Coumarin-derived imino sulfonate 5h ameliorates cardiac injury induced by myocardial infarction via activating Sirt1/Nrf2 signaling pathway

doi:10.21203/rs.3.rs-1986700/v1

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Coumarin-derived imino sulfonate 5h ameliorates cardiac injury induced by myocardial infarction via activating Sirt1/Nrf2 signaling pathway

https://doi.org/10.21203/rs.3.rs-1986700/v1

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Myocardial infarction (MI) is an irreversible damage caused by ischemia and hypoxia in coronary arteries accompanied with elevated levels of catecholamine, leading to the accumulation of free radicals. Our previous study discovered coumarin-derived imino sulfonates as a novel class of potential cardioprotective agents possessing strong anti-oxidative effect in cardimyocytes. Therefore, the identification of the compound with the highest cardioprotective activity, 5h, and the mechanism involved was necessary. As a kind of catecholamine, isoproterenol can clinically induce myocardial infarction injury similar to the symptoms of myocardial infarction patients. Our experiments explored the underlying mechanism of this effect of compound 5h by assessing cardiac function, infarct size, histopathological changes, and downregulation of Sirt1 by transfection of adenovirus in vitro and by administering Ex527, a specific inhibitor of Sirt1, in vivo. Compound 5h exhibited strong cardioprotective actions in vivo and in vitro via improving cell survival, cardiac function and decreasing the cellular oxidative stress and cardiac infarct size against MI. Furthermore, compound 5h significantly enhanced cardiac expression of Sirt1, subsequently activating the Nrf2/NQO1 signaling pathway. However, adenovirus induced Sirt1 downregulation or Sirt1 specific inhibitor largely blocked such beneficial effects of 5h in vitro and in vivo, respectively. Taken together our results demonstrated, for the first time, that the cardioprotective action of 5h against MI was mediated by reducing oxidative stress and apoptosis through the Sirt1/Nrf2 signaling pathway. Our findings proposed novel insights in the development and evaluation of coumarin-derived imino sulfonate compounds as epigenetics-targeted drug therapy for MI.

Myocardial infarction

Coumarin-derived imino sulfonate

Oxidative stress

Sirt1

Nrf2

Acute myocardial infarction (MI), a serious life-threatening disease caused by coronary artery occlusion, mainly increases the mortality rate of patients with cardiovascular disease (Chen & Kunsch, 2004). The increase of cardiac oxidative stress, especially reactive oxygen species (ROS), is dramatically accumulated during the pathological process of acute MI, leading to the cardiac dysfunction (Doulamis et al., 2017; Gandhi et al., 2009). Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a major redox-sensitive transcription factor that acts as an antioxidant sensor to regulate cellular redox state (Chen & Kunsch, 2004) by controlling the expression of various cytoprotective genes. Oxidative stress promotes the nuclear translocation of Nrf2, which binds to antioxidant response elements (AREs) within target gene promoters to regulate gene expression (Jin, Li, Li & Xiao, 2019). Therefore, targeting Nrf2 has been considered as the potential therapeutic strategy for treatment and prevention for acute MI.

Coumarin, a naturally present compound in various vegetables and herbs and spices (e.g., tonka bean, cinnamon), were used as popular flavors in the U.S (Rahim, Saad, Osman, Hashim, Yahya & Talib, 2011; Wang, Avula, Nanayakkara, Zhao & Khan, 2013).Meanwhile, coumarin also can be found in cereals (e.g., wheat, maize, barley, and oat) or several derived products, such as corn flakes, flour, malt and beer(Dall’Asta et al., 2004). Notably, it plays an important role in the drug discovery process due to its physiological and pharmacological activities with a stable structure, especially for anti-oxidative stress property (Raters & Matissek, 2008). Studies have shown that the widely concerned coumarin derived compounds have various pharmacological activities, such as anticoagulant, anti-inflammatory(Garg, Gupta, Sharma & Sahu, 2020), antibacterial, antioxidant, antitumor (Wu et al., 2020a), antiviral and enzyme inhibitory effects(Ozalp, Danış, Yuce-Dursun, Demir, Gündüz & Ogan, 2020), without toxic actions on normal cells. The basic skeleton of coumarin, a general term for cis-o-hydroxycinnamic acid lactones, has the parent nucleus of benzoα-pyrone. Notably, coumarin derivatives have been reported as Nrf2 activator previously (Yao, Zhang, Zhang, Li, Hua & Li, 2020). Our previous study reported that pretreatment of H9c2 cells with coumarin-derived imino sulfonate compound 5h with the highest activity and low cytotoxicity could dramatically reduce the oxidative stress induced by hydrogen peroxide (H₂O₂) via activating the Nrf2 pathway(Wei et al., 2019). However, the exact mechanism underlying such beneficial effect of compound 5h on activating Nrf2 remains largely to be elucidated.

