reperfusion injury

Journal Pre-proof Cardioprotective effects of galectin-3 inhibition against ischemia/reperfusion injury Dan Mo, Wen Tian, Hui-Nan Zhang, Ying-Da Feng, Yang Sun, Wei Quan, Xiao-Wei Hao, Xue-Ying Wang, Xiao-Xiao Liu, Chen Li, Wei Cao, Wen-Juan Liu, Xiao-Qiang Li PII:

S0014-2999(19)30653-3

DOI:

https://doi.org/10.1016/j.ejphar.2019.172701

Reference:

EJP 172701

To appear in:

European Journal of Pharmacology

Received Date: 16 February 2019 Revised Date:

25 September 2019

Accepted Date: 26 September 2019

Please cite this article as: Mo, D., Tian, W., Zhang, H.-N., Feng, Y.-D., Sun, Y., Quan, W., Hao, X.W., Wang, X.-Y., Liu, X.-X., Li, C., Cao, W., Liu, W.-J., Li, X.-Q., Cardioprotective effects of galectin-3 inhibition against ischemia/reperfusion injury, European Journal of Pharmacology (2019), doi: https:// doi.org/10.1016/j.ejphar.2019.172701. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Cardioprotective effects of galectin-3 inhibition against ischemia/reperfusion injury Dan Mo a , Wen Tian a , Hui-Nan Zhang a, Ying-Da Feng a, Yang Sun a, Wei †

†

Quan a, Xiao-Wei Hao a, Xue-Ying Wang a, Xiao-Xiao Liu a, Chen Li a, Wei Cao ab

, Wen-Juan Liu b, Xiao-Qiang Li a*

a

Key Laboratory of Gastrointestinal Pharmacology of Chinese Materia Medica

of the State Administration of Traditional Chinese Medicine, Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi'an, China b

Shaanxi Key Laboratory of Natural Products & Chemical Biology, School of

Chemistry & Pharmacy, Northwest A&F University, Yangling, China †

The first two authors contributed equally to this work.

*

Corresponding author at:

Prof. Xiao-Qiang Li ([email protected]), Key Laboratory of Gastrointestinal Pharmacology of Chinese Materia Medica of the State Administration of Traditional Chinese Medicine, Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, 169 Changle West Road, Xi'an, Shaanxi 710032, China. Tel: +86 29 84772221, fax: +86 29 84772221.

1

Abstract Myocardial ischemia/reperfusion (IR) injury is caused by the restoration of the coronary blood flow following an ischemic episode. Accumulating evidence suggests that galectin-3, a β-galactoside-binding lectin, acts as a biomarker in heart disease. However, it remains unclear whether manipulating galectin-3 affects the susceptibility of the heart to IR injury. In this study, RNA sequencing (RNA-seq) analysis identified that Lgals3 (galecin-3) plays an indispensable role in IR-induced cardiac damage. Immunostaining and immunoblot assays confirmed that the expression of galectin-3 was markedly increased in myocardial IR injury both in vivo and in vitro. Echocardiographic analysis showed that cardiac dysfunction in experimental IR injury was significantly attenuated by galectin-3 inhibitors including pectin (1%, i.p.) from citrus and binding peptide G3-C12 (5.0 mg/kg, i.p.). Galectin-3 inhibitor-treated mice exhibited smaller infarct sizes and decreased tissue injury. Furthermore, TUNEL staining showed that galectin-3 inhibition suppressed IR-mediated cardiomyocyte apoptosis. Mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) levels were well-preserved and IR-induced changes of mitochondrial cyto c, cytosol cyto c, caspase-9, caspase-3, Bcl-2 and Bax in the galectin-3 inhibitor-treated groups were observed. Our findings indicate that the pathological upregulation of galectin-3 contributes to IR-induced cardiac dysfunction and that galectin-3 inhibition ameliorates myocardial injury, highlighting its therapeutic potential. 2

Key word: galectin-3; heart; ischemia/reperfusion; apoptosis.

3

1. Introduction Ischemic heart disease (IHD) is found to be worldwide a leading cause of death, and a serious threat to human health. Amongst IHD, myocardial ischemia/reperfusion (IR) injury manifests as decreased blood pressure, cardiac dysfunction, and sudden death during the recovery of blood flow (Eltzschig et al. 2011). Studies of acute myocardial infarction have shown that in animal models, fatal reperfusion injury accounts for 50% of the infarct size (Yellon et al. 2007). Previous studies indicate that the mechanisms of IR injury include oxidative stress, inflammation, calcium overload and metabolic disorders (Murphy et al. 2008). Although several strategies to ameliorate lethal IR injury have been proposed (Xia et al. 2016), translating these findings to the clinic remains challenging (Bolli et al. 2004, Heusch et al. 2017) and no effective therapies are available. New and improved treatments are therefore urgently required. Galectins have characteristic carbohydrate-recognition domains (CRD) and the ability to bind β-galactosides. They are evolutionarily conserved and influence distinct cell functioning (Delacour et al. 2009). Since their discovery in 1976, 15 mammalian galectins have been described (Delacour et al. 2009). Galectins specifically bind to extracellular or intracellular ligands and modulate cellular development, immune responses, and cancer progression (Balan et al. 2010, Dong et al. 2018). Lgals3 on chromosome 14 encodes galectin-3, also known as Mac-2 or GBP (Liu et al. 2016, Coppin et al. 2017). Galectin-3 is 4

