Exploring Cardioprotective Potential of Esculetin Against Isoproterenol Induced Myocardial Infarction in Rats: In Vivo and in Vitro Evidence

All are as mean ± SEM and n=6. Data analysis done by one way analysis of variance followed by the Tukey multiple comparison tests. # p<0.05, indicate disease control compared with normal. ** p<0.05, indicate Esculetin compared with disease control group.


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Cardiovascular diseases (CVDs) comprise disorders of the heart and blood vessels and still represent a major cause of death globally. CVDs have shown to be responsible for approximately 17.9 million deaths each year, which accounts for 31% of all deaths worldwide. Among the various CVDs, myocardial infarction (MI) is a major cause of mortality and morbidity across the world. Any blockade in the coronary artery leads to insu cient blood supply to heart, causing the heart muscle that is being supplied by the artery to get infarcted ultimately resulting in ischemic tissue necrosis in addition to other pathological and structural changes. The pathogenesis of MI includes hyperlipidemia, oxidative stress, peroxidation of membrane lipids, and loss of plasma membrane integrity [1].
Isoproterenol (ISO), a synthetic catecholamine and β-adrenergic agonist is well-known to cause severe stress in the myocardium by generating free radicals which in turn stimulates lipid peroxidation and perhaps the major contributing factor for the irreversible damage to the myocardial membrane [2]. ISO upon administration causes increase in heart rate leading to increased oxygen demand, high calcium burden and accumulation besides causing alterations in the morphology and membrane integrity of the myocardium with elevated cAMP levels in the myocardial cells [3].
Induction of myocardial infarction was previously performed by surgical procedures, but it has incidence of morbidity, mortality and animals were prone to pneumothorax infections [4]. Isoproterenol-induced myocardial infarction is simple and non-invasive model, considered as one of the most widely used experimental model to study the bene cial effects of many drugs and cardiac function [2] and it is similar to those pathophysiological changes observed in human myocardial infarction [5].
Natural products have high universal demands due to their claimed advantage in terms of both safety and e cacy against various diseases like MI. Plant based coumarins are low-molecular weight phenolic compounds that has been used for the prevention and treatment of various thromboembolism MI and stroke [6]. Esculetin (6, 7-di hydroxyl coumarin) is a natural coumarin derivative isolated from many plants such as Artemisia capillaries, Citrus limonia, Solanum surrettence and Euphorbia lathyris [7] with multiple pharmacological & biochemical properties [8]. There are numerous treatment approaches that have been developed to attenuate the risk of myocardial infarction, but most of them fail when translated from the bench to the bedside. Therefore, there is a need to nd new drugs which hostile MI as well as be used as suitable therapeutic candidate and also can be translated to clinical use in the future [9].
The present study was intended to investigate whether esculetin can provide protection against free radical generation by ISO and its associated alterations in the membrane bound enzymes and myocardial lysosomal enzyme activities in experimental rats. Further, we also tried to explore the probable mechanism of action of the esculetin for its cardio protection in H9C2 cell lines.

Experimental Procedures
Chemicals Isoproterenol (CAS Number 5984-95-2), and Esculetin (CAS Number 305-01-1) was purchased from Sigma Aldrich. Co, St. Louis, USA. All the chemicals used in the present study were of analytical grade and indigenous.
In vitro studies 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity The antioxidant activity of esculetin was measured using DPPH radical scavenger assay in triplicate. Brie y, the stock solution (1mg/mL) of esculetin was prepared in methanol. Concentration of 5, 25, 50 and 100µg/mL were used along with ascorbic acid as reference standard. The test were then incubated with 50µL of 0.1mM DPPH solution and made up to the nal volume to 3mL with methanol. A blank was prepared using DPPH solution and methanol. The reaction mixture was incubated for 30 min at room temperature in the dark followed by measuring absorbance at 517nm [10].
H9C2 cell culture H9C2 myoblast cells from rat's myocardium were acquired from National Centre for Cell Sciences, Pune, India. The myoblast cells were cultured in Dulbecco's modi ed Eagle's Medium (DMEM) medium supplemented with 10% FBS and 10 ml/l100 × antibiotic-antimycotic solution containing 10,000 units of penicillin and 10 mg/ml streptomycin in 0.9% normal saline in a humidi ed atmosphere of 95% air and 5% CO 2 at 37•C.