Importantly, silent information regulatory transcript 1 (Sirt1), a nicotinamide adenine dinucleotide (NAD⁺)-dependent histone deacetylase, is involved in multiple cellular metabolisms, especially for the oxidative stress(Yang et al., 2016). Studies have reported that Sirt1 can regulate important transcription factors such as Nrf2, which is important for antioxidant, anti-inflammatory and cytoprotective effects(Yang et al., 2018b). Our previous work has demonstrated the role of Sirt1 in activating Nrf2 during myocardial ischemia/reperfusion(Xu et al., 2021). A hypothesis that is made more plausible that whether the cardioprotective role played by 5h is related to the regulation of Nrf2 by Sirt1.

Therefore, this study aimed to explore the cardioprotective effect and the exact mechanism of 5h against myocardial infarction in vitro and in vivo. In the present study, our results identified, for the first time, that the cardioprotective action of 5h against myocardial infarction was mediated by reducing oxidative stress and apoptosis through the Sirt1/Nrf2 signaling pathway. Our findings provided a novel insights in the development and evaluation of coumarin-derived imino sulfonate compounds as epigenetics-targeted drug therapy for myocardial infarction.

2.1. Materials

Compound 5h was synthesized as our previous work(Wei et al., 2019). Luteolin (purity > 98%) was obtained from Aladdin Industrial Co. (Shanghai, China). Ex527 (purity 99.87%) was purchased from Med Chem Express (MCE, USA). Biochemical assay kits of creatine kinase-MB (CK-MB)(H197-1-1), lactate dehydrogenase (LDH)(A020-2-2), aspartate aminotransferase (AST)(C010-2-1), superoxide dismutase (SOD)(A001-3-2), catalase (CAT)(A007-1-1), malondialdehyde (MDA)(A003-1-1) and glutathione (GSH)(A006-2) were purchased from Nanjing Jiancheng (Nanjing, China). Terminal deoxynucleotidyl transferase-mediated Dutp Nickel End Labeling (TUNEL)(G002-1-2) Fresh Red Blood Cell Apoptosis Detection Kit was supplied from Vazyme Biotech (Nanjing, China). BCA Reagent test kit Purchased from Solebao Technology Co.

2.2. Cell culture and treatment

H9c2 cell (ATCC Cat#CRL-1446, RRID:CVCL_XE65) were purchased from the American Type Culture Collection (Shanghai, China). Cells were cultured in Dulbecco's Modified Eagle Media: Nutrient Mixture F-12 (DMEM/F12) with 10% FBS (GIBCO, Carlsbad, California, USA) and 100IU·mL^− 1 penicillin and 100 µg·mL^− 1 streptomycin at 37°C in a humidified atmosphere of 5% CO₂. Cells were fed every 2–3 days and sub-cultured when they reached 70–80% confluence. H9c2 cells were seeded at a density of 5\(\times\)10³ cells/well in 96-well plates (100µL/well) and adhered overnight in an incubator at 37°C until confluency ≥ 80%.

For treatment, H9c2 cells were seeded at a density of 5×10³ cells per well in 96-well plates overnight. After reaching about 70–80% confluency, cells were starved for 12 hin DMEM/F12 supplemented with 0.5% FBS. Thereafter, 5, 10, 20 µmol·L^− 1 of coumarin derived compound 5 h were pretreated with (dissolved in 0.1% DMSO) cells for 24 h, followed with or without 300 µmol·L^− 1 of H₂O₂ for 1.5 h (Cui, et al., 2021).

2.3. Adenoviral-mediated gene transfer

Antisense Sirt1 adenoviruses (Ad. Sirt1-AS) or control Ad. GFP were obtained from Biocan BioNTech (Shenzhen, China). Adenoviruses were amplified in HEK293 cells, purified with ViraKit from Bijapur and tittered, according to the standard procedure of Adeno XTM rapid titer kit from BIOMIGA (San Diego, CA, USA). After 2 h of plating, H9c2 cells were infected with Ad. Sirt1-AS or Ad. GFP at a multiplicity of infection (MOI) of 400 for 48 h before the addition of a suitable volume of complete DMEM medium. The efficiency of adenoviral gene transfer was evaluated in cultured H9c2 cells by fluorescence microscopy (Nikon Eclipse Ti-S; Nikon Ltd., Tokyo, Japan). Nearly 100% of cells appeared infected at 400 MOI by 48 h. The cell phenotype and morphology remained similar among non-infected and adenoviral-infected groups after 48 h of infection. The cardiomyocytes were then treated with compound 5h in the presence or absence of H₂O₂ for indicated time, washed with PBS and harvested for quantitative immunoblotting, or cellular injury assays.

2.4. Cell viability and LDH determination

In order to evaluate the protective effect of compound 5h, the Control group, model group and treatment group were set up respectively; H9c2 cells were exposed to different concentrations of compound 5h (5, 10, 20 µM) for 24 h, and then treated with 300 µM H₂O₂ for 1.5 h. All experimental groups of H9c2 cells were treated with MTT solution and incubated for 4 h at 37°C in the dark. Finally, discard the supernatant from the 96-well plate, dissolve the purple crystals with 150 µL of dimethyl sulfoxide (DMSO), and measure cell viability at 490 nm.