ubiquitously distributed and localizes to the cytoplasm, nucleus, and plasma membrane, and can be secreted into the blood (Chen et al. 2016, Diaz-Alvarez et al. 2017). Recent studies indicate that higher circulating levels of galectin-3 contribute to an increased risk of cardiomyopathy (Nguyen et al. 2018). As a biomarker of cardiac disease (McCullough 2014, Venkatraman et al. 2018), galectin-3 levels correlate with inflammation and fibrosis which play key roles in the development and progression of heart failure, and cardiac remodeling (Yu et al. 2013). Decreased galectin-3 expression is associated with cardioprotective functions during IR injury (Liu et al. 2018) and galectin-3 is known to cause neonatal hypoxic-ischemic brain injury (Doverhag et al. 2010). However, despite the evidence that galectin-3 participates in IR injury, its potential pathogenic effects are poorly characterized. In this study, we report that galectin-3 is significantly upregulated in cardiomyocytes challenged with IR. Using two independent galectin-3 inhibitors including pectin from citrus and the binding peptide G3-C12, we investigated their therapeutic potential in the protection of IR-induced cardiac dysfunction.

2. Materials and methods 2.1. Animals From the Laboratory Animal Center of the Fourth Military Medical University (FMMU), male C57BL/6J mice (8-10 weeks old) weighing between 5

20 and 22 g were purchased. All experiments were approved by the Ethics Committee of FMMU. The protocol was performed according to the Guide for the Care and Use of Laboratory Animals. During the entire experiment procedures, the mice were exposed to 12/12 h of light/darkness and given free access to normal food and water. Mice were subjected to 30 min/ 24 h IR model using method as described previously(Gao et al. 2010).

2.2. RNA-seq analysis Male C57BL/6J mice were randomly assigned into two groups including the sham and IR, three animals per group. All the mice were anesthetized with pentobarbital sodium and killed after the IR modeling. RNA-seq analysis was performed on the area at risk (AAR) parts of the cardiac regions below ligated left coronary artery of the heart, as previous study reported (Lyu et al. 2018). Left ventricular tissue was separated and frozen in liquid nitrogen. During the sampling process, RNase contamination was avoided. RNA-seq was performed by using an Illumina platform, as previously described (Giudice et al. 2014). The RNA samples of cardiac tissue were sequenced by the Illumina HiSeq 4000 instrument (California, USA) and carried out by running 150 cycles. Trimmed reads (pass FastQC (version 0.11.5) filter, Cambridge, UK) were aligned to mouse reference genome (GenCode mm10) as well as mouse transcriptome (GenCode mm10) with Hisat2 software (version 2.0.5, Maryland, USA). Transcriptional abundance estimation was completed via StingTie 6

software (version 1.3.1c, California, USA). The gene & transcript expression level (FPKM value) and major changes in gene & transcript expression were measured by Ballgown (version 2.8.4, Washington, USA).

2.3. Reagents Pectin was obtained from Sigma Aldrich (Missouri, USA). G3-C12 (Sequence: ANTPCGPYTHDCPVKR) was purchased from GenScript (Nanjing, China). Male C57BL/6J mice were randomly intravenously injected with 1% pectin (the final concentration of DMSO was 0.1% (v/v) ) or 1.0, 2.5 or 5.0 mg/kg G3-C12 (dissolved in normal saline, NS, respectively) each day for 3 days before IR treatment. The dose and schedule were carried out as described previously (Lu et al. 2017, Sun et al. 2017).

2.4. Echocardiographic measurements Echocardiographic views were acquired with the Vevo2100 (Visual Sonics Int., Toronto, Canada) 24 h after surgery. Mice were anesthetized with 2% isoflurane inhalation with an isoflurane delivery system (Viking Medical, Medford, NJ) during echocardiographic examination. At M-Mode, left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameters (LVESD), posterior wall diastolic thickness (PWT, d), and posterior wall systolic thickness (PWT, s) were measured (Gardin et al. 2002). GraphPad Prism 5 software was used to calculate LV fractional shortening 7

(FS), ejection fraction (EF), and mass (Gardin et al. 2002). Technicians who performed and read echocardiography were blind to the groups allocation.

2.5. Myocardial infarct size. Briefly, to obtain samples for infarct size analysis, mice hearts were perfused with 3% Evans Blue in normal saline and then removed. Subsequently, the LV tissue was frozen and cut into 6 slices along the horizontal axis. The heart sections were then incubated with 1% TTC in phosphate buffer (pH 7.4, 37℃) for half an hour in the dark. After staining by TTC, red parts in the heart indicated ischemic but viable tissue, while pale areas represented infarcted myocardium. Images of Evans Blue and TTC dyeing results were taken photos with a macro lens. Infarct size areas were determined using Image-Pro Plus 6.0 software. The size of infarction area was calculated as infarct area divided by area at risk (IF/AAR). The weight of the heart was also taken into account as previous study reported (De Meyer et al. 2012, Zhang et al. 2016).

