SEM; n = 3–7 rats. ∗P < 0.05, ∗∗P < 0.01 compared with controls.
      To examine the mechanism for the reduction in the of SOCE-induced contractile response in high-salt diet rats, we compared the expression levels of STIM1 and Orai1 in coronary arteries derived from high-salt diet rats and controls. Our western blotting results showed that the level of expression of STIM1 in the coronary arteries of the 4% high-salt diet rats was significantly lower than that of the control group (Fig. 4C and G, P < 0.05). Although Orai1 expression levels also appeared to be decreased in the 4% high-salt diet rats compared with controls, this decrease was not statistically significant (Fig. 4D and H, P > 0.05).

      3.4 Changes in plasma ET-1 concentration in high-salt diet rats

      Because the expression levels of the ETA receptor and the ETB receptor in the smooth muscle layer of coronary artery segments were significantly decreased in high-salt diet rats compared with controls, we next measured the concentration of plasma ET-1 by using the ELISA method. Our results showed that the concentration of plasma ET-1 in high-salt diet rats was significantly higher than that in control rats (55.8 ± 4.7 pg/mL vs. 44.9 ± 5.8 pg/mL respectively; P < 0.05). These data suggest that increased plasma ET-1 levels in high-salt diet rats may contribute to cardiovascular impairment.

      3.5 Increased collagen deposition in the media layer of coronary arteries in salt-induced hypertensive rats

      ET-1 promotes fibrosis. In Masson staining, the coronary smooth muscle cells were stained red and the collagen fibers were stained blue. Compared with the control group, the deposition of collagen in the media layer of coronary arteries in high-salt intake rats was increased significantly (Figs. 5A, B, E). Sirius red staining method was used to detect the distribution of collagen type I and III in media area of coronary arteries. Collagen type I was observed red/yellow and collagen type III was observed green with polarized light microscope. Compared with the control group, the density of collagen type III was decreased but the density of collagen type I was increased in the coronary arteries of 4% high-salt diet rats (Fig. 5C and D). The ratios of the percentages and areas of collagen I to collagen III expression were increased in the coronary arteries of 4% high-salt diet rats (Fig. 5F and G). These results indicated that abnormal collagen deposition and fibrosis lead to the remodeling of the coronary artery.
      Figure 5
      Figure 5Expression levels of collagen in the media tunica of the coronary artery in 4% high-salt diet rats vs. controls. Representative images of Masson's trichrome staining of the distribution of collagen in coronary artery ring segments derived from rats fed a regular diet (control; A) or a high-salt diet (B). The smooth muscle of the coronary artery is stained red and the collagen fibers are stained blue. Summary data (E) of the collagen volume fraction. Representative picrosirius red–stained images of collagen type I and type III in sections of coronary artery ring segments derived from control (C) or 4% high-salt diet rats (D). Collagen I appears yellow/red and collagen III appears green under a polarized light microscope. Summary data (F, G) of the ratios of the percentages of the areas and of the percentages of the integrated optical densities (IOD) of collagen type I to collagen type III in coronary arteries derived from controls vs. those from 4% high-salt diet rats. The values are shown as mean ± SEM; n = 6 rats. ∗∗P < 0.01 compared with control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