In addition, the cytotoxicity test kit was used to detect the LDH release rate according to the manufacturer's instructions (Nanjing Jiancheng Institute of Bioengineering, China), and then quantified by the absorbance at 490nm with a microplate reader.

2.5. Detection of ROS

Intracellular levels of ROS were assessed using conversion of non-fluorescent dihydroethidium (DHE) to fluorescent ethidium bromide (Wei et al., 2018). Briefly, H9c2 cells were plated in a 6-well plate (2.5×10⁵ cells/well) and incubated with the designated doses of compound 5h with or without 300µM H₂O₂. Cells were then washed in PBS and incubated with 5 µM DHE at 37°C for 30 min according to the manufacturer's instructions. Ethidium fluorescence (excitation at 495 nm, emission at 529 nm) was examined by flowcytometry (Accuri C6, BD, San Jose, CA, USA) immediately. 1× 10⁴ cells were collected and analyzed. The superoxide anion levels were calculated by the FlowJo software. Additionally, morphological analysis was performed through fluorescence microscopy (Nikon Eclipse Ti–S, Nikon Ltd, Japan). For calculation of relative changes in ROS level, values of individual samples were divided by the mean value of samples from the control or Ad. GFP group, respectively.

2.6. Animals

Our experimental reports follow the ARRIVE guidelines (Percie et al., 2020) Specific pathogen-free (SPF) male Sprague-Dawley rats weighing 200-250g, 7–8 weeks old, were obtained from the Henan Provincial Laboratory Animal Center (NO. 410975221100019174, Zhengzhou, China). All animal experiments were conducted in

accordance with the guidelines of the Chinese Council on Animal Care. Rats were housed in groups of 3 to 4 per cage under standard circadian light conditions (12/12 h) at a controlled ambient temperature of 25 ± 2°C for 7 days prior to the experiment. Make every effort to minimize animal distress and the number of animals required to obtain reliable results according to the rules of replacement, improvement, or reduction (3Rs).

2.7. Preparation of rat acute MI model

Following acclimatization for 1 week, animal experiments were conducted. Luteolin, demonstrated to possess significant anti-ischemic effects in previous studies (Rezvan, 2017), was employed as the positive control, The specific ani-mal experiment process was shown in the supplementary material (Figure S1).

Experiment protocol I: Rats were randomized to six groups (n = 12): the control (Control), myocardial infarction model (MI), low-dosage of compound 5h (5 mg·kg^− 1) L + MI (5hL + MI), middle-dosage of compound 5h (10 mg·kg^− 1) M + MI (5hM + MI), high-dosage of compound 5h (20 mg·kg^− 1) H + MI (5hH + MI), and positive control luteolin (20 mg·kg^− 1) + MI (LUT + MI) group.

Experiment protocol II: Rats were randomized to five groups (n = 12) Control, MI, 5hM + MI, Ex527 + MI and Ex527 + 5hM + MI group.

The compound 5h was administered Intraperitoneally in rats from 5hL, 5hM, and 5hH groups, respectively, once a day for two weeks. Rats from the Con group were orally administered 1mL of normal saline once a day. In the LUT + MI group, rats were administered with luteolin (20 mg·kg^− 1) intraperitoneally once a day for two weeks. In theEx527 + MI and Ex527 + 5hM + MI groups, Ex527 (5 mg·kg^− 1) or the same volume of vehicle was injected intraperitoneally (48 h interval, 4 consecutive times) before ISO injection. All rats excluding those in Control group, were subcutaneously injected by ISO (100 mg·kg^− 1) with a 24 h interval for two days for the induction of MI(Cui, Shi, Xu, Zhao, Zhang & Wei, 2021).

2.8. Electrocardiogram

Electrocardiograms (ECG) detected the types with alteration (ST-segment depression or elevation). Then, each rat was anesthetized with 2–3% isoflurane (by inhalation) at 12 h after the final injection of ISO in the inducing chamber. At the same time, the adequacy of anesthesia was calculated on the basis of the disappearance of the pedal withdrawal reflex. Subsequently, the lead II electrocardiograph was obtained with the use of the multi-channel physiological signal acquisition and processing system (RM6240, Chengdu instrument factory, China).

2.9. Echocardiographic assessment of cardiac function

Left ventricular function was assessed using an MS-250, 16.0–21.0 MHz imaging transducer connected to an echocardiography system (FUJIFILM Visual Sonics Vevo 2100, Inc., Toronto, Ontario, Canada), designed for small animals. Under anesthesia, the rat's chest was shaved and two-dimensional echocardiography was performed. Images were obtained by identifying the ventricular profile and posterior wall of the left ventricle. Left ventricular fractional shortening (FS%) and ejection fraction (EF%) were automatically calculated by the echocardiographic system. Each parameter was evaluated by calculating the average of five cardiac cycles.