2.6. H&E and immunohistochemistry Formalin-fixed heart tissues were paraffin-embedded and cut into 4 µm sections. H&E and anti-galectin-3 antibody (1:100, Abcam, Massachusetts, USA) were both applied. Photos were taken with a Nikon Eclipse 80i microscope. The immune staining results were evaluated by immunoreactive 8

score (IRS), a semi-quantitative scoring system, according to previous studies (Specht et al. 2015).

2.7. TUNEL Staining Using the One Step TUNEL apoptosis assay kit (Beyotime, Shanghai, China), apoptotic cell nuclei were stained as the manufacturer's instructions described. The paraffin-embedd LV tissue sections were counterstained with mouse anti-α-sarcomeric actin (#BM0001) to specifically mark cardiomyocytes. DAPI staining was performed to visualize total nuclei. TUNEL positive cells were quantified with Image-Pro Plus 6.0.

2.8. Cell treatment paradigm The mouse cardiac muscle HL-1 cell line was purchased from Shanghai Huzhen, China. The culture medium contains DMEM (Hyclone, Utah, USA) added with 10% fetal bovine serum (Hyclone, Utah, USA), 100 µg/ml streptomycin (Solarbio, Beijing, China) and 100 U/ml penicillin at 37℃ in a 5% CO2/95% air humidified incubator. To determine the effects of inhibiting galectin-3 on simulated IR injury, HL-1 cells were incubated with either normal culture medium, 0.01% pectin, 10 µM G3-C12, 25 µM G3-C12 or 50 µM G3-C12 for 40 min and were then subjected to a lethal episode in an air-tight hypoxic chamber for 12 h followed by 1 h reoxygenation as previous studies have reported (Ong et al. 2010, Horstkotte et al. 2011). 9

2.9. Cell Death Assay Cell death was assessed by trypan blue staining and by analyzing lactate dehydrogenase (LDH) release as a marker of cell damage. Cell viability was determined using trypan blue (0.04% final concentration) exclusion assay and a total of 500 cells were evaluated under the microscope. Measurement of LDH leakage in culture medium was performed using the lactate dehydrogenase assay kit (Nanjing Jiancheng, China). Spectrophotometer (wave length of 440 nm) was used to detect the absorbance changes. The experiment was repeated at least three times.

2.10. Measurement of MDA and SOD In order to demonstrate whether oxidative stress was affected upon galectin-3 inhibition, measurement of MDA and SOD levels were taken as index of lipid peroxidation. For the MDA and SOD assay, cells were collected and crushed with cold PBS and a homogenate was prepared. The homogenate was centrifuged at 3500 rpm for 15 min at 4℃, and the supernatant was used for biochemical analyses. The MDA concentration in the homogenate was determined using a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) based on thiobarbituric acid (TBA) reactivity. Briefly, MDA reacts with TBA at 90-100℃ and acidic condition. The developed red color of the resulting reaction was measured at 532 nm with a spectrophotometer. Other procedures were carried out following the 10

manufacturer’s protocols. The SOD activity in the homogenate was assessed using a commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) based on the hydroxylamine (xanthine-xanthine oxidase) method. SOD could specifically inhibit superoxide ions. The xanthine-xanthine oxidase system produced superoxide ions, which reacted with hydroxylamine to form a red formazan dye, and the absorbance at 550 nm was determined.

2.11. Measurement of MMP To elaborate the effects of inhibiting galectin-3 on mitochondrial membrane potential (MMP) in H/R injury, experimental groups cells were pretreated with either 0.01% pectin or (10, 25, 50 µM) G3-C12 for 40 min. Hypoxia was induced for 12 h followed by reoxygenation (1 h). The cells were collected and then resuspended with fresh media. MMP was accessed using the mitochondrial membrane potential assay kit with JC-1 (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Measurement of fluorescence ratio was evaluated by flow cytometry. The ratio of aggregates (red in the web version, 590 nm) to monomer (green in the web version, 525 nm) was calculated as an indicator of MMP. Three independent experiments were repeated at least.

2.12. Mitochondrial isolation Mitochondrial isolation was carried out by using a Cell Mitochondria 11

Isolation Kit (Beyotime, Beijing, China) according to the manufacturer’s instruction. Briefly, Cardiac HL-1 cells were collected and then washed twice with cold PBS. Cells were resuspended in mitochondrial isolation buffer (provided in the kit) with 1 mM phenylmethanesulfonyl fluoride on ice for 15 min and homogenized with 15 strokes of a homogenizer. Cell homogenates were centrifuged at 1000× g at 4°C for 10 min. Supernatant was transferred to a new tube and centrifuged at 3500× g, 10 min at 4°C, and the precipitation from this process was mitochondrial fraction. Then, the supernatant was transferred to another new tube and centrifuged at 12,000× g, 10 min at 4°C to obtain cytosolic fraction. Both mitochondrial and cytosolic fractions were boiled in protein sample buffer and analyzed by western blot.

