Sulindac attenuates isoproterenol-induced myocardial injury: In vivo and molecular docking evidence

Poonam Sanjay Patil; Janavie Patel; Akshata Pahelkar; Doshi Gaurav Mahesh

doi:10.25259/IJPP_29_2026

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Original Article

ARTICLE IN PRESS

doi:

10.25259/IJPP_29_2026

Sulindac attenuates isoproterenol-induced myocardial injury: In vivo and molecular docking evidence

Poonam Sanjay Patil¹, Janavie Patel¹, Akshata Pahelkar², Doshi Gaurav Mahesh^1,

1Department of Pharmacology, SVKM’s Dr Bhanuben Nanavati College of Pharmacy, Mumbai, Maharashtra, India.

2Department of Pharmaceutical Analysis, SVKM’s Dr Bhanuben Nanavati College of Pharmacy, Mumbai, Maharashtra, India.

*Corresponding author: Doshi Gaurav Mahesh, Department of Pharmacology, SVKM’s Dr Bhanuben Nanavati College of Pharmacy, Mumbai, Maharashtra, India. gaurav.pharmacology@gmail.com

Received: 2026-01-18, Accepted: 2026-07-07, Epub ahead of print: 2026-09-01,

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This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

How to cite this article: Patil PS, Patel J, Pahelkar A, Mahesh DG. Sulindac attenuates isoproterenol-induced myocardial injury: In vivo and molecular docking evidence. Indian J Physiol Pharmacol. doi: 10.25259/IJPP_29_2026

Abstract

Objectives:

Myocardial infarction (MI) arises when blood flow to a particular region of the heart is obstructed, leading to myocardial injury and characteristic electrocardiographic (ECG) abnormalities. Dysregulation of the Wnt/β-catenin signalling pathway has been implicated in post-MI cardiac remodelling. This study aimed to evaluate the cardioprotective potential of sulindac, a β-catenin inhibitor, in an isoproterenol (ISO)-induced rat model of MI.

Materials and Methods:

Myocardial infarction was induced in Wistar rats by subcutaneous administration of ISO. Sulindac was administered orally at doses of 2, 4, and 10 mg/kg. ECG changes, blood pressure, heart rate, oxidative stress markers, cardiac biomarkers, histopathological alterations, and β-catenin levels were evaluated. Molecular docking was performed to investigate the interaction of sulindac with tumour necrosis factor-alpha (7JRA), β-catenin (1A9U), and human interleukin-6 (1ALU) using a validated docking protocol.

Results:

Sulindac treatment significantly reduced heart rate, cardiac biomarkers, oxidative stress, and β-catenin levels while improving blood pressure and antioxidant enzyme activity in a dose-dependent manner. Histopathological examination demonstrated reduced inflammatory cell infiltration and myocardial fibrosis in sulindac-treated animals. Molecular docking revealed favourable interactions of sulindac with TNF-α, β-catenin, and human interleukin-6, supporting its potential multi-target activity.

Conclusion:

Sulindac attenuated ISO-induced myocardial injury and was associated with reduced β-catenin levels and favourable molecular docking interactions with inflammatory mediators and β-catenin. These findings suggest that sulindac may exert cardioprotective effects, potentially through modulation of the Wnt/β-catenin signalling pathway. However, further molecular studies are required to confirm the underlying mechanism and support its therapeutic potential in myocardial infarction.

Keywords

Cardiac biomarkers

Electrocardiogram

Myocardial infarction

Sulindac

β-catenin

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INTRODUCTION

Cardiovascular diseases (CVDs) are considered the leading cause of death.^[1] In CVDs, myocardial infarction (MI) emerges as a major cause of death despite tremendous advancements in cardiovascular care, necessitating further understanding of its aetiology and creative treatment strategies.^[2] MIs are caused by underlying coronary artery disease, where the heart muscle is deprived of oxygen due to coronary artery blockage. Prolonged hypoxia in the heart can lead to myocardial cell death and necrosis.^[3] Prolonged oxygen deprivation from coronary obstruction results in irreversible myocardial injury, often manifested by chest pain radiating to adjacent areas and supported by diagnostic electrocardiogram (ECG) changes and elevated cardiac biomarkers such as troponins.^[4,5]

