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Abstract

The present study demonstrated the protective effects of arbutin (ARB) on hyperlipidemia, mitochondrial, and lysosomal membrane damage and on the DNA damage in rats with isoproterenol (ISO)-induced myocardial infarction (MI). Rats were pretreated with ARB (25 and 50 mg/kg body weight (bw)) for 21 days. After pretreatment with ARB, MI was induced by subcutaneous injection of ISO (60 mg/kg bw) for two consecutive days at an interval of 24 h. The levels of TC, TG, and FFA were increased and decreased the level of PL in the heart tissue of ISO-induced MI rats. Very-low-density lipoprotein cholesterol and low-density lipoprotein cholesterol were increased while high-density lipoprotein cholesterol was decreased in the plasma of ISO-administered rats. A heart mitochondrial fraction of the ISO rats showed a significant decrease in the activities of mitochondrial enzymes isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase. The activities of lysosomal enzymes (β-glucosidase, β-glucuronidase, α-galactosidase, β-galactosidase, cathepsin-B, and cathepsin-D) were increased significantly in the heart tissue homogenate of disease control rats. In ISO-induced MI, rat’s significant increase in the percentage of tail DNA and tail length, and a decrease in the level of head DNA were also observed. ARB administration to MI rats brought all these parameters to near normality, showing the protective effect of ARB against MI in rats. The results of this study demonstrated that the 50 mg/kg bw of ARB shows higher protection than 25 mg/kg bw against ISO-induced damage.

Keywords

Arbutin isoproterenol myocardial infarction mitochondrial enzymes lysosomal enzymes

Introduction

Myocardial infarction (MI) is one of the main causes of morbidity and mortality in the developed and most of the developing countries. Although public awareness is raised, clinical care is improved, and health innovations are widely used, MI remains the most important cause of death worldwide.¹ MI is an acute condition of necrosis of myocardium that occurs due to the imbalance between coronary blood supplies to any part of the heart, resulting in the death of cardiac tissue (myocardial necrosis). Consequences of MI include peroxidation of membrane lipids, loss of plasma membrane integrity, and hyperlipidemia.²

Isoproterenol [1-(3,4-dihydroxyphenyl 2-isopropylamino ethanol) hydrochloride] (ISO)-induced MI serves as a well-standardized model to study the cardioprotective effects of many drugs.³ ISO is a synthetic catecholamine and β-adrenergic agonist that has been found to cause severe stress in the myocardium leading to an infarction in the heart muscle.⁴ ISO induces damage in cardiac myocytes through the hypoxia, calcium overload, coronary hypotension, excessive production of free radicals, and depletion of energy due to its oxidative metabolism.⁵ Catecholamine oxidation forms quinonoid compounds giving rise to the production of hydrogen peroxide and superoxide anions, which damage the lipids, proteins, and DNA in the cell.⁶ ISO also increases the levels of myocardial and circulatory lipids, representing its hyperlipidemic effect. Elevated levels of blood cholesterol and its accumulation in heart tissue are well associated with cardiovascular damage.⁷

The blood cholesterol level can generally be regulated by the cholesterol biosynthesis, the absorption of dietary cholesterol, and the excretion of cholesterol via fecal sterol or bile acid. Alterations in the lipid metabolism directly reflect the composition of lipoproteins in ISO-induced MI rats. Recent studies have revealed that lipid-associated disorders are not only attributed to the total serum cholesterol but also its distribution among different lipoproteins.⁸ Lipids and lipoproteins play a major role in the pathogenesis of ISO-induced MI. The low-density lipoprotein cholesterol (LDL-C) are the major carriers of cholesterol to the tissues having atherogenic potential, while the high-density lipoprotein cholesterol (HDL-C) transfers the cholesterol from peripheral tissues to the liver, thus giving protection against various cardiac problems. Administration of ISO into the rats increases LDL-C level and decreases HDL in the blood, which causes the build-up of harmful deposits in the arteries and thus favors MI.⁹

Mitochondria are the important subcellular organelles for cellular oxidative processes and also the most important source of reactive oxygen species (ROS) in the cell. Normal cardiac functions depend on sufficient supply of oxygen and oxidizable substrate to produce adequate ATP to meet the energy demand of the organ. This process is achieved through several metabolic pathways, including oxidative phosphorylation and tricarboxylic acid (TCA) cycle, which directly involved in the production of ATP.¹⁰ The diminished oxygen supply during MI impairs energy production through the oxidative phosphorylation.¹¹ Mitochondria are the major source of cellular energy, and a number of substances and conditions can affect the mitochondrial functions. A number of studies have reported that the increased oxidative stress causes mitochondrial damage resulting in the modification in mitochondrial enzyme activity.¹² ISO administration also causes the alterations in the permeability of the membrane, which brings about a loss of integrity and function of myocardial membranes.¹³ This leads to the disruption of the mitochondria with the inactivation of the enzymes concerned with the energy metabolism of myocardium, which is a prelude to MI.¹⁴