2.13. Calcium retention capacity measurement (CRC) According to a modified protocol by Matas et al (Matas et al. 2009), calcium retention capacity (CRC) was taken as the total amount of Ca2+ taken by mitochondria prior to the Ca2+ pulse triggering Ca2+ release. The incubation medium containing, in mM: 150 sucrose, 50 KCl, 2 KH2PO4, 20 Tris/HCl, 5 succinate-Tris and 0.25 µM Calcium Green-5N was used to measure extra-mitochondrial Ca2+ with a spectrofluorometer (excitation: 506 nm; emission: 530 nm).

Isolated mitochondria (250 µg protein) were added in 2

ml of this incubation medium. After 2 min of incubation, the CRC was measured by adding Ca2+ pulses every minute until mPTP opening. In a 12

parallel assay, additional application of 1 µM cyclosporine A (CsA) at the onset of reperfusion delayed mPTP opening. The CRC was expressed as nmol Ca2+ mg protein−1.

2.14. mPTP assay Mitochondrial permeability transition pore (mPTP) colorimetry assay kit (GENMED, Shanghai, China) was applied for mPTP detection. Spectrophotometer was used to detect the changes in the volume (expansibility) of mitochondria, so as to analyze and observe the opening state of mitochondrial membrane channel, that is, when the mitochondrial membrane channel is open, the mitochondrial volume increases (mitochondrial expansion), and the light scatter ability decreases. Corresponding wells of a 96-well plate was used, and 20 µl mitochondrial samples were added before 170 µl reagent buffer according to the manufacturer's instructions. Immediately the samples were placed in the microplate reader at a standard wavelength of 540 nm. Then 10 µl inducing solution (20 mM CaCl2) from the mPTP colorimetry assay kit was added in, and the plate was immediately placed in the microplate reader (wavelength 540 nm). Change was obtained at 18 min. The absorbance ratio (A540/ initial A540) was calculated to characterize the opening conditions of mPTP.

2.15. Western blot 13

Western blot was conducted in accordance with the standard protocols. BCA protein detection kit (Beijing Leagene Biotechnology, China) was used to determine the protein concentration. Then, protein lysates were separated into equivalent amounts by SDS-PAGE and transferred to the polyvinylidene fluoride membranes (PVDF) membranes (Millipore, Billerica, USA). Immunoreactive proteins were detected by enhanced chemiluminescence (ECL) and band densities were quantified with Image-Pro Plus 6.0 software according to the manufacturer's instructions. Three independent experiments were repeated at least. Proteins were normalized to the tubulin expression every time respectively. "Fold change" shown in the y-axes means that result in the NX group was normalized as the control. The antibody against galectin-3 (#ab53082) was purchased from Abcam (Massachusetts, USA). The antibody against COX IV (#A01060-1) was purchased from Abbkine (California, USA). The antibodies against Bax (#50599-2-Ig), Bcl-2 (#12789-1-AP), caspase 3 (#19677-1-AP), caspase 9 (#66169-1-Ig), cyto c (#10993-1-AP), β-actin (#66009-1-Ig) and tubulin (#10094-1-AP) were all purchased from Proteintech (Chicago, USA).

2.16. Statistics The results are reported as the mean ± S.E.M. for at least three independent experiments. A student's t-test, Mann Whitney test, two-way ANOVA or one-way ANOVA with Tukey's post-hoc tests were given to analyze 14

data difference. Graph Pad Prism version 5 soft was used for group comparison. P value < 0.05 was considered statistically significant.

3. Results 3.1. Galectin-3 is up-regulated in myocardial IR injury Using RNA-seq, we identified 886 differentially expressed genes (DEGs) (P < 0.05, |log2 (Fold Change)| > 0.585) between IR and sham groups. Fig. 1A showed 724 up-regulated genes and 162 down-regulated genes in the IR group compared to the sham group. Fig. 1B shows scatter plots of the average FPKM values of sample genes in each group. Fig. 1C shows volcano plots which evaluate the DEGs under two screening conditions (log2 (Fold Change) & -log10 (p value)). Gene Ontology (GO) enrichment analysis was applied to annotate the DEGs as three GO categories, including “biological process” “cellular components” and “molecular functions” (Fig. 2A-C). The top 10 enriched GO terms for DEGs upregulated in the IR group were shown using the GO database. As shown in Fig. 2D, Venn diagrams depicted common or unique genes in the top 1 subset with highest enrichment scores from the three mentioned GO terms. Eight genes were found to be significant in all three terms, 72 of which were unique to “immune system processes”, 195 were specific to the “extracellular exosome” and 229 were expressed only in “protein binding”. As shown in Fig. 2E, 8 genes (Lgals3, Lcn2, Fgr, Anxa1, Cd300a, Havcr2, Alcam, Lyn) expressed in the IR group were significantly up-regulated. 15

Lgals3, encoding galectin-3, was the top DEG in response to IR, showing a 22-fold increase. We reasoned that Galectin-3 may therefore play an important role in the pathogenesis of IR injury. To validate the upregulation of Lgals3 (galectin-3) by RNA-seq, male C57BL/6J mice were subjected to myocardial IR. Formalin-fixed, paraffin-embedded tissue sections showed a notable increase of galectin-3 in IR-induced injury mice compared to sham groups (Fig. 3A). To confirm these observations in vitro, we used the HL-1 cell line in cardiac cell H/R experiments mimicking IR injury (Ong et al. 2010, Horstkotte et al. 2011). Fig. 3B shows elevated galectin-3 expression, in agreement with the RNA-seq data.