MI is divided into various categories according to its aetiology, including Type 1: Ischemia caused by a primary coronary event that results in spontaneous MI; Type 2: Ischemia to either a decrease in oxygen supply (such as coronary artery spasm, arrhythmia or hypotension) or an increase in oxygen demand (such as hypertension); Type 3 is linked to sudden unexpected cardiac mortality.^[6] Type 4a is linked to percutaneous coronary intervention; Type 4b is linked to documented stent thrombosis; Type 5 is linked to coronary artery bypass grafting. A severe myocardial localised necrosis may occur from any invisible occlusion of many large epicardial coronary channels that last for 20–40 min. An obstruction that is typically thrombotic is caused by a plaque that bursts within the coronary artery. The myocardium’s blockage of oxygen exhaustion causes myofibril pressure and sarcolemmal collapse.^[7] The main ultrastructural alterations that take place during MI are described above; specific patterns are seen in the mitochondrial alterations that follow damage caused by isoproterenol (ISO). Long-term ischemia eventually causes liquefactive degradation in the heart tissue. Both the subepicardium and the subendocardium are damaged.^[8] It is accepted that since the subepicardium has more collateral circulation, its passage is deferred. Depending on the area of localised necrosis, cardiac tissue is impaired.^[7] Owing to the restricted capacity of the myocardium for regeneration, scar tissue patches the infarcted zone. This regularly occurs during the rebuilding of the heart, which incorporates dilatation, segmental hypertrophy of the surviving tissue and cardiac remodelling.

Risk factors related to MI include advanced age, tobacco use, irregular lipid profile/blood apolipoprotein, diabetes, metabolic disorder and family history of heart disease, which significantly increase MI susceptibility.^[9,10] Parallel to clinical advances, molecular research has underscored the importance of the wingless-related integration site (Wnt) signalling pathway in cardiac remodelling post-MI. It was initially discovered in Drosophila and mouse tumour studies. The wingless genes of Drosophila and integrase-1 in mouse breast cancer are the primary sources of the Wnt gene.^[11] The Wnt signalling pathway includes both non-canonical and canonical pathways. Non-canonical Wnt pathways, such as the Wnt/Ca²⁺ pathway and non-canonical Wnt planar cell polarity,^[12] are free of β-catenin and T-cell factor (TCF)/lymphoid enhancer-binding factor.^[13] The canonical Wnt/β-catenin pathway involves the atomic translocation of β-catenin and enactment of two essential pathways:^[14] The canonical Wnt pathway, which fundamentally controls cell multiplication, and the non-canonical Wnt pathways, which control cell extremity and migration, forms a common regulatory organisation.^[15-18]

Recent evidence highlights β-catenin as a critical regulator of cardiac repair and remodelling following MI. Beyond its classical role as a downstream effector of canonical Wnt signalling, β-catenin independently influences cardiomyocyte survival, inflammatory response and fibrotic remodelling. Dysregulated accumulation of β-catenin has been observed in experimental models of myocardial injury and is associated with adverse cardiac remodelling.^[19] While modulation of upstream Wnt signalling has been shown to affect post-MI outcome in preclinical studies, conflicting reports persist regarding the therapeutic benefit of pathway activation or inhibition. These discrepancies underscore the importance of focusing on β-catenin itself as a central signalling node.^[14] Notably, the translational relevance of Wnt-targeted strategies remains limited due to interspecies variability and experimental heterogeneity, emphasising the need for further studies to clarify the role of β-catenin-associated signalling in post-MI cardiac remodelling.

Sulindac is a non-steroidal anti-inflammatory drug (NSAID) belonging to the arylalkanoic acid class and is widely recognised for its anti-inflammatory, analgesic and chemopreventive properties. As a prodrug, sulindac undergoes reversible metabolic activation in the liver to its sulphide form, which is primarily responsible for its cyclooxygenase (COX) inhibitory activity.^[20] In addition to its classical mechanism of COX inhibition, sulindac has garnered significant attention for its ability to modulate key molecular pathways, notably the Wnt/β-catenin signalling cascade. The Wnt/β-catenin pathway is critical for cell proliferation, differentiation and survival, and its dysregulation has been implicated in cancer, fibrosis and cardiac remodelling after MI. Sulindac results in inhibiting β-catenin signalling by promoting β-catenin degradation and suppressing β-catenin/TCF-mediated transcriptional activity independent of COX inhibition.^[21] This dual action makes sulindac a promising candidate for targeting pathological Wnt modulation by reducing β-catenin levels in various disease states, including cardiovascular conditions, where Wnt signalling is aberrantly activated following myocardial injury.^[22] To better contextualise the cardioprotective effects of sulindac observed in this study, it is important to compare its pharmacological profile with that of other commonly used NSAIDs. While most NSAIDs primarily act through COX inhibition, only a few, including sulindac, significantly reduced β-catenin levels in myocardial tissue.