Measurement of cell viability by MTT assay
The cells were seeded in 96-well culture plates at a density of 7×104 cells/well. When the cells reached 80% con uence, they were treated with 1‰ dimethyl sulfoxide (DMSO) solution, and 5, 25 and 50 μg/mL of esculetin at 37 o C for 24 h. The cells were then incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) (0.5 mg/mL) solution for 4h, and the resulting formazan was solubilized with 150 μL of DMSO for 30 min. The absorbance of each well was measured at 570 nm, and the absorbance of control cells was considered to indicate 100% cell viability [10,11].

Intracellular Reactive Oxygen Species (ROS) measurement
The generation of intracellular reactive oxygen species (ROS) was measured by using the ROS-sensitive uorescence indicator called Dichloro uorescin diacetate (DCFH-DA) as per our previous protocol. H9C2 cells grown in 96 well plates to ~75% con uence were treated in triplicate. The cells were treated with esculetin at 0.1, 1, 5 and 10µM. After 12h, 10µM arsenic was added to the plate. The cells were incubated for an additional 30 min at 37°C and after PBS wash cells were incubated with 20µM DCFH-DA 30min at 37°C in the dark. After, Cells were washed, and analyzed by ow cytometer. The orescence intensity was calculated using the FAC Suite software [10].
RNA isolation, cDNA synthesis and qPCR to assess mRNA expression of TNF-α, IL-6, NF-κb After arsenic and esculetin treatment, the total RNA was isolated from H9C2 cells by using Trizol reagent (Thermo Fisher Scienti , Inc.). The isolated RNA was quanti ed by using a nano-drop spectrophotometer and complementary DNA (cDNA) was synthesized from 1µg of RNA was used for reverse transcription reaction using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed by using the SYBR green reagent according to the manufacturer's protocol (MilliporeSigma).
In vivo study Animals Male wistar rats weighed between 230-280 g were used in this study. Rats were housed under standard conditions and fed with standard pellet with drinking water ad libitum. The animals were kept in polypropylene cages and maintained at a room temperature of 25±2 0 C with 55±5% relative humidity and

Induction of myocardial infarction
Myocardial infarction was induced by dissolving isoproterenol (100 mg/kg) in normal saline and injected subcutaneously to rats for last two consecutive days of the experimental schedule [14].

Experimental schedule
The treatment schedule was xed for 28 days and the rats were divided into ve groups of six each. Rats of group I received the normal saline and served as normal control, group II was received ISO (100mg/kg body weight) for last two consecutive days of the study and served as disease control. Groups III and IV received esculetin 10 and 20 mg/kg body weight respectively, once a day orally for 28 days of the study along with ISO for last two consecutive days of the study and serve as test groups.

Blood sample collection and analysis
At the end of treatment blood was collected from retro orbital plexus by anesthetizing the rats with thiopental sodium (35 mg/kg body weight, intra peritoneal) [15] and serum was separated by centrifugation at 2000 rpm. Serum was used to analyze various biochemical parameters such as determinations of cardiac biomarkers lactate dehydrogenize (LDH), and creatinine kinase MB (CK-MB) by using commercial diagnostic kits (Agappe Pvt. Ltd, Kerala, India).