3.2. Galectin-3 inhibition ameliorates IR-induced cardiac dysfunction To explore whether galectin-3 affects IR cardiac responses, mice were grouped into sham; pectin + sham; 5.0 mg/kg G3-C12 + sham; IR; pectin + IR; 1.0 mg/kg G3-C12 + IR; 2.5 mg/kg G3-C12+IR; and 5.0 mg/kg G3-C12 + IR. Pectin and G3-C12 doses were selected based on previous studies (Lu et al. 2017, Sun et al. 2017). After IR treatment, echocardiographic examinations were used as non-invasive methods to detect mouse cardiac function. Normal cardiac function can be evaluated by ejection fraction (EF) (EF≥55%) (Sweitzer et al. 2008) and fractional shortening (FS) (FS≥25%) (Gardin et al. 2002). As shown in Fig. 3C-D, representative images of the M-mode echocardiography indicated that the IR group showed impaired cardiac 16

function compared to the sham group. No significant differences between sham-operated animals treated with galectin-3 inhibitors and sham groups were observed. galectin-3 inhibitors Inhibiting galectin-3 markedly increased the levels of these indicators, particularly following G3-C12 treatment. These data suggest that galectin-3 inhibitors have significant cardioprotective effects on IR injury model mice (Table 1).

3.3. Galectin-3 inhibition prevents heart infarction and histopathological scores of IR-injured mice To further evaluate the effects of galectin-3 inhibition on myocardial IR injury, we measured LV infarct size (IF). Evans Blue and TTC staining were performed to determine the extent of myocardial IR injury. Fig. 4A shows that the infarct areas increased in IR groups (36.3 ± 4.49%) compared to sham groups (0.333 ± 0.33%), whilst infarct areas decreased following galectin-3 inhibition (19.3 ± 2.96% in the pectin + IR group, 18.7 ± 3.18% in the 5.0 mg/kg G3-C12+IR group). Importantly, the area at risk (AAR), which is the potential ischemic area in the LV, remained unchanged amongst all groups. Furthermore, we measured myocardial histomorphology by H&E staining. Inflammatory infiltration and the disappearance of regular cardiac striated muscle structures in the IR group were observed. These histopathological changes improved following pectin and G3-C12 treatment (Fig. 4B). Histological scores were measured using the Carraway double-blind method 17

(Chen et al. 2017) and showed comparable results. These data indicate that the inhibition of galectin-3 attenuates IR injury.

3.4. Galectin-3 inhibition prevents IR-induced cardiomyocyte apoptosis Apoptosis is a major cause of cardiomyocyte death in cardiovascular disease (Whelan et al. 2010). Galectin-3 is known to trigger apoptosis (Fukumori et al. 2003, Gao et al. 2017, Xue et al. 2017) which was examined using TUNEL staining. Increased cell death was observed in the IR group (1.0 ± 0.04 as control) which markedly decreased in pectin and G3-C12 treatment groups (0.41 ± 0.07 in the pectin + IR group, 0.46 ± 0.07 in the 2.5 mg/kg G3-C12+IR group, 0.27 ± 0.09 in the 5.0 mg/kg G3-C12+IR group) in the infarcted myocardium of mice (Fig. 4C). We therefore conclude that inhibiting galectin-3 decreases IR induced apoptosis.

3.5. Galectin-3 inhibition prevents H/R injury in cardiac HL-1 cells To elucidate the mechanism(s) by which Galectin-3 protects against myocardial IR injury, we set up a H/R model using cardiac HL-1 cells. Cells were treated with either normal culture medium, 0.01% pectin, 10 µM G3-C12, 25 µM G3-C12 or 50 µM G3-C12 for 40 min. Hypoxia was induced for 12 h as previously described (Ong et al. 2010, Horstkotte et al. 2011). Hypoxic cells were reoxygenated whilst normoxia cells were maintained in a normal environment for 1 h. Trypan blue staining and lactate dehydrogenase (LDH) 18

release were assessed to evaluate cell injury. As shown in Fig. 5A-C, H/R treatment induced significant decreases in cell vitality (43.5 ± 5.8%). Pectin and G3-C12 at 10 µM, 25 µM and 50 µM reversed cellular viability to 70.8 ± 4.3%, 46.0 ± 6.7%, 63.3 ± 8.0% and 78.5 ± 5.6%, respectively. No significant differences between normoxia-conditioned galectin-3 inhibitor groups and control groups were observed (Fig. 5B). H/R treatment markedly increased cell LDH leakage, whilst cells treated with pectin and 50 µM G3-C12 were protected against H/R-induced damage to a greater extent (Fig. 5C). We further assessed MMP, changes to which represent early cell apoptosis. Inhibiting galectin-3 was profoundly beneficial to MMP levels (54.9 ± 5.7% in pectin + H/R groups and 73.7 ± 5.3% in 50 µM G3-C12+H/R groups vs. 27.2 ± 4.0% in the H/R group, respectively, P < 0.05) (Fig. 5D-E). mPTP levels were also reduced by G3-C12 and pectin (Fig. 5F). Galectin-3 inhibition increased the calcium retention capacity in HL-1 cells upon H/R in the absence and presence of CsA (Fig. 5G). In addition, H/R treatment significantly increased the content of MDA and reduced SOD activity. Galectin-3 inhibition reduced the content of MDA and increased SOD activity (Fig. 5H-I).