MATERIALS AND METHODS

Drugs and chemicals

ISO was procured from TCI Chemicals, metoprolol succinate was received from CTX Life Sciences Pvt. Ltd (Gujarat) and sulindac was procured from Dhamtec Pharma and Consultants (Mumbai).

Experimental animals

48 male Wistar rats (6–8 weeks old, weighing 160–200 g) were obtained from the National Institute of Bioscience, Pune, Maharashtra. The animals were maintained in cages under standard laboratory conditions (12:12 h light–dark cycle with lights on at 07:00 am, room temperature 20–22°C). They were provided ad libitum access to food and water. All animals were acclimatised for 1 week before the initiation of the experimental procedures. The study was approved by the Institutional Animal Ethics Committee (IAEC) (Approval No. CPCSEA/IAEC/P-89/2022). This study was conducted and reported in accordance with the ARRIVE 2.0 (Animal Research: Reporting of in vivo Experiments) guidelines.

Preparation of drug solutions

ISO at a dose of 85 mg/kg was dissolved in normal saline and administered subcutaneously (S.C) on the 13^th and 14^th day. Metoprolol succinate (10 mg/kg) was dissolved in 10 mL of distilled water. Sulindac (2 mg/kg, 4 mg/kg and 10 mg/kg) was prepared in 0.5% carboxymethyl cellulose. All solutions were freshly prepared before dosing and used within 1 h.

Methodology

The animals were allocated into six experimental groups (n = 48) before the initiation of treatment. No formal randomisation method was employed for group allocation. The animals were dosed orally for 14 days. For the induction of MI, ISO (85 mg/kg) was administered S.C to the negative control, positive control (metoprolol 10 mg/kg), test group 1, test group 2 and test group 3 (sulindac 2 mg/kg, 4 mg/kg and 10 mg/kg)^[23] on 13^th and 14^th day, except the normal control. On the 14^th day, the animals were anaesthetised with urethane (1.25 g/kg), and the electrodes were placed to record the ECG and heart rate (HR). The left ventricle was cannulated to record blood pressure. After the completion of test drug administration and recording of the required parameters, blood was collected for biochemical parameters, animals were sacrificed and their hearts were removed for further analysis. On the 14^th day, the animals were subjected to evaluation parameters and anaesthetised to collect blood samples for further biochemical evaluation. Hearts were isolated and stored in 10% buffered formalin for histopathological studies.

Molecular docking

Ligand preparation

The 3D structure of sulindac was imported into molecular operating environment (MOE) and prepared before docking. Preparation included protonation at physiological pH (7.4), assignment of partial charges, 3D hydrogen addition and geometry optimisation using the MMFF94x force field. The ligand structure was minimised until energy convergence was achieved to remove unfavourable torsions and optimise spatial orientation for docking placement.

Protein preparation

Molecular docking was performed to investigate the potential interactions of sulindac with key proteins implicated in the pathogenesis of MI. β-catenin (Protein Data Bank [PDB] ID: 1A9U) was selected because it is the principal effector of the canonical Wnt/β-catenin signalling pathway, which plays a critical role in cardiac remodelling and fibrosis following MI. Tumour necrosis factor-alpha (TNF-α; PDB ID: 7JRA) and human interleukin-6 (IL-6; PDB ID: 1ALU) were selected because they are major pro-inflammatory cytokines involved in myocardial inflammation, cardiomyocyte injury and adverse ventricular remodelling. These targets were chosen to evaluate the potential of sulindac to modulate both inflammatory and Wnt/β-catenin-mediated pathways associated with myocardial injury. The crystal structures of TNF-α (7JRA), β-catenin (1A9U) and human IL-6 (1ALU) were processed in MOE before docking. Preparation involved removal of co-crystallised water molecules, heteroatoms and non-participating chains, followed by protonation, 3D hydrogen correction and energy minimisation using the MMFF94x force field until a root mean square gradient of 0.01 kcal/mol/Å was reached. Active-site cavities were predicted using the site finder module in MOE. For docking validation, native co-crystallised ligands were re-docked into their respective binding pockets and superimposed on the experimental pose to compute root mean square deviation (RMSD)_ref, ensuring workflow reliability.