Na + /K + ATPase activity of myocardial membrane
The myocardial membrane Na + /K + ATPases activity was determined according to procedure done by Periyathambi and Ponnian 2007. The incubation mixture contained 10mM of Tris buffer, 20mM of potassium chloride, 125mM of sodium chloride, 1mM of EDTA and 3mM of ATP. To the incubation mixture, the reaction was initiated by the addition of 0.2mL of tissue homogenate and the contents were incubated at 37°C for 15 min. To stop the reaction of 10% trichloro acetic acid (TCA) was added. The tubes were centrifuged and supernatant was used for the estimation of liberated P i . 1.0mL of supernatant was made up to 4.3mL with distilled water and added 1.0ml 3mM of ammonium molybdate reagent. The tubes were incubated at room temperature for 10 minutes, and later 0.4ml of amino naptholsulphonic acid reagent was added to develop the color and the P i released recorded using a standard P i graph [16].

Preparation of lysosomal sub cellular fractions
Lysosomal subcellular fractions were isolated according to the method of Venkatachalem et al.,2003. The heart tissue sample was cut open and placed in isotonic saline to remove the blood. Then the heart tissue was rinsed in ice cold 0.25 M sucrose, blotted, weighed and minced. The enzyme extracts were prepared by homogenizing the tissue samples in 0.25 M sucrose at 4°C. The portion of the homogenate was subjected to differential centrifugation, and the different fractions were separated as follows: structural proteins, nucleus, and cell debris at 600×g for 10 min; mitochondria at 5000×g for 10 min; lysosomes at 15,000×g for 10 min. Myocardial sub-fractions were treated with Triton X-100 ( nal concentration 0.2% v/v) in ice for 15 min prior to the determination of enzymatic activity [17] .

Determination of tissue antioxidants
At the end of the experimentation hearts were excised from rats and homogenate in 0.1M Tris buffer (pH 7.4) and the separated homogenates were used for estimation of heart antioxidants like super oxide dismutase (SOD) [21], Reduced glutathione (GSH) [22], Catalase [23] and lipid peroxidation (LPO) [24].
Histopathological studies of heart After removal of myocardial tissue immediately washed with ice cold saline to remove all the blood and xed in 10% buffered neutral formalin solution. After xation was complete, tissues were embedded in para n and serial sections were cut in to 0.5µm. Each section was stained with hematoxylin and eosin. The sections were examined under light microscope and histograms were taken.

Statistical analysis
Results were expressed as mean± standard error mean multiple comparisons of the signi cant analysis of variance (ANOVA) followed by the Dennett's test as post parametric test using computer based tting program (Prism graph pad 5.0). A p value of <0.05 was considered as statistically signi cant.

Results
DPPH radical scavenging activity of esculetin DPPH assay is the simple and sensitive choice to assess the free radical scavenging activity of any natural origin compounds. DPPH accepts an electron or hydrogen radical to become a stable diamagnetic molecule. Esculetin exhibited signi cant radical scavenging activity at all the working concentrations (Fig 1) nevertheless, the maximum scavenging activity was at 50µM compared to control.

Esculetin Inhibits Intracellular ROS production in H9c2 cell
In the study, we investigated the inhibitory effect of esculetin on arsenic induced ROS generation in H9c2 cells by using DCFH-DA a ROS sensitive non-uorescent agent (Fig 1). The DCF stained cells were analyzed by ow cytometer. During incubation period a signi cant ROS generation is noticed in cells placed with arsenic alone. Whereas cells treated with esculetin at 25 and 50µg/mL concentrations exhibited dose-dependent inhibitory effects with IC 50 value of 125M.
Effect of esculetin on H9c2 cell viability MTT assay was used to determine the effect of esculetin on H9C2 cell viability at 5, 25 and 50μg/mL concentration. After the effective treatment with esculetin H9C2 cells observed as viable with no signi cant cytotoxicity at all the concentrations.
Esculetin attenuates mRNA expression of TNF-α, IL-6, and NF-κB against arsenic induced ROS in H9C2 cells The mRNA expression of pro-in ammatory cytokines TNF-α, IL-6 and NF-κB were measured using RT-qPCR method against arsenic induced ROS generation in H9C2 cells (Fig 2). The mRNA expression TNFα, IL-6 and NF-κB were signi cantly (p≤ 0.05) elevated, approximately 9 folds compared to control cells. While the cells treated with esculetin at 25 and 50μg/mL concentration in the presence of arsenic attenuated the expression of mRNA at 4 to 6 folds (p≤ 0.05) lesser than the control. No differences were observed in the levels of these pro-in ammatory cytokines between the control and esculetin alone groups. The above results indicated that pre-treatment with esculetin could reduce the production and expression of pro-in ammatory cytokines in TNF-α, IL-6 and NF-κB in arsenic induced ROS in H9C2 cells.