2.10. Blood sample and tissue processing

At the indicated time points, rats were sacrificed by overdose with sodium pentobarbital (3%, 80 mg·kg^− 1) anesthesia, blood was collected from the carotid artery, and cardiac specimens were obtained. Serum samples were collected in dry tubes without anticoagulant and centrifuged at 3000 rpm and 4°C for 10 min. Immediately, heart tissues were removed, minced and placed on ice. Residual serum and heart tissue samples were kept at − 80°C for further analysis.

2.11. Detecting of cardiac infarct size

Cardiac infarct size was determined using 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich, ST. Louis, MO) staining. Rat hearts were rapidly dissected, frozen at − 20°C, and cross-sectioned (2mm thick). Quarters were stained with 1% TTC buffer pH 7.4 in the dark at 37°C. Red staining areas suggest ischemic but viable tissue, while negative staining areas (white or pale) suggest myocardial infarction. Regions were segmented and assessed digitally using Image-Pro Plus 6.0. The cardiac infarction area was the ratio of the infarcted myocardium to the left ventricle area × 100%.

2.12. Detecting of biochemical indexes

Activities of CK-MB, LDH, and AST in serum were assayed using commercial kits purchased from Jiancheng Institute of Biotechnology (Nanjing, China).

2.13. Histopathological analysis of heart tissue

Heart tissues were fixed in 10% buffered formalin and embedded in paraffin. For the histological assessment, paraffin embedded tissue sections of heart (4µm) were stained with hematoxylin-eosin (H&E), and examined microscopically (×200).

2.14. TUNEL staining of heart section

The heart paraffin sections were fabricated and dyed by adopting the TUNEL apoptosis detection kit (KeyGen Biotech, Nanjing, China) for revealing apoptotic cells, following the manufacturer’s instructions. Apoptotic positive cells were observed by light microscopy or fluorescence microscopy. The experiment was repeated for five different sections of each sample. Ten random fields (×400) were analyzed per section. The average percentage of apoptotic cells was then calculated.

2.15. Western blot assay

For cell samples, cell lysates were obtained using commercial RIPA lysis buffer (Beyotime, China). For cardiac samples, they were homogenized with commercial RIPA lysis buffer. The protein content was determined by BCA kit (Solobo, China). Then, proteins were separated by SDS-PAGE and transferred to PVDF membranes. Western blots were blocked in 5% nonfat dry milk-TBS-0.1% Tween 20 for 2 h. Then, the membrane was incubated with the primary antibody overnight at 4°C. Horseradish peroxidase-conjugated secondary antibody rabbit or mouse antibody (1:10,000; Cell Signaling Technology Co., Ltd, MN, USA) was incubated for 2 h. Immunoreactivity was detected by a gel imaging system (Protein Simple, Santa Clara, California, USA) and enhanced chemiluminescence detection reagent super Signal West Pico chemiluminescence reagent (34,079, Pierce, Thermo Scientific, Rockford, IL, USA). Quantification of protein bands was performed using the Image J program (RRID:SCR_003070). Primary antibodies used were monoclonal against the oxidative stress proteins Sirt1 (Cell Signaling Technology cat. no. 9475, RRID: AB_2617130), Nrf2 (Abcam cat. no. ab137550, RRID: AB_2687540), NQO1 (Abcam cat. no. ab80588, RRID: AB_1603750). Antibody. Meanwhile, GAPDH (Cell Signaling Technology Cat#5174, RRID:AB_10622025) was used as a loading control.

2.16. Statistical analysis

Through randomization and blinded analysis, the study aimed to establish groups of equal size. The sample size n defines the number of biologically independent replicates used for statistical analysis. Comparisons between multiple groups were performed by Tukey's post hoc test or nonparametric Kruskal-Wallis test, followed by Bonferroni test (from GraphPad Prism 7.0, RRID: SCR_000306) ANOVA was used for multiple group comparisons. Quantitative data were presented as mean and standard deviation (SD), and results were considered statistically significant at P < 0.05.

3.1. Compound 5h inhibited the injury induced by H₂O₂ in H9c2 cells

To determine the effect of compound 5h on cell viability in H₂O₂-treated H9c2 cells, MTT and LDH leakage assays were selected. We found that compound 5h up to 100 µM did not exhibit any significant cytotoxicity in H9c2 cells (Fig. 1A). Therefore, compound 5h at 5, 10, 20 µM was chosen in the experiments for subsequent experiments. Pretreatment with compound 5h (5, 10, 20 µM) for 24 h significantly increased the cell viability of H9c2 cells exposed to H₂O₂ (Fig. 1B). Membrane integrity by measuring LDH release was also monitored. Cells exposed to H₂O₂ significantly increased the LDH release. Whereas, pretreatment with compound 5h (5, 10, 20 µM) profoundly decreased the LDH release in a dose-dependent manner (Fig. 1C). Additionally, compared with the control group, H9c2 cells morphologically atrophied and died after H₂O₂ treatment, while H₂O₂-induced damage was significantly recovered by pretreatment with compound 5h at different concentrations (Fig. 1D).