3.6. Galectin-3 inhibition preserves mitochondrial homeostasis and attenuates HL-1 apoptosis induced by H/R We next explored whether galectin-3 inhibition is beneficial to H/R-induced mitochondrial dysfunction and cell apoptosis. As shown in Fig. 19

6A-B, galectin-3 inhibition prevented the release of cyto c from the mitochondria in cardiomyocytes subjected to H/R. Western blot analysis indicated that H/R significantly increased the expression of Bax (1.57 fold change), cleaved caspase-3 (3.57 fold change) and cleaved caspase-9 (3.2 fold change) in HL-1 cells, which was suppressed by treatment with galectin-3 inhibitors (Fig. 6C-D). In addition, H/R-stimulated the degradation of Bcl-2, a vital anti-apoptotic protein, which was attenuated following galectin-3 inhibition (2.13 to 2.97-fold compared to the H/R group). Taken together, these data suggest that galectin-3 inhibitors significantly protect cardiac cells subjected to H/R injury.

4. Discussion Cardiovascular disease is a major cause of morbidity and mortality, and is responsible for more than 15 million deaths each year (Mortality et al. 2016). Myocardium IR injury directly relates to the loss of blood flow and the duration of ischemic insult, resulting in imbalances between energy supply and demand (Lesnefsky et al. 2017). Previous studies suggest that galectin-3 participates in various physiological and pathological processes, including cell growth, differentiation, adhesion, and apoptosis (Liu et al. 2009). However the relationship between galectin-3 and myocardial IR injury is poorly defined. In this study, RNA-seq analysis of mice left ventricular samples subjected to IR revealed a 22-fold upregulation in Lgals3 compared to sham groups (Fig. 20

1-2), which was confirmed by immunohistochemical staining and western blot analysis (Fig. 3A-B). Galectin-3 is a biomarker for the severity of heart failure (de Boer et al. 2014). Increased expression of cardiac galectin-3 at the mRNA and protein level prompted us to explore whether alterations in galectin-3 affect the susceptibility of the heart to IR injury. We targeted galectin-3 and used pharmaceutical inhibitors to ameliorate myocardial damage. It has been shown that pectin from citrus binds to galectin-3 and promotes its regulation and biological activity through recognition of its carbohydrate recognition domain (de Boer et al. 2014, Lu et al. 2017). G3-C12 is also a promising ligand specifically binds to galectin-3 (Sun et al. 2017). In this study, we verified at the whole animal level that the inhibition of galectin-3 protects the heart against IR injury. During reperfusion, tissue responses that promote reactive oxygen species production, endoplasmic reticulum stress, inflammatory infiltration, and calcium overload lead to tissue injury. These pathologic events lead to extensive cardiomyocyte death and affect the development of coronary heart disease (Sun et al. 2015). Galectin-3 inhibition is also beneficial for aldosterone-induced cardiac and renal injury (Calvier et al. 2015). Pharmacological inhibition of the mitochondrial NADPH oxidase 4/PKCa/Gal-3 pathway reduces left ventricular fibrosis following myocardial infarction and offers new therapeutic options for adverse cardiac remodeling (Asensio-Lopez et al. 2018). These data are consistent with the hypothesis that galectin-3 inhibition confers cardioprotective effects, demonstrated by improved cardiac 21

function, decreased infarct size and improved histopathological scores in vivo (Fig. 4). Inhibiting galectin-3 also increased cell viability and decreased LDH leakage in vitro (Fig. 5). These findings were consistent with those of Doverhag and colleagues who indicated that Galectin-3 is involved in neonatal hypoxic-ischemic brain injury by regulating inflammatory responses (Doverhag et al. 2010). Mitochondria are central players and final effectors in cell death, and are the focus of many cardioprotective signaling mechanisms (Zhang et al. 2015, Dong et al. 2016, He et al. 2017). Cardiomyocyte mitochondria lead to cell damage during IR injury as they fail to provide energy and excessive reactive oxygen species production. These results clearly indicate that galectin-3 treatment results in well-preserved MMP and mPTP levels (Fig. 5D-G), which prevent the release of cyto c from the mitochondria to the cytoplasm (Fig. 6A-B). Mitochondria are not only solely responsible for energy metabolism, but are likely to act as important signaling organelles for apoptotic induction (Zhou et al. 2017). Yoshihiro and colleagues reported that galectin-3 exhibits pro-apoptotic activity in mast cells through oxidative stress and mitochondrial permeability transition through its binding to putative surface receptors via CRD (Suzuki et al. 2008). Al-Salam and Hashmi indicated that galectin-3 has anti-apoptotic effects during myocardial IR injury. Western blot analysis revealed that the levels of cleaved-caspase 3, cytc and BAX in IR models were significantly higher in galectin-3-KO mice compared to WT mice, 22