      3.6 Echocardiographic parameters and non-invasive LV hemodynamics in high-salt diet rats

      Echocardiography is of great value in the assessment of cardiac function and structure in patients with cardiovascular disease. We used echocardiography to assess the following measures of cardiac structure and non-invasive LV hemodynamics: IVSd, IVSs, LVIDd, LVIDs, LVPWd, LVPWs, LVFS and LVEF. Our analyses showed that IVSd, IVSs, LVPWd and LVPWs were significantly increased in high-salt diet rats compared with controls. These results indicated that the interventricular septum and left ventricular posterior wall were significantly thickened in high-salt diet rats. Compared with that in controls, heart rate appeared increased in high-salt diet rats, but this increase was not statistically significant. No significant differences in LVEF and LVFS were observed between high-salt diet rats and control rats (Table 1). Taken together, these results suggested that left ventricular hypertrophy had developed in high-salt diet rats and that cardiac function was likely in the compensatory stage.
      Table 1Measures of cardiac structure and left ventricular function in high-salt diet rats vs. controls.
      MeasureControl (n = 5)4% High-salt (n = 5)
      IVSd (mm)1.76 ± 0.102.10 ± 0.06∗∗
      LVIDd (mm)6.24 ± 0.346.14 ± 0.53
      LVPWd (mm)1.70 ± 0.171.98 ± 0.07∗
      IVSs (mm)2.58 ± 0.162.78 ± 0.07∗
      LVIDs (mm)3.50 ± 0.283.34 ± 0.48
      LVPWs (mm)2.48 ± 0.122.66 ± ±0.08∗
      HR (beats per min)394.40 ± 29.34418.00 ± 26.97
      SV (mL)0.47 ± 0.060.45 ± 0.11
      LVEF (%)83.40 ± 5.9883.40 ± 4.27
      LVFS (%)47.68 ± 7.0447.84 ± 4.79
      Abbreviations: HR, heart rate; IVSd, interventricular septum thickness in diastole; IVSs, interventricular septum thickness in systole; LVEF, left ventricle ejection fraction; LVFS, left ventricle fractional shortening; LVIDd, left ventricle internal diameter in diastole; LVIDs, left ventricle internal diameter in systole; LVPWd, left ventricle posterior wall thickness in diastole; LVPWs, left ventricle posterior wall thickness in systole. SV, stroke volume. Values represent the mean ± SEM. ∗P < 0.05, ∗∗P < 0.01 compared with control.