Lysosomes are cytoplasmic organelles present in animal tissues, which contain hydrolytic enzymes capable of degrading the cellular constituents. The lysosomal membrane is a possible site for oxidative-free radical attack, and its membrane leakage can play an important role in the initiation of apoptosis.¹⁵ Cathepsin-D is a lysosomal aspartic protease, plays a significant role in the inflammation, and its release is commonly used as an indicator for loss of lysosomal membrane integrity.¹⁶ Increased activities of lysosomal hydrolases are responsible for tissue damage and infarcted myocardium in ISO-induced rats.¹⁷ Alterations in the lysosomal enzyme activity have been observed in patients with MI.¹⁸ The leakage of the enzymes from the enclosed sac due to the membrane destabilization induced by ISO leads to autolysis of myocardial cells that causes MI. The leakage of the lysosomal enzymes may be prevented by inhibiting the membrane peroxidation.¹⁹

The alkaline single-cell gel electrophoresis (comet assay) has been extensively used to determine the DNA damage, including alkali-labile sites, DNA cross-linking, strand breaks, and incomplete excision repair sites in mammalian cells.²⁰ In cardiomyocytes, the β-adrenoceptor over stimulation can activate NAD(P)H oxidase to produce ROS and to induce endoplasmic reticulum stress, and increased oxidative stress can also lead to DNA damage. DNA fragments migrate farther in response to an electric field, as a result, the nucleotides look like a “comet” with a bright fluorescent head and tail region.²¹ This is a rapid, sensitive, simple, and reliable biochemical technique for evaluating DNA damage caused by free radicals. Free-radical-mediated oxidative stress is an important cause of DNA damage and it has been recognized as a major cause of cell death in all aerobic organisms.

Since ancient times, medicinal plants are believed to exhibit antioxidant activity and established to play a major role in the management of different diseases in humans, including cardiovascular diseases (CVDs).²² Arbutin (ARB) is a glycosylated hydroquinone, found in various plant species, such as Bergenia crassifolia (Saxifragaceae), leaves of bearberry (Ericaceae), and pear trees (Rosaceae).²³ ARB has been used in numerous skin whitening and depigmenting cosmetics and is effective for the treatment of urinary tract infections, prevents asthma, and relieves cough.²⁴ In vitro and in vivo experiments of the previous studies have confirmed that ARB is effective against inflammation, urinary stones, and high blood pressure.^25,26 Additionally, ARB also has antioxidant, antihyperlipidemic, antihyperglycemic, and bactericidal effects.^27

–30 These numerous advantages have rapidly raised the demand for ARB in recent years. Till date, no study has been carried out regarding the effect of ARB on mitochondrial and lysosomal alterations, lipid profile, and DNA damage induced by ISO. So, an attempt was made to evaluate the cardioprotective activity of ARB in ISO-induced MI. The structure of ISO and ARB is shown in Figure 1.

Figure 1.

Chemical structure of (a) isoproterenol [l-β-(3, 4-dihydroxyphenyl)-α-isopropyl amino ethanol hydrochloride] and (b) arbutin [4-hydroxyphenyl-β-d-glucopyranoside].

Materials and methods

Chemicals

ISO hydrochloride and ARB were purchased from Sigma-Aldrich (St Louis, Missouri, USA). The kit used for the assay of cardiac lipid profile was obtained from Agappe Diagnostics (Kochi, Kerala, India), Qualigens Diagnostics (Mumbai, Maharashtra, India), and Roche Diagnostics (Risch, Switzerland). All other chemicals used in this study were of analytical grade obtained from E. Merck and HiMedia (Mumbai, Maharashtra, India).

Experimental animals

The study was carried out with male albino Wistar rats weighing 160–180 g obtained from Biogen (Bangalore, Karnataka, India). Rats were maintained as per the principles and guidelines of the ethical committee for animal care, Annamalai University, in accordance with the Indian National Law on Animal Care (160/PO/ReBi/S/1999/CPCSEA, dated November 25, 1999, Pro. No. 1127). The animals were housed in plastic cages with paddy husk for bedding at a temperature of 27 ± 2°C with 12-h light:12-h dark cycles. The experiments were conducted in accordance with the “Guide for the Care and Use of Laboratory Rats.”

Experimental induction of MI

MI was induced by subcutaneous (s.c.) injection (thigh muscle) of ISO (60 mg/kg body weight (bw) dissolved in physiological saline) for the two consecutive days.³¹

Experimental design

A total number of 30 rats were used for this study, and after acclimatization, the animals were randomly divided into the five groups consisting of six rats each:

Group 1: Animals received standard laboratory diet and drinking water ad libitum for 23 days and served as a control.