3.2. Compound 5h protected against acute MI induced by ISO in rat

To determine the effect of compound 5h against acute MI in vivo, myocardial infarction was established in rats by more subcutaneous injection of ISO. Electrocardiogram was monitored and recorded after the second subcutaneous injection of ISO. Compared with the rats in the Control group, the ST-segment of the ISO rats was markedly elevated, indicating that the establishment of the MI model was successful. However, pretreatment with compound 5h at different concentration significantly reduced the ST-segment of ISO treated rats (Fig. 2A). In addition, cardiac infarct size was assessed by TTC staining assay (Fig. 2B), and increased cardiac infarct size was observed in MI rats up to 68.79% compared to Control group. However, pretreatment with compound 5h (5, 10 or 20 mg·kg^− 1) significantly reduced infarct size to 45.33%, 29.70%, and 6.30%, respectively, as compared with the MI rats (Fig. 2C). In addition, three sensitive indexes of myocardial enzymes in serum were analyzed (Fig. 2D-F). The activities of CK-MB, AST and LDH in the serum of the MI group were significantly higher than those of the Control group. Whereas, values of these indexes were significantly declined in compound 5h (5, 10 or 20 mg·kg^− 1) pretreated rats. Although the lowest dose of compound 5h (5 mg·kg^− 1) only showed a trend of reversing the increase in serum AST activity and no statistical difference was found, both the middle and high doses of compound 5h (10 or 20 mg·kg^− 1) could significantly reverse the myocardial zymogram in serum.

Also during MI, oxidative stress leads to lipid peroxidation, leading to the cell membrane rupture. Compared with the Control group, the serum activities of CAT, GSH, and SOD in the MI group were significantly decreased (Fig. 2G, H, J), while the level of MDA was dramatically decreased (Fig. 2I). Notably, pre-administration with compound 5h significantly enhanced the activity of these enzymatic antioxidant defense systems in a dose-dependent manner. Specifically, compound 5h increased CAT levels to about 3 folds, GSH about 2 folds of those in MI rats. Meanwhile, compound 5h dose-dependently declined MDA serum level, relative to the MI group (Fig. 2I).

3.3. Compound 5h ameliorated cardiac function against acute MI induced by ISO in rat

Cardiac function was assessed by echocardiography (Fig. 3A). As indicated by (Fig. 3C-D), cardiac function was dramatically impaired by ISO induced acute MI, compared with the Control group, as demonstrated by the significant decrease in EF% to 59.58% and FS% to 42.52%, respectively. However, pretreatment with 5h (5, 10 or 20 mg·kg^− 1) significantly improved EF% to 77.06%, 83.14%and 91.24%, respectively, and elevated FS% to 52.15%, 61.06% and 65.46%%, respectively. The heart rate of the MI group was also markedly reduced, relative to that in Control group. As compared with the Control rats, MI injury was also confirmed by a decrease in hemodynamic functional recovery, demonstrated by the significantly decreased LVSP (Fig. 3F), left ventricular maximum and minimum pressure rate (LV dp/dt_max and LV dp/dt_min) (Fig. 3H), along with the remarkably elevated LVEDP (Fig. 3G). Notably, compound 5h pretreatment surprisingly reversed these changes back to near normal values (Fig. 3E), indicating that compound 5h possessed the strong cardioprotective effect against acute MI injury in vivo.

Moreover, microscopy of heart tissue sections from different experimental groups revealed clear alterations among groups, as depicted in Fig. 3B. Hearts from the Control group exhibited marked myocardium integrity, normal myocardial fibers, without any infarction, inflammatory infiltration, or cardiac necrosis. However, the MI group had obvious signs of leukocyte infiltration, large-area of cardiac necrosis and severe edema. All these pathological changes were reversed after pretreatment with compound 5h (Fig. 3B). In addition, the number of apoptotic cells was significantly increased in rat exposed to large dose of ISO. In contrast, pretreatment with compound 5h (5, 10, 20 mg‧kg^− 1) showed fewer apoptotic cells, respectively (Fig. 3I-J). Importantly, cardiac apoptosis was lower in middle and high dosage of compound 5h, than those in the positive control group.

3.4. Knockdown of Sirt1 blocked the cardioprotective effect of compound 5h against H₂O₂ induced injury in H9c2 cells.

Previous studies have shown that compound 5h could protect H9c2 cells from H₂O₂-induced damage by up-regulating Nrf2(Wei et al., 2019). Given the predomitory role of Sirt1 in activating Nrf2, we investigated the potential effect of compound 5h on Sirt1 protein expression in ISO induced acute MI in vivo (Fig. 4A-D). Compared with the Control group, the level of Sirt1 in the MI group was significantly decreased. Whereas, compound 5h (5, 10, 20 mg·kg^− 1) treatment profoundly increased the cardiac expression of Sirt1, which was even higher than that in the Control group. In addition, protein expressions of Nrf2 and NQO1 were markedly up-regulated by administration of compound 5h (Fig. 4A-D).