Effect of esculetin on cardiac biomarkers in ISO induced myocardial toxicity
The alterations in serum heart biomarkers like CK-MB and LDH were tabulated in Table 1. The concentration of CK-MB and LDH were signi cantly (p < 0.05) increased in isoproterenol alone treated rats compared to normal control rats. Animals treated with esculetin brought these cardiac markers near normalcy (p < 0.05) compared to isoproterenol alone treated rats. Table 1 Esculetin effect on CK-MB, LDH and myocardial Na + /K + ATPase activity in ISO induced myocardial toxicity.

Effect of esculetin on membrane bound Na + /K + ATPase activity in ISO induced myocardial toxicity
The effect of Esculetin on membrane bound Na + /K + ATPase activity has shown in Table 1, signi cant (p<0.05) decrease of myocardial membrane bound Na + /K + ATPase in rats treated with control when compared with normal rats. Whereas, the same Na + /K + ATPase signi cantly (p < 0.05) increased in rats pretreated with esculetin for four weeks when compared with control rats.
Effect of Esculetin on lysosomal membrane destabilization in ISO induced myocardial toxicity Table 2, shows that alterations of activities of lysosomal hydrolases enzymes like β-glucuronidase, βgalactosidase, β-glucosidase, and acid phosphatase. Activity of these enzymes were signi cantly (p<0.05) increased in heart tissue homogenates of rats treated with isoproterenol alone when compared with normal rats. Animals pre-treated with esculetin at doses of 10 and 20 mg/kg body weight for 28 days brought these enzyme activities signi cantly (p<0.05) low and near to normal when compared with isoproterenol alone treated rats. Table 2 Esculetin effect on myocardial lysosomal enzyme's activity in ISO induced myocardial toxicity.

Effect of esculetin on tissue antioxidants in ISO induced myocardial toxicity
The changes in heart antioxidants are presented in Table 3. In ISO induced rats, there was a signi cant (p<0.05) decrease in GSH, Catalase, SOD and a signi cant increase in LPO compared to normal control group and pre-treatment with esculetin at doses of 10 and 20mg/kg, respectively, brought the elevated heart antioxidants near normal. Table 3 Esculetin effect on myocardial tissue antioxidant enzyme's activity in ISO induced myocardial toxicity. Histopathological observations (Fig 3) of normal control rat's heart revealed normal cardiac muscle bundles without any in ammation, whereas myocardium of rats treated with isoproterenol alone has shown a marked in ammatory signs like membrane damage and cellular in ltration along with focal myonecrosis. Rats pre-treated with esculetin at doses of 10 and 20mg/kg body weight has shown reduction in in ammatory signs and myonecrosis compared to disease control.