Group 2: Animals received ARB (50 mg/kg bw, orally) treatment for the first 21 days.

Group 3: Animals received ISO (60 mg/kg bw, s.c.) at an interval of 24 h on 22nd day and 23rd day.

Group 4: Animals received ARB (25 mg/kg bw, orally) for the first 21 days and ISO (60 mg/kg bw, s.c.) on 22nd day and 23rd day.

Group 5: Animals received ARB (50 mg/kg bw, orally) for the first 21 days and ISO (60 mg/kg bw, s.c.) on 22nd day and 23rd day.

The total experimental duration was 23 days. After 24 h treatment, the animals were anesthetized between 8:00 am and 9:00 am using ketamine (24 mg/kg bw, intramuscular injection) and euthanized by cervical dislocation. The blood was collected in a heparinized centrifuge tube, centrifuged at 2000 r/min for 10 min, and the plasma was separated. The separated plasma was used for estimations. The tissues (heart and liver) were excised, washed in ice-cold isotonic saline, and blotted with a filter paper. The remaining portion of the tissues was weighed and homogenized in 0.1 M Tris-HCl buffer (pH 7.4) and the homogenate was used for the estimations. A small portion of the tissues was stored in 10% formalin for histological analysis.

Analysis of plasma and heart lipid profile

Plasma and tissue lipids were extracted by the method of Folch et al.³² To a known volume of plasma or tissue homogenate, 10.0 mL of chloroform–methanol (2:1 v/v) mixture was added and mixed well for 30 min and was filtered through Whatman filter paper (no. 42, pore size: 8 μm) into a separating funnel. The filtrate was mixed with 0.2 mL of physiological saline and the mixture was kept undisturbed overnight. The lower phase containing the lipid was drained off into preweighed beakers. The upper phase was re-extracted with more of chloroform–methanol mixture; the extracts were pooled and evaporated under vacuum at room temperature.³⁰

The lipid extract was redissolved in 3.0 mL of chloroform–methanol (2:1) mixture and aliquots were taken for the estimation of lipids. Total cholesterol (TC), triacylglycerol (TG), free fatty acids (FFA), and phospholipids (PLs) were estimated by the methods of Allain et al.,³³ McGowan et al.,³⁴ Falholt et al.,³⁵ and Zilversmit and Davis,³⁶ respectively. HDL-C and LDL-C were estimated by a standard commercial kit purchased from Agappe Diagnostics. The very-low-density lipoprotein cholesterol (VLDL-C) in the plasma was calculated as VLDL-C = TG/5 and LDL-C = Total cholesterol − (HDL-C + VLDL-C), respectively.

Isolation of mitochondria from the heart tissue and the assessment of mitochondrial enzymes activities

Mitochondrial fraction was isolated from the heart tissue of normal experimental animals by differential centrifugation. Briefly, a 20% (w/v) homogenate was prepared in 0.25 M sucrose containing 0.05 M Tris-HCl buffer and 5.0 mM ethylenediaminetetraacetic acid. To remove cell debris, tissue fragments, and cell nuclei (nuclei pellet), the homogenate was centrifuged at 600 × g for 10 min, the supernatant fraction was separated and centrifuged in a refrigerated centrifuge at 10,000 × g for 5 min at 4°C to bring down the mitochondrial pellet. After centrifugation, the supernatant was poured off while the loose upper part of the mitochondrial pellet may come off as well. Most of the pellets containing healthy mitochondria were dense enough to remain behind. The white foamy material, near the top of the tube, consists of lipids, which were removed by wiping the inside of the tube with cotton. After using a Pasteur pipette to remove the last bit of liquid, the remaining mitochondrial pellet was resuspended in a specific volume of potassium chloride and used for the estimation of the activities of heart mitochondrial enzymes, such as isocitrate dehydrogenase (ICDH),³⁷ succinate dehydrogenase (SDH),³⁸ malate dehydrogenase (MDH),³⁹ and α-ketoglutarate dehydrogenase (α-KGDH)⁴⁰ were analyzed.

Isolation of lysosomal fraction from the heart tissue and the assessment of activities of lysosomal enzymes

The lysosomal enzymes, such as β-glucuronidase using the method of Kawai and Anno,⁴¹ β-glucosidase, α- and β-galactosidase using the method of Conchie et al.,⁴² cathepsin-B using the method of Barrett,⁴³ and cathepsin-D using the method of Sapolsky et al.,⁴⁴ were analyzed.