To determine the role of Sirt1 in compound 5h of injured H9c2 cells, cells were infected with adenovirus Ad. Sirt1-AS (antisense) to knockdown Sirt1 as previous research(Xu et al., 2021), followed by H₂O₂ exposure. The cell viability of Ad. GFP + H₂O₂ and Ad. Sirt1-AS + H₂O₂ groups was significantly decreased to 50.17% and 46.79%, respectively, compared with Ad. GFP + CON group (Fig. 4E). The LDH release rate of Ad. Sirt1-AS + H₂O₂ cells was supprisingly increased up to 94.03%, which was higher than that of the Ad. GFP + H₂O₂ group (Fig. 4F). Pretreatment with compound 5h alone markedly restored the abnormalities caused by H₂O₂ in H9c2 cells. However, the cardioprotective effect of compound 5h was significantly blocked in Ad. Sirt1-AS infected cells. ROS levels were also measured using DHE as the fluorescent probe. Compared with the Ad.GFP + CON group, H₂O₂ treatment induced significant ROS accumulation in H9c2 cells. Notably, compound 5h significantly decreased the ROS accumulation in Ad. GFP + H₂O₂ cells (Fig. 4G-H). However, downregulation of Sirt1 almost completely abolished such actions of compound 5h.

Next, the protein levels of Sirt1 and Nrf2/NQO1 signaling pathway were detected. In Ad. GFP-infected H9c2 cells, upon H₂O₂ injury, pretreatment with compound 5h significantly up-regulated the expression of Sirt1, subsequently elevated the levels of Nrf2 and NQO1 (Fig. 4I-L). However, down-regulation of Sirt1 remarkably blocked such beneficial effects of compound 5h on Nrf2/NQO1 signaling pathway. These findings suggested that Sirt1 indeed played a critical role in activating Nrf2.

3.5. Compound 5h ameliorated acute MI injury dependent on Sirt1 in vivo

To further confirm the involvement of Sirt1 in the cardioprotective effect of compound 5h in vivo, the following experiments were performed by applying Ex527, the specific inhibitor of Sirt1 in vivo. Experiments found that compared with the Control group, a significant ECG ST-segment elevation was observed in the MI group, while the compound 5hM treated group (10 mg·kg^− 1) reduced ST-segment elevation. However, inhibition of Sirt1 expression by Ex527 significantly reversed the effect of compound 5h on ST-segment elevation (Fig. 5A). Furthermore, a dramatic increase in cardiac infarct size up to 60.87% was observed in the MI group. Compared with MI group, 5hM (10 mg·kg^− 1) significantly reduced infarct size to only 30.03% (Fig. 5B-C). Notably, inhibition of Sirt1 significantly reversed the beneficial effect of that in 5hM treatment group, which was similar to that in MI group. Furthermore, compared with MI group, the serum indexes CK-MB, LDH, AST all showed an obvious downward alteration with the decrease of 38.4%, 9.5% and 41.09% respectively (Fig. 5D-F). After the administration of Ex527, these indexes rose back to the similar level as those in MI rats. Moreover, Ex527 significantly inhibited the adjust of activities or levels of CAT, GSH, SOD and MDA in serum by compound 5h (Fig. 5G-J). These data indicated that Ex527 significantly inhibited the protective effect of compound 5h against myocardial infarction in rats.

3.6. Compound 5h improved MI-injured cardiac function dependent on Sirt1 in vivo

As previously described, left ventricular function was assessed by echocardiography (Fig. 6A), and cardiac function was impaired after MI compared with the Control group. Administration with compound 5h (10 mg·kg^− 1), significantly elevated the cardiac function in MI rats. However, such improvement was significantly inhibited by Ex527, demonstrated by the declined EF% to 60.27%, FS% to 39.20%, relative to those in compound 5hM + MI group (Fig. 6C-D). Likewise, relative to the MI group, values of LVSP, LVEDP and the maximum and minimum left ventricular pressure rate (LV dp/dt_max and LV dp/dt_min) of the Ex527 + MI group were almost the same as those of the MI group (Fig. 6F-H). Ex527 significantly abolished the hemodynamic functional recovery alteration induced by compound 5h, demonstrated by markedly reduced LVSP to 70.75%, LV dp/dt_max to 53.32%, and elevated LVEDP to 18.96%, LV dp/dt_min to 56.11% of those in compound 5hM + MI rats. Similarly, the heart rate of the Ex527 + 5hM + MI group was the same as that in the MI group (Fig. 6F-H).