whilst Bcl-2 levels decreased. From immunohistochemical analysis, cleaved-caspase 3 and cytc levels in galectin-3-KO mice were also significantly higher in the IR model compared to WT mice. Annexin V assays showed that IR-induced apoptosis in the cardiomyocytes of galectin-3-KO mice was higher than WT mice (Al-Salam et al. 2018). In this study, when galectin-3 was inhibited, mitochondrial homeostasis was maintained. Meanwhile, the levels of Bax, cleaved caspase-3, and cleaved caspase-9 decreased, whilst Bcl-2 levels increased following Galectin-3 inhibition. The formation of the apoptosome was reduced and apoptosis was inhibited. The possible reasons for these differences were that galectin-3-KO mice were used for all previous experimental studies. In our animal experiments, galectin-3 inhibitors (G3-C12, pectin), previously demonstrated to have high affinity for galectin-3, were used to transiently reduce activity and function (Sun et al. 2015, Sun et al. 2017). Higher levels of circulating galectin-3 are associated with major adverse cardiovascular events including heart failure (HF), arrhythmias, arterial stiffening, re-hospitalization post-HF discharge, diastolic dysfunction, and the severity of atrial fibrosis and mortality (Nguyen et al. 2018). The upregulation of galectin-3 has been shown in failing human hearts and is a powerful predictor of poorer prognosis (Yu et al. 2013). Under normal conditions, galectin-3 is widely distributed including in the heart, and plays a variety of important physiological roles including cell growth, adhesion and cell-cell interactions. Galectin-3 specifically binds to extracellular or 23

intracellular ligands and modulates cell development, immune responses, and signal transduction by binding to intracellular ligands and participating in intracellular signaling (Balan et al. 2010). Galectin-3 acts as a physical barrier and biological sensor by regulating the entry of various pathogens through its surveillance of the extracellular milieu. It also associates with various receptor kinases to cause alterations in intracellular biological processes. Moreover, galectin-3 lattices regulate metabolic homeostasis through alterations in glucagon receptor and solute transporter expression (Suthahar et al. 2018). The accumulation of this evidence suggests that galectin-3 inhibition has protective effects against myocardial IR injury. This was consistent with previous studies in which downregulating galectin-3 expression was associated with cardioprotective functions (Liu et al. 2018). Galectin-3 gene knockouts may cause harmful effects due to physiological dysfunction. Nguyen and colleagues found that increased galectin-3 levels were associated with etiology and mediated by different mechanisms in cardiomyopathy (Nguyen et al. 2018). Further studies are now required to elucidate these mechanisms and explore the clinical application of galectin-3 inhibitors. In conclusion, galectin-3 inhibition significantly affects the development of myocardial IR injury. These studies suggest a novel pharmacological strategy for cardioprotection in which galectin-3 can be used as a new biological target for specific interventions.

24

Conflict of interest statement The authors declare that there is no conflict of interest.

Acknowledgments This work was supported by National Natural Science Foundation of China No. 81970245, 81770432, to X. Q. Li., No.81473329 to W. Cao, No.81601151 to H.N. Zhang, the Innovation Capacity Support Project of Shaanxi Province in China No.2019PT-23 to X. Q. Li., and the Major Project of Science, Technology and Innovation in Yangling demonstration area No.2017CXY-19 to W. Cao.

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Fig.1. Differentially expressed genes in myocardial IR according to RNA-seq. (A) Heatmap of whole differentially expressed genes between sham and IR groups. (B-C) Scatter plots and volcano plots. Red dots represent up-regulated DEGs; green dots represent down-regulated DEGs; grey dots represent genes without significant changes.

Fig.2. DEG Gene Ontology (GO) analysis shows a vital role of galectin-3 in IR-induced cardiac injury. (A) GO terms in biological process (BP). (B) GO terms in cellular components (CC). (C) GO terms in molecular function (MF). (D) Intersection outcomes showing commonly and uniquely expressed genes among the three GO terms with the highest enrichment scores. (E) Heatmap of the top 8 DEGs.

Fig.3. Inhibition of galectin-3 decreases IR-induced cardiac dysfunction. (A) Representative images of immunohistochemistry stained mouse heart sections. Scale bars: 100 µm. Whole sections are shown on the right. (B) Quantification of galectin-3 expression level in HL-1 cells subjected to H/R. Beta-actin was used as a loading control. Data are the mean ± S.E.M., n = 3, **P < 0.01. (C) Representative echocardiographs showing cardiac function from the various groups. (D) Ejection fractions (EF) and fractional shortening (FS) measured by echocardiography. n = 5, *P < 0.05, **P < 0.01 versus sham 32

group. ##P < 0.01 versus IR group.

Fig.4. Galectin-3 inhibition contributes to the protection from IR-induced damage. (A) Representative images of Evans Blue and TTC stained sections. Dashed lines indicate lesion borders. Scale bar, 2 mm. n = 3. Size of the infarction area calculated as infarct area divided by the area at risk (IF/AAR). LV, left ventricular. (B) H&E staining of heart sections and histologic scores. n = 4 areas, 3 mice per group. Scale bar, 100 µm. Yellow arrows indicate myocardial damage. (C) Representative micrographs and TUNEL staining in IR injured heart sections. TUNEL-positive cells are indicated with arrows. Scale bar: 100 µm. Data are the mean ± S.E.M.. **P < 0.01 versus sham group, #

P < 0.05, ##P < 0.01 versus IR group.