      4. Discussion

      In the present study, we compared changes in the contractility of coronary arteries in high-salt diet rats vs. control rats fed a normal diet and further explored the mechanisms underlying the observed changes. Our key findings included the following: compared with control rats, (1) the interventricular septum and posterior wall of high-salt diet rats were significantly thickened; (2) ET-1 and U46619-induced vasocontraction were significantly decreased in endothelium-denuded coronary artery ring segments from high-salt diet rats; SOCE-induced vasocontraction of coronary arteries was also reduced in high-salt diet rats; (3) plasma concentrations of ET-1 were markedly elevated in high-salt diet rats; while ET receptor distribute on coronary artery smooth muscle layer decreased; (4) coronary artery fibrosis were most sever in high-salt diet rats; (5) Echocardiography suggested left ventricular hypertrophy without obvious impaired function. These findings indicated that the vascular contractile response was decreased and the degree of fibrosis was increased in the coronary arteries of high-salt diet rats. Left ventricular hypertrophy had developed in high-salt diet rats and that cardiac function was likely in the compensatory stage.
      ET-1 plays an important role in coronary artery spasm and promotes cardiac fibroblasts to synthesize matrix protein, aggravating myocardial fibrosis [
      • Barton M.
      • Yanagisawa M.
      Endothelin: 30 Years from discovery to therapy.
      ]. Our study also found that the concentration of plasma ET-1 was significantly increased in high salt diet rats. The high concentration of plasma ET-1 observed in the high-salt diet rats in the present study may contribute to increased coronary artery fibrosis and reduced coronary artery flow. Given the involvement of coronary artery contraction abnormalities in the progression of myocardial ischemia [
      • Collins H.E.
      • Zhu-Mauldin X.
      • Marchase R.B.
      • Chatham J.C.
      STIM1/Orai1-mediated SOCE: current perspectives and potential roles in cardiac function and pathology.
      ,
      • Pande J.
      • Dimmers G.
      • Akolkar G.
      • Skelley L.
      • Samson S.E.
      • Grover A.K.
      Store operated Ca2+ entry dependent contraction of coronary artery smooth muscle: inhibition by peroxide pretreatment.
      ], we examined changes in coronary artery contractile responses in high-salt diet rats. Changes in coronary artery tension associated with a high-salt diet remain controversial. Panth et al. found that coronary vessel vasodilatation dysfunction and cardiac hypertrophy occurred in pigs fed a high-salt diet [
      • Panth N.
      • Park S.H.
      • Kim H.J.
      • Kim D.H.
      • Oak M.H.
      Protective effect of salicornia europaea extracts on high salt intake-induced vascular dysfunction and hypertension.
      ]. By contrast, Simõeset al. found that a high-salt diet did not cause further damage to coronary vessel vasodilatation in elderly spontaneously hypertensive rats [
      • Simoes M.R.
      • Furieri L.B.
      • Forechi L.
      • Baldo M.P.
      • Rodrigues S.L.
      • Salaices M.
      • et al.
      High salt intake does not produce additional impairment in the coronary artery relaxation of spontaneously hypertensive aged rats.
      ]. Therefore, the effects of high-salt intake on coronary artery contraction tone are still unclear. Disfunction of the coronary arteries smooth muscles is responsible for abnormal contractile tension. The changes of endothelium-denuded coronary artery vascular tension under different stimulation factors were measured included TXA2 analogue, U46619 ET-1 and KCl. It was found that there was no significant difference in the changes of coronary artery tension between the two groups under high potassium stimulation, while the coronary artery contractile tension of rats in the high-salt diet group was significantly decrease than that in the control group under the action of TXA2 analogues U46619 and ET-1. It is interesting. Clinically, previous studies suggests that a high-salt diet is more likely to impairs endothelial function and increase arterial stiffness [
      • Dickinson K.M.
      • Clifton P.M.
      • Burrell L.M.
      • Barrett P.H.
      • Keogh J.B.
      Postprandial effects of a high salt meal on serum sodium, arterial stiffness, markers of nitric oxide production and markers of endothelial function.
      ,
      • Dickinson K.M.
      • Clifton P.M.
      • Keogh J.B.
      Endothelial function is impaired after a high-salt meal in healthy subjects.