Additionally, it was observed that the hearts from the Control group showed marked myocardium integrity, normal myocardial fibers without any infarction, inflammatory infiltration, or cardiac necrosis. Significant leukocytes infiltration, extensive cardiac necrosis and severe edema were observed in the MI rats and Ex527 + 5h + MI group (Fig. 6B). While ratsadministered only with compound 5h showed significant improvement on the histopathological alterations induced by ISO. The number of apoptotic positive cells in heart tissues was significantly increased in rats subjected to Ex527 + MI, which showed no significant difference with the MI rats. In contrast, pretreatment with compound 5h (10 mg·kg^− 1) showed much less apoptotic cells. However, Ex527 blocked such wonderful effect of compound 5h on the reduction of apoptotic cardiomyocytes in MI rat heart tissues. The expressions of Sirt1, Nrf2 and NQO1 were determined by quantitative immunoblotting with or without treatment with Sirt1 specific inhibitor Ex527. As expected, Ex527 significantly reversed the up-regulation of Sirt1 induced by compound 5h (Fig. 6K-N). In addition, cardiac Nrf2 and NQO1 expressions were subsequently down-regulated by Ex527, which further blocked the active effect of compound 5h on Nrf2/NQO1 signaling pathway during acute MI.

Coumarin derivatives, modified from chalcone derivatives, reduce cardiomyocyte death through their antioxidant properties, possibly due to their role in free radical scavenging. The present study employed large doses of ISO to induce myocardial infarction model in vivo and H₂O₂ to induce cardiac injury in vitro to determine the effect of compound 5h on cardiac oxidative stress during acute MI, and to further investigate the exact mechanism underlying such strong cardioprotective action.

As our previous work, the results of MTT experiments showed that compound 5h had no toxic effect on H9c2 cardiomyocytes. Compound 5h (5, 10, 20 µM) pretreatment exerted a powerful cardioprotective effect by significantly increasing cell viability, reducing LDH leakage rate and improving cell morphology in H₂O₂-induced injury in vitro. In addition, compound 5h (5, 10, 20 mg·kg^− 1) pretreatment significantly improved myocardial membrane stability compared with MI rats, as demonstrated by decreased serum levels of myocardial zymogram and improved cardiac histopathological changes. Excitingly, we found that compound 5h not only alleviated the impaired left ventricular systolic dysfunction and elevated ST-segment induced by acute MI, but also reduced the cardiac infarct size in vivo. Therefore, pre-administration for compound 5h indeed exerted a strong cardioprotective effect against acute MI injury both in vitro and in vivo.

Antioxidative stress has been proposed to play a key role in alleviating myocardial crest obstruction(Wu et al., 2020b). Notably, increased levels of antioxidant proteins can protect cardiomyocytes from oxidative stress-induced cellular apoptosis and death, which are major conquences caused by ROS under stress conditions (Rezvan, 2017). Sirtuins, the class III deacetylases, are nicotinamide adenine dinucleotides, including seven members in human. The most characteristic and unique member of these is sirtuin1 (Sirt1), which shuttles between the nucleus and cytoplasm(Wu et al., 2020b). Apart from its epigenetic regulation of deacetylation-translation of lysine residues in histones, Sirt1 mediates the activity of several other key cellular proteins associated with cardioprotection, among which was a key regulator of Nuclear factor erythrocyte 2-related factor 2 (Nrf2), the dominant regulator of endogenous antioxidative stress system(D'Onofrio et al., 2020; Takizawa et al., 2013). Nrf2 is a nuclear transcription factor that binds to and activates a cis-acting enhancer sequence called an antioxidant response element (ARE), in the induction of endogenous antioxidant enzymes against oxidation(Xu et al., 2021) To date, the protective role of Nrf2 in the heart has been demonstrated in numerous studies (Guan, Liang & Wang, 2018; Yang et al., 2018a). Nrf2 acts as a transcriptional regulator, and the loss of Nrf2 promotes the rapid progression to heart failure after myocardial infarction(Li et al., 2016). Furthermore, Nrf2 is involved in the protective effect of neural Sirt1 against focal cerebral ischemia in rats(Ding et al., 2016). Interestingly, our previous study has demonstrated the direct interaction between Sirt1 and Nrf2 activation during myocardial ischemia/reperfusion(Xu et al., 2021).

Our current data have confirmed that compound 5h significantly increased Sirt1 expression, as well as elevated the antioxidant enzyme, such as CAT and SOD in serum, leading to markedly declined MDA concentrations in MI rats. Meanwhile, compound 5h pretreatment significantly promoted the cardiac expression of Nrf2, decreased cardiac apoptosis against acute MI injury in vivo. These data demonstrated the beneficial effects of compound 5h may be related to the Sirt1/Nrf2 activation. Using adenovirus for antisense transfection of Sirt1, beneficial effects of compound 5h against H₂O₂-induced injury of H9c2 cells were dramatically abolished by down-regulation of Sirt1, as demonstrated by decreased cell viability, increased LDH leakage rate and ROS level. Most importantly, knock down of Sirt1 significantly down-regulated the activation of Nrf2/NQO1 signaling pathway induced by compound 5h. Furthermore, Ex527, a specific inhibitor of Sirt1, also significantly blocked the cardioprotective effects of compound 5h on acute MI injury in vivo, as evidenced by the augmentation of serum myocardial enzymogram and cardiac apoptotic cells, declination of cardiac function, aggravation of histopathological alterations, eventually leading to the increased cardiac infarct size in MI rats. In parallel with the result in vitro, Ex527 markedly inhibited the up-regulation of Nrf2/NQO1 induced by compound 5h. Therefore, taking into account the results obtained in vivo and in vitro, it was clear that up-regulation of Sirt1 would indeed be a major contributor to cardioprotective action of compound 5h against acute MI.