Docking studies

Docking simulations were executed in MOE using the triangle matcher placement algorithm to generate 100 initial poses per complex. The poses were refined through force-field-based scoring and rescored using the Generalized born volume integral/weighted surface area ΔG (GBVI/ WSA dG) function (E_score2). The top 10 energetically feasible conformations were retained for interaction analysis. Final docked complexes were exported in PDB format and visualised using BIOVIA Discovery Studio for 2D interaction mapping, 3D pose representation and electrostatic surface pocket visualisation without altering docking geometry.

Evaluation parameters

Morphometric parameter

The (cardiac hypertrophy index [CHI]) is a quantitative parameter used to assess the extent of heart enlargement (hypertrophy), particularly in experimental animal models. Animals were euthanised and their hearts were isolated after measurement of haemodynamic parameters and perfused with saline. To calculate CHI, the heart weight of each rat was divided by the body weight measured on day 14.

Haemodynamic parameters

Blood pressure

Blood pressure was measured using direct arterial cannulation of the right carotid artery under anaesthesia. Rats were anaesthetised with urethane (1.25 g/kg, intraperitoneally) to ensure minimal cardiovascular interference. Once a surgical plane of anaesthesia was achieved, the neck region was shaved and sterilised with 70% ethanol and povidone-iodine. A midline cervical incision was made to expose the right common carotid artery, which was carefully isolated from surrounding tissues and nerves using blunt dissection. Two silk ligatures were placed, one proximally (toward the heart) and another distally (toward the head). A small incision was made between the ligatures, and a pre-heparinised polyethylene catheter (polyethylene-50 tubing) filled with heparinised saline (50 U/mL) was gently inserted into the artery and secured with the ligatures. The catheter was connected to a pressure transducer linked to a data acquisition system (Power Lab, AD Instruments) to monitor systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial blood pressure. After stabilisation, the values were recorded for analysis. Throughout the procedure, body temperature was maintained using infrared lamps, and sterile conditions were observed to minimise the risk of infection.

ECG

The ST elevation, R amplitude and QTc interval were determined using three lead electrodes inserted S.C in the rats, and the ECG was recorded using a data acquisition system.

HR

HR of rats was also recorded in a similar manner to ECG.

Cardiac biomarkers estimation

Creatinine kinase-myoglobulin binding (CK-MB), creatine kinase-N-acetyl cysteine (CK-NAC) and lactate dehydrogenase (LDH) levels were estimated using ERBA diagnostic kits.

Estimation of anti-oxidative enzymes

Antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD) and malondialdehyde (MDA), were estimated from heart tissue homogenates. CAT activity measurement principle was based on the consumption or decomposition of hydrogen peroxide (H₂O₂) by the sample. 23 µL of 30% H₂O₂ was diluted with phosphate buffer 50 mM to prepare 10 mM H₂O₂.^[24] The reaction was initiated by adding H₂O₂ to the supernatant of the heart tissue homogenate to start the reaction. The absorbance was measured for 3 min at 240 nm using a ultraviolet spectrophotometer. Changes in absorbance were calculated to estimate CAT activity by determining µmol H₂O₂ consumption/min/mg protein.^[25] 2.5 mL of carbonate buffer solution (0.05M, pH 10.5), 0.5 mL of epinephrine solution (3 × 10⁻⁴ mM) and 0.5 mL of ethylenediaminetetraacetic acid solution (0.1 mM) were added to 50 µL of the tissue supernatant.^[26] The change in absorbance was measured at 480 nm for the auto-oxidation of epinephrine to adrenochrome at pH 10.5. One unit of activity of SOD correlates with 50% inhibition of the autooxidation of epinephrine to adrenochrome. Units of SOD activity were expressed as SOD activity/mg of tissue protein.^[27] Thiobarbituric acid (TBA)-reactive substances were expressed as MDA equivalents. MDA forms adducts with TBA in a ratio of 1:2. Next, 2 mL of 10%w/v trichloroacetic acid was added to 100 µL of the tissue supernatant and kept in an ice bath. The supernatant was separated from the mixture by centrifugation.^[28] Then, 2 mL of 0.7% w/v TBA was added to 2 mL of the supernatant and incubated at 95°C for 60 min. The mixture was cooled and centrifuged at 4000 rpm for 10 min. The supernatant was separated, and the absorbance was measured at 530 nm.^[29]