Fig.5. Galectin-3 inhibition prevents H/R-induced cardiomyocyte injury. (A) Representative images of cardiomyocytes. Scale bar: 100 µm. (B) Cell viability detected by trypan blue staining. (C) Measurement of lactate dehydrogenase (LDH) release in different cardiomyocyte culture medium. LDH release in the NX group was normalized to controls. (D) Flow cytometry fluorescence ratios showing MMP levels by JC-1. Percentages in B2 and B4 areas show higher or lower MMP levels, respectively. (E) MMP level analysis. MMP in the NX group was normalized to controls. (F) Changes in mPTP opening. (G) Calcium retention capacity in the absence and presence of 33

cyclosporine A (CsA). (H) MDA content. (I) SOD activity. Data are the mean ± S.E.M., n = 3. *P < 0.05, **P < 0.01 versus NX group, #P < 0.05, ##P < 0.01 versus H/R group.

Fig.6. Inhibition of galectin-3 protects cardiomyocytes from H/R damage. (A) Representative immunoblots of mitochondrial cyto c and cytosolic cyto c. (B) Quantification of the indicated proteins. Cytochrome c oxidase IV (COX IV), a marker for mitochondria, was used as a loading control for the mitochondrial fraction. Tubulin served as a loading control for the cytosolic fraction. (C) Representative immunoblots of Bax, Bcl-2, cleaved caspase-3 and cleaved caspase-9. (D) Quantification of the indicated proteins. Tubulin expression was used as a loading control. Data are the mean ± S.E.M., n = 3. *P < 0.05, **P < 0.01 versus NX group, #P < 0.05, ##P < 0.01 versus H/R group.

34

Table

1.

Echocardiographic

parameters

of

LV

function

in

control mice and in mice after IR induction

Sham

(%)

LV Mass (mg)

Heart Rate (bpm)

44.93 ±6.13

57.31 ±6.55

495.40

80.98

46.29

479.00

1.07

±7.09 75.30

±8.18 38.29

60.20 ±8.80

LVEDD

LVESD

PWT,d

PWT,s

EF

FS

(mm)

(mm)

(mm)

(mm)

(%)

2.83 ±0.10

1.56 ±0.18

0.73 ±0.06

1.09 ±0.05

80.90 ±5.33

0.63 ±0.01

1.07 ±0.06

0.78

2.95

1.59

5.0 mg/kg

±0.10 2.73

±0.25 1.69

G3-C12

±0.05

±0.15

±0.07

±0.05

±4.97

IR

3.32 ±0.23

2.71 ±0.23

0.61 ±0.02

0.77 ±0.03

Pectin+IR

2.95 ±0.06

1.84 ±0.12

0.8 ±0.06

3.83 ±0.12

2.97 ±0.17

3.37 ±0.15 3.02 ±0.09

1.0 mg/kg G3-C12+IR 2.5 mg/kg G3-C12+IR 5.0 mg/kg G3-C12+IR

±13.56

48.15

±12.07 506.33

±4.43

±4.00

±11.49

45.72 b ±3.64

18.59 b ±1.83

55.49 ±8.79

476.25

1.2 ±0.07

74.75 c ±3.93

37.56 c ±3.49

64.77 ±7.01

469.40

0.51 ±0.03

0.67 ±0.06

52.57 b ±5.76

22.48 a ±3.01

70.57 ±6.61

483.40

2.25 ±0.12

0.69 ±0.08

0.99 ±0.16

69.76 c ±2.82

33.14 c ±2.09

70.91 ±11.32

479.00

1.89 ±0.19

0.61 ±0.04

1.00 ±0.09

74.18 c ±5.73

37.73 c ±4.71

50.4 ±2.91

474.60

±16.26 ±15.82 ±18.59

±15.34 ±16.35

LVEDD: left ventricular end-diastolic diameter; LVESD: left ventricular end-systolic diameters; PWT,d: posterior wall thickness in diastole; PWT,s: posterior wall thickness in systole; EF: ejection fraction; FS: fractional shortening; LV mass: left ventricular mass. Data are presented as the mean ± S.E.M., n=5. aP < 0.05, bP < 0.01 versus sham group; cP < 0.01 versus IR group.

1

Dear Editor, We undersigned a statement that this manuscript entitled “Cardioprotective effects of galectin-3 inhibition against ischemia/reperfusion injury” is original. It has not been published before and is not currently being considered for publication elsewhere. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We understand that the Corresponding Author is the sole contact for the Editorial process. He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. Signed by all authors as follows: Dan Mo, Wen Tian, Hui-Nan Zhang, Ying-Da Feng, Yang Sun, Wei Quan, Xiao-Wei Hao, Xue-Ying Wang, Xiao-Xiao Liu, Chen Li, Wei Cao, Wen-Juan Liu, Xiao-Qiang Li. Thank you and best regards. Yours sincerely, Dan Mo Corresponding author: Name: Xiao-Qiang Li E-mail: [email protected]