Discussion
The present study validates to reduce the acuteness of isoproterenol induced myocardial infarction by stabilizing the myocardial membrane integrity. The results of experiment bring a new outcome that, pretreatment with esculetin in experimental rats protects against myocardial infarction. Reactive oxygen species generation by oxidative stress plays an important role in the development of myocardial infarction both experimental and clinically [25].
Antioxidant compounds, especially polyphenol compounds from plants, are capable of counteracting the harmful effects caused by ROS and therefore it can prevent chronic diseases related to oxidative stress [26]. In the present study reveals that, esculetin has shown that direct radical quenching effect in DPPH radical scavenging assay. In addition, it also reduced the intracellular ROS production induced by arsenic [27] in H9C2 cell lines. Treatment with esculetin in H9C2 cells, a noticeable reduction of DCFDA uorescence intensity was observed this indicates that, esculetin may interact with cellular anti-oxide enzymes. Thus, esculetin has the ability to alleviate free radicals ions both in vitro and in vivo. This was further con rms by tissue antioxidant data of present study.
Anti-oxidant enzymes such as catalase, superoxide dismutase (SOD), glutathione peroxidase and glutathione-S transferase (GSH) are the rst line of cellular defense against oxidative injury by superoxide anion radical and H 2 O 2 before interacting to form the more reactive hydroxyl radical [28]. Auto-oxidation of ISO produces highly cytotoxic free radicals like quinines which in addition with superoxide anion and potent hydroxyl radicals damages the polyunsaturated fatty acids of myocardial membrane [29,30]. The elevation of these free radicals brings an imbalance between tissue bound cellular scavenging enzymes like SOD, GSH and Catalase and lipid peroxidase [31].
The elevation of free radical including superoxide, hydroxyl ions and hydrogen peroxide ions was the reason for decreasing of tissue bound antioxidant enzymes [32,33]. It is in line with previous studies, that tissue anti-oxidant enzymes were signi cantly decreased where the LPO levels were increased in the hearts of ISO alone treated rats [34,35]. Pre-treatment with esculetin brought the elevated levels of tissue anti-oxidant enzymes like SOD, GSH and Catalase near base line. Our results are agreement with several previous ndings in which esculetin is a potential naturally occurring antioxidant both in vitro and in vivo [36,37].
Probably, the anti-oxidant effect of esculetin could by hydrogen donating ability of esculetin [38], the catechol structure of esculetin contributes the stronger antioxidant activity [36]. The available data of the present study con rms with existing literature that, esculetin has the ability to activate anti-oxidant enzymes or and might combat the excess ROS formed with in the cell [39,40].
Arsenic one of the major component to induce intra-cellular ROS [27]. Cytokines such as IL-1β, TNF-α and IL-6 are pro-in ammatory markers play a vital role in various in ammatory pathways like JNK, NF-κB and NLRP [41]. NF-κB is a multiple transcription factor that regulates transcription of various genes involved in the pathogenesis of myocardial infarction like TNF-α and IL-6 [42,43]. These pro-in ammatory cytokines and in ammatory signaling pathways could promote the development of myocardial infarction. Intracellular ROS promotes the production and release of these pro-in ammatory cytokines from cardiomyocytes. TNF-α, IL-1b, and IL-6, are early predictors of organ dysfunction and, causes to activate cardiomyocytes apoptosis [44]. In our study we investigated for the change in the mRNA expression levels of ROS sensitive transcription factor NF-κB along with its pro-in ammatory cytokines TNF-α and IL-6 in esculetin treated H9C2 cells and noticed a dose dependent decline in the mRNA expressions of above said markers. This denotes that esculetin could effectively inhibited the expression of mRNAs of pro-in ammatory cytokines, which are the prominent markers of in ammation and myocardial infarction, this effect may prevent the myocardium to undergo apoptosis or cell necrosis. The present ndings are in line with earlier research saying esculetin could effectively down regulated the TNF-α, NF-κB and IL-6 levels upon various stimulus including ROS [13,45,46].
CK-MB and LDH are indicator biomarker to diagnose the severity of myocardial infarction and number of necrotic cells [47]. These enzymes are present in cardiac muscle, upon the injury release into the blood stream [48]. The available data [49,50], revels that isoproterenol administration signi cantly elevates the CK-MB and LDH levels in the serum; in our study also the same. In the in vitro model [28], esculetin improved the cell viability and reduces the release of LDH, in the same line, pre-treatment with esculetin reduce the ISO induced raise of CK-MB and LDH to protect the myocardium, and it also maintained the cell viability in H9C2 cell lines. This implying that esculetin has the potentiality to protect pathological and morphological changes in the rats in a dose-independent manner.