Alkaline single-cell gel electrophoresis (comet assay)

DNA damage was estimated by alkaline single-cell gel electrophoresis according to the method of Singh et al. A layer of 1% normal melting agarose was prepared on microscope slides. Myocardial cells (50 µL in 1 × 10⁶ cells) were mixed with 100 µL of 1% low melting agarose. The suspension was pipetted out onto the precoated slides. Slides were immersed in cold lysis solution at pH 10 and kept at 4°C for 60 min. After lysis, the slides were placed in the alkaline electrophoresis buffer at pH 13 and left for 25 min. Subsequently, slides were transferred to an electrophoresis tank with fresh alkaline electrophoresis buffer and electrophoresis was performed on field strength of 25 V and 300 mA for 25 min at 4°C. Slides were neutralized in 0.4 M Tris, pH 7.5 for 5 min, and stained with 20 g/mL ethidium bromide. For visualization of DNA damage, observations were made using a 40× objective on an epifluorescent microscope equipped with an excitation filter of 510–560 nm and a barrier filter of 590 nm. Images were captured with a digital camera with networking capability and analyzed by image analysis software, CASP (Ver. 1.2.2: Zbigniew Koza, Poland).⁴⁵

Histopathology

Masson’s trichrome is commonly used for staining collagen and detecting cardiac fibrosis. Heart tissue was fixed in 10% formalin, routinely processed, and embedded in paraffin. Paraffin sections (3 mm) were cut on glass slides, stained with Masson’s trichrome stain by the method of Masson, and examined under a light microscope to find myocardial fibrosis.⁴⁶

Statistical analysis

Values are expressed as means ± standard deviation. The data were statistically analyzed by one-way analysis of variance followed by Duncan’s multiple range test (DMRT) using a statistical package program (SPSS 17 for Windows). The p values <0.05 were considered as statistically significant.

Results

Plasma lipid profile

The plasma TC, HDL-C, LDL-C, and VLDL-C of normal and experimental rats are shown in Figure 2. Significant elevation in the level of TC, LDL-C, and VLDL-C and a significant reduction in the level of HDL-C were observed in ISO-administered rats. Treatment with ARB brought all these lipids to near normal.

Figure 2.

Effect of ARB on TC, HDL-C, LDL-C, and VLDL-C in the plasma of control and ISO-induced rats. Values are given as means ± SD for six rats in each group. (a–e) Values not sharing a common superscript (a, b, c, d) differs significantly at p ≤ 0.05 (DMRT). ARB: arbutin; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; VLDL: very-low-density lipoprotein cholesterol; SD: standard deviation; DMRT: Duncan’s multiple range test.

Myocardial and hepatic tissue lipids

In Figure 3(a) and (b), the levels of TC, FFA, TG, and PL in tissues of heart and liver of control and ISO-induced rats are depicted. ISO-administered rats showed a significant increase in tissue TC, TG, and FFA with a reduction in PL. Improvement in the levels of these lipids to near normal was observed in rats treated with ARB.

Figure 3.

Effect of ARB on TC, TG, FFA, and PL levels in (a) heart and (b) liver of control and experimental rats. Values are given as means ± SD for six rats in each group. (a–e) Values not sharing a common superscript (a, b, c, d, e) differs significantly at p ≤ 0.05 (DMRT). The values were expressed as mg/dL of heart and liver. ARB: arbutin; SD: standard deviation; DMRT: Duncan’s multiple range test.

Effect of ARB on the activities of mitochondrial TCA cycle enzymes in the heart of control and ISO rats

Figure 4 exhibits the activities of the mitochondrial TCA cycle enzymes in the normal and experimental rats. The activities of these TCA cycle enzymes, such as ICDH, α-KGDH, SDH, and MDH, were significantly decreased in the heart mitochondrial fractions of MI rats when compared with those in the control group, and these enzymes are required for the aerobic oxidation of pyruvate in mitochondria. Pretreatment with ARB (50 mg/kg bw) increased these enzyme activities significantly when compared to the ISO group.

Figure 4.

Effect of ARB on the activities of mitochondrial TCA cycle enzymes in the heart mitochondrial fraction of control and ISO-induced rats. Values are given as means ± SD for six rats. Values not sharing a common superscript differ significantly at p < 0.05 (DMRT). Units: nmol of α-ketoglutarate liberated/h for ICDH; nmol of ferrocyanide formed/h for α-KGDH; nmol of succinate oxidized/min for SDH; nmol of NADH oxidized/min for MDH. ARB: arbutin; SD: standard deviation; ISO: isoproterenol; DMRT: Duncan’s multiple range test; ICDH: isocitrate dehydrogenase; KGDH: α-ketoglutarate dehydrogenase; SDH: succinate dehydrogenase; MDH: malate dehydrogenase; NADH: nicotinamide adenine dinucleotide.

Effect of ARB on the activities of lysosomal enzymes in the heart of control and ISO rats

Figure