β-catenin levels

Hearts were isolated from animals and washed with phosphate buffer (PBS, pH 7.4) to remove excess blood. Heart tissue was minced and homogenised in PBS (pH 7.4) using a glass homogeniser in an ice bath at a temperature of 2–8°C. The heart tissue homogenate was centrifuged at 3000 rpm for 20 min to obtain a clear supernatant. Aliquots of the supernatant were stored at −80°C for further analysis. To determine the expression of this heart tissue homogenate, a standard procedure was used as provided in enzyme-linked immunosorbent assay (ELISA) kits (Krishgen Biosystem). The optical density of each well was measured at 450 nm wavelength.

Histopathological examination

The isolated hearts were immediately stored and fixed in a 10% formalin-buffered solution at room temperature. Heart sections (5 µm) were cut and embedded in paraffin. These sections were then stained using haematoxylin and eosin (H&E) and Masson’s trichrome stain.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 8.0.2 (263), 64-bit Windows software. One-way analysis of variance followed by Tukey’s post hoc multiple comparison test was used to evaluate the data. A p < 0.05 was considered statistically significant.

RESULTS

CHI

ISO-induced cardiac hypertrophy in the negative group showed a significant increase in CHI compared with the normal group (^###p < 0.001). Metoprolol-treated rats showed a significant decrease in CHI compared to the negative group (**p < 0.01). In addition, animals treated with 2 mg, 4 mg and 10 mg sulindac showed a significant decrease in CHI as compared to the negative group (*p < 0.05) (**p < 0.01) [Figure 1a-d].

Effect of sulindac on cardiac hypertrophy index (a and b) SBP; (c) heart rate and (d) DBP. T1, T2 and T3 – Dose levels of sulindac (2 mg/kg, 4 mg/kg and 10 mg/kg, respectively. The values are expressed as mean ± standard deviation. Data was analysed by one-way analysis of variance followed by post hoc Tukey’s multiple comparison test. ##p < 0.01. ###p < 0.001 versus Normal control *p < 0.05; **p < 0.01, versus negative control. SBP: Systolic blood pressure, DBP: Diastolic blood pressure.

Blood pressure and HR

Negative group showed a significant decrease in SBP [Figure 1b] and DBP [Figure 1d], and a significant increase in HR [Figure 1c] compared to the normal group (^##p < 0.01) (^###p = 0.001). Animals pre-treated with metoprolol (10 mg/kg) showed a marked increase in DBP (p < 0.05) and a decrease in HR (p < 0.01); while no significant improvement was observed in SBP compared to the negative group. Sulindac (2 mg/kg) showed a significant improvement in HR (*p < 0.05), while no significant difference was observed in SBP and DBP compared to the negative control group. Sulindac (4 mg/kg) significantly improved HR, SBP and DBP (*p < 0.05) compared to the negative control group. Sulindac (10 mg/kg) significantly improved HR (*p < 0.05), SBP (*p < 0.05) and DBP (**p < 0.01) compared to the negative group.

ECG pattern

The normal group showed a normal ECG pattern, whereas the negative group showed a marked decrease in R-amplitude and ST-segment elevation, indicating MI. The positive group showed an improved ECG pattern compared to the negative group. The pre-treated groups (2 mg/kg, 4 mg/kg and 10 mg/ kg) represented significantly decreased ISO-induced ST-elevation and a remarkable increase in R-amplitude [Figure 2].

Sulindac treatment improves the electrocardiogram pattern.