      ]. The decrease of coronary contractile tension after endothelium removal may be a compensatory effect on the increase of ET-1 secretion. We further studied this presumed compensatory effect and found a significant reduction of coronary vasoconstriction occurs in normal control group, but not in high salt diet group after Bosentan administration. The purpose of using bosentan was to detect the activity of ET-1 receptor. However, ET-1 receptor is decreased in the coronary arteries of rats on high salt diet, so bosentan pretreatment failed to cause obvious inhibition in the coronary arteries of rats on high salt diet. Therefore, we carried out a follow-up verification on the expression of endothelin receptors on coronary artery smooth muscle. Consistent with our hypothesis, the expression of ETA and ETB on vascular smooth muscle of rats in the high-salt diet group were significantly reduced. The effect of endothelin receptors on the contractile function of vascular smooth muscle is not only related to the above-mentioned receptor-dependent effects, but also plays an indispensable role through the change of intracellular Ca2+ handling [
      • Calderon-Sanchez E.M.
      • Avila-Medina J.
      • Callejo-Garcia P.
      • Fernandez-Velasco M.
      • Ordonez A.
      • Smani T.
      Role of Orai1 and L-type CaV1.2 channels in Endothelin-1 mediated coronary contraction under ischemia and reperfusion.
      ,
      • Horinouchi T.
      • Terada K.
      • Higashi T.
      • Miwa S.
      Endothelin receptor signaling: new insight into its regulatory mechanisms.
      ], among which SOCE related signal pathway plays an important role [
      • Dominguez-Rodriguez A.
      • Diaz I.
      • Rodriguez-Moyano M.
      • Calderon-Sanchez E.
      • Rosado J.A.
      • Ordonez A.
      • et al.
      Urotensin-II signaling mechanism in rat coronary artery: role of STIM1 and Orai1-dependent store operated calcium influx in vasoconstriction.
      ]. Other researchers have shown the importance of the SOCE signaling pathway in serotonin [
      • Deng C.Y.
      • Yang H.
      • Kuang S.J.
      • Rao F.
      • Xue Y.M.
      • Zhou Z.L.
      • et al.
      Upregulation of 5-hydroxytryptamine receptor signaling in coronary arteries after organ culture.
      ] and urotensin II-induced [
      • Dominguez-Rodriguez A.
      • Diaz I.
      • Rodriguez-Moyano M.
      • Calderon-Sanchez E.
      • Rosado J.A.
      • Ordonez A.
      • et al.
      Urotensin-II signaling mechanism in rat coronary artery: role of STIM1 and Orai1-dependent store operated calcium influx in vasoconstriction.
      ] coronary vasocontraction. Therefore, in our experiments, we depleted the intracellular Ca2+ store, and then added Ca2+ to active Orai channel to explore the role of SOCE signaling pathway in coronary systolic function [
      • Tang X.
      • Qian L.L.
      • Wang R.X.
      • Yao Y.
      • Dang S.P.
      • Wu Y.
      • et al.
      Regulation of coronary arterial large conductance Ca2+-activated K+ channel protein expression and function by n-3 polyunsaturated fatty acids in diabetic rats.
      ]. Our results showed that agonist-induced and SOCE-mediated coronary contractile functions of rats in the high-salt diet group were weakened. Orai1 is a pore forming subunit of store-operated Ca2+ channels [
      • Hogan P.G.
      • Rao A.
      Store-operated calcium entry: mechanisms and modulation.
      ], which is responsible for a selective SOCE. STIM1 is an important protein that senses the concentration of Ca2+ in the endoplasmic reticulum and participates in the calcium influx process (store-operated calcium entry, SOCE) [
      • Bolotina V.M.
      Orai1, STIM1, and iPLA2β determine arterial vasoconstriction.
      ]. When extracellular agonists bind to receptors on the cell membrane, it causes intracellular calcium signal transduction and the releases of Ca2+ in the endoplasmic reticulum. When the concentration of Ca2+ in the endoplasmic reticulum decreases, the expression of STIM1 increases and binds to Orai protein on the cell membrane to form a microdomain to allow an influx of external calcium [
      • Bolotina V.M.
      Orai1, STIM1, and iPLA2β determine arterial vasoconstriction.
      ]. There were evidences showed that ET receptor can participate in the activation of STIM1/Orai1-mediated SOCE process [
      • Chuang T.Y.
      • Au L.C.
      • Wang L.C.
      • Ho L.T.
      • Yang D.M.
      • Juan C.C.
      Potential effect of resistin on the ET-1-increased reactions of blood pressure in rats and Ca2+ signaling in vascular smooth muscle cells.
      ,
      • Snow J.B.
      • Kanagy N.L.
      • Walker B.R.
      • Resta T.C.
      Rat strain differences in pulmonary artery smooth muscle Ca(2+) entry following chronic hypoxia.
      ,
      • Liu X.R.
      • Zhang M.F.
      • Yang N.
      • Liu Q.
      • Wang R.X.
      • Cao Y.N.
      • et al.
      Enhanced store-operated Ca2+ entry and TRPC channel expression in pulmonary arteries of monocrotaline-induced pulmonary hypertensive rats.
      ]. In our experiment, ET-1 increased after the high-salt diet, the same as Dr Marcela Herrera and Dr Yuhui Yang previously reported [
      • Yang Y.
      • Liu X.
      • Liu Y.
      • Fu H.
      • Gao Y.
      • Liu X.
      • et al.
      The development of salt-sensitive hypertension regulated by PSGL-1 gene in mice.
      ,
      • Herrera M.
      • Garvin J.L.