The functioning ability of the membrane bound enzymes are depends on the stability of plasma membrane. Under normal cell physiology Na + -K + ATPase will balances the Na + and K + ions across the membrane, upon its disruption may leads to ionic inequality and cell death. Therefore, the determination of membrane bound enzyme activities will indicate any alteration to the membrane physiology under pathological conditions [51]. ISO liberated free radicals bind to the membrane lipids to cause injure, thereby inhibits the membrane associated enzymes function including ATPases [2]. The loss of ATPase activity in the ischemic state may be responsible for causing functional damage and reversible necrotic changes in the involved myocardial cell [52].
Palanivel Karthika et al., has found that, pre-treatment with esculetin has effectively increased the Na + -K + ATPase activity, this could be due to the ability of esculetin to protect SH group of Na + -K + ATPase enzyme from oxidative damage through inhibition of peroxidation of membrane lipids [53]. It is in line that, the same circumstances were observed in the present study with pre-treatment with esculetin appreciably restored the membrane bound Na + -K + ATPase near to normalcy.
Lysosomes are surrounded by phospholipid enriched membranes, and are a potential site for free-radical attack including ISO produces quinines [54]. Therefore, radical ions causing to loss the membrane stability and leads the release of hydrolytic enzymes from its sacs to augments cell necrosis [55], [56].
Hence, signi cant attention has been raised on the involvement of lysosomal enzymes in myocardial damage. From our previous report it was observed that, administration of the ISO to the rats causes a signi cant elevation in the activities of the lysosomal enzymes [30]. In the present study same effect observed.
Stabilization of myocardial cell membranes, mainly the lysosomal membranes, may extend the viability of myocardial cells to prevent MI. Pre-treatment with esculetin could effectively inhibit the release of lysosomal enzymes from their sacs, and protects the myocardium from necrosis. This provides the rst in vivo evidence that esculetin protects myocardial from ISO-induced injury by lysosomal membrane stabilization.
Biochemical ndings that proves the cardio protective effect was further supported by histopathological examination. Myocardial tissue sections of normal rats depicted clear integrity of the myocardial cell membrane whereas the sections of hearts treated with ISO showed necrosis of muscle bers with in ammatory cell in ltration, edema and fragmentation of muscle bers, which indicated involvement of oxidative stress and in ammatory processes. Pre-treatment with esculetin showed the integrity to the structure of myocardium from ISO injury near to the normal myocardium denotes its cardio protective action. The limitation of the study was a clear mechanism of action by which pathway esculetin interfering to reducing the myocardial necrosis has not done.

Conclusion
Based on the results of this study, it can be concluded that Esculetin ameliorates isoproterenol associated pathological features like attenuating the oxidative stress, stabilization of lysosomal, myocardial membrane and modulation of pro-in ammatory markers TNF-α, NF-κB and IL-6 cytokines to ascertain its cardioprotective potentials. Understanding the molecular mechanisms involved in the lysosomal enzyme leakage may prove bene cial measures in the prevention of myocardial infarction. Thus, esculetin may consider as a better therapeutic option for treatment of myocardial infarction. Further work need to be done in order to establish speci c molecular mechanisms by which esculetin prevent myocardial infarction and possibility for its clinical application. Author's contribution CPP has designed and prepared the manuscript, VKN performed all in vitro and cell line work and in the preparation of manuscript. SR performed animal activities and biochemical assays, NKGV analyzed the data and results interpretation and TK edited the complete manuscript and made ready for communication.

Funding
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Availability of the data and materials
Corresponding author will provide the data used in present work upon the request.

Ethics approval and Declaration
All methods including animal experimental procedures were adopted in the study were performed in accordance with ARRIVE guidelines. Animal experimental protocols used in the present study were and procedures adopted in the study are in agreement with appropriate guidelines and regulations.

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