In conclusion, the inhibitory effect of the novel series of coumarin-derived imino sulfonate compound 5h against acute MI induced oxidative stress and apoptosis was mainly exerted by the enhancement of cardiac Nrf2/NQO1 signaling pathway via Sirt1 activation both in vivo and in vitro. Our findings proved the important for the future design of coumarin-derived structurally related Sirt1 activator to up-regulate the endogenous anti-oxidative stress system.

AST

aspartate transaminase

CAT

catalase

CK-MB

creatine kinase – muscle and brain (subunits)

ECG

electrocardiogram

ejection fraction

fractional shortening

GSH

glutathione

hematoxylin-eosin

H₂O₂

hydrogen peroxide

LAD

left anterior descending coronary artery

LDH

lactate dehydrogenase

LUT

luteolin

LV dp/dt_max

maximum rates of developed left ventricular pressure

LV dp/dt_min

minimum rates of developed left ventricular pressure

LVEDP

left ventricular end-diastolic pressure

LVSP

left ventricular systolic pressure

MDA

malondialdehyde

myocardial infarction

MOI

multiplicity of infection

SOD

superoxide dismutase

Author contribution

Xueli Xu: Investigation, Data curation, Writing - original draft, Writing - review & editing. Yangyang Shi: Resources and software. Qingwen Yu: Methodology. Yajie Peng: Data curation. Fei Zhao: Project administration. Jing Cui and Yajing Chen: Visualization. Lingxu Liu , Yiran Zhang and Ji Zhang: Resources. Bo We: Funding acquisition. Supervision.

Funding

This work was funded by the National Natural Science Foundation of China (Grant No. U1904154), the Young Talent Enterprise Cooperative Innovation Project of Zhengzhou University (Grant No. 32320445), and the Science and Technology Breakthrough Project of Henan province (Grant No. 222102310030, NO. 222102310023).

Data availability

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.

Declaration

Humman ethics Not applicable.

Competing interests The authors declare no competing interests.

Ethics approval Animal experiments were approved by the Animal Ethics Committee of Zhengzhou University (Approval number: AW410975221100019174) and strictly enforce According to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Consent to participate Not applicable.

Consent for publication All authors have read and agreed to the published version of the manuscript.

Confict of interest The authors declare no competing interests.

Supplementary data

Supplementary Data in Supplementary Materials.

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No competing interests reported.

FigureS1.pdf
Experimental protocol to study the effect of coumarin-derived iminosulfonates on MI injury in rats for 5 h. (A) In vivo study (1). (B) In vivo study (2).

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Coumarin-derived imino sulfonate 5h ameliorates cardiac injury induced by myocardial infarction via activating Sirt1/Nrf2 signaling pathway

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Materials

2.2. Cell culture and treatment

2.3. Adenoviral-mediated gene transfer

2.4. Cell viability and LDH determination

2.5. Detection of ROS

2.6. Animals

2.7. Preparation of rat acute MI model

2.8. Electrocardiogram

2.9. Echocardiographic assessment of cardiac function

2.10. Blood sample and tissue processing

3. Results

3.1. Compound 5h inhibited the injury induced by H₂O₂ in H9c2 cells

3.2. Compound 5h protected against acute MI induced by ISO in rat

3.3. Compound 5h ameliorated cardiac function against acute MI induced by ISO in rat

3.5. Compound 5h ameliorated acute MI injury dependent on Sirt1 in vivo

3.6. Compound 5h improved MI-injured cardiac function dependent on Sirt1 in vivo

4. Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Coumarin-derived imino sulfonate 5h ameliorates cardiac injury induced by myocardial infarction via activating Sirt1/Nrf2 signaling pathway

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Materials

2.2. Cell culture and treatment

2.3. Adenoviral-mediated gene transfer

2.4. Cell viability and LDH determination

2.5. Detection of ROS

2.6. Animals

2.7. Preparation of rat acute MI model

2.8. Electrocardiogram

2.9. Echocardiographic assessment of cardiac function

2.10. Blood sample and tissue processing

3. Results

3.1. Compound 5h inhibited the injury induced by H2O2 in H9c2 cells

3.2. Compound 5h protected against acute MI induced by ISO in rat

3.3. Compound 5h ameliorated cardiac function against acute MI induced by ISO in rat

3.5. Compound 5h ameliorated acute MI injury dependent on Sirt1 in vivo

3.6. Compound 5h improved MI-injured cardiac function dependent on Sirt1 in vivo

4. Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

3.1. Compound 5h inhibited the injury induced by H₂O₂ in H9c2 cells