      A high-salt diet stimulates thick ascending limb eNOS expression by raising medullary osmolality and increasing release of endothelin-1.
      ], but ETB, the receptor of ET-1 and STIM1 were decreased. It may be due to the binding of ET-1 and ETB decreased after the high-salt diet, the calcium signal transmitted to the cells decreased, and the SOCE process weakened. Through Western blot experiment, we found that the expression of STIM1 in the high-salt diet group was significantly lower than that in the normal control group, while there was no significant difference in the expression of Orai1. STIM1 is a sensor that activates store-operated Ca2+ channels, which can response to the depletion of Ca2+ stored in the ER [
      • Feldman C.H.
      • Grotegut C.A.
      • Rosenberg P.B.
      The role of STIM1 and SOCE in smooth muscle contractility.
      ]. Therefore, the decreased coronary artery contraction may be caused by the reduced expression of SOCE proteins in high-salt diet rats.
      A high-salt diet is an independent cardiovascular risk factor in myocardial hypertrophy and interstitial fibrosis [
      • Ferreira D.N.
      • Katayama I.A.
      • Oliveira I.B.
      • Rosa K.T.
      • Furukawa L.N.
      • Coelho M.S.
      • et al.
      Salt-induced cardiac hypertrophy and interstitial fibrosis are due to a blood pressure-independent mechanism in Wistar rats.
      ]. Previous studies had shown that high-salt diet can induces LVH and fibrosis through activation of the cardiac renin-angiotensin system and a change in myocardial diastolic function and later with abnormal systolic function [
      • Le Corvoisier P.
      • Adamy C.
      • Sambin L.
      • Crozatier B.
      • Berdeaux A.
      • Michel J.B.
      • et al.
      The cardiac renin-angiotensin system is responsible for high-salt diet-induced left ventricular hypertrophy in mice.
      ,
      • He F.J.
      • Burnier M.
      • Macgregor G.A.
      Nutrition in cardiovascular disease: salt in hypertension and heart failure.
      ,
      • Waeber B.
      • Weber R.
      • Brunner H.R.
      Physiopathologie de l'hypertrophie ventriculaire gauche [Physiopathology of left ventricular hypertrophy].
      ]. In this experiment, ET-1 and vascular collagen synthesis increased after high-salt diet, suggesting that LVH may also be the result of collagen formation induced by ET-1 and its signal transduction. Therefore, we speculated that the phenomenon of LVH may be the synergistic effect of ET-1 and high-salt diet. Resent work found that salt substitution can reduce the incidence of stroke and coronary heart disease [
      • Neal B.
      • Wu Y.
      • Feng X.
      • Zhang R.
      • Zhang Y.
      • Shi J.
      • et al.
      Effect of salt substitution on cardiovascular events and death.
      ]. Our study also found that high salt diet caused left ventricular hypertrophy, which is an independent risk factor for coronary heart disease. Therefore, a high-salt intake may be responsible for the development of coronary artery remodeling in rats. In the present study, we observed the collagen content of the media tunica was significantly increased in isolated coronary arteries, as identified by Masson's trichrome staining and picrosirius red staining. Our results indicated that fibrosis had occurred in the middle layer of the coronary artery in high-salt diet rats to increase coronary artery stiffness and cause coronary artery remodeling then led to coronary flow reducing and exacerbate myocardial ischemia.
      In summary, we showed that coronary artery vasoactive responses were impaired in high-salt diet rats. The reduced coronary artery contractility may be caused by the down-regulated ET receptor–mediated SOCE signaling pathway. The decreased expression of ET receptors in the coronary artery smooth muscle layer was likely a compensatory mechanism responding to the elevated concentration of ET-1 in the plasma. A high ET-1 level in plasma also triggered coronary artery fibrosis, leading to coronary vessel wall stiffness in high-salt diet rats. Our study provides new evidence for a role of the endothelin receptor pathway in understanding the underlying mechanisms of high-salt intake–induced coronary artery dysfunction.

      Author contributions

      H.X., H.L., and R.Z. designed experiments.
      H.X., H.L., K.H., M.D., and R.Z. wrote and revised the manuscript.
      K.H., H.L., M.D., Y.X., and Z.J. performed the experiments and analyzed data.
      R.Z. supervised the project.
      All authors contributed to the article and approved the final manuscript.

      Funding

      The present study was supported by Natural Science Foundation of China (Grant No. 81970446 ). The funder had no role in the study design; collection, analysis and interpretation of data; writing of the report; and decision to submit the article for publication.

      Declaration of competing interest

      The authors declare that they have no conflict of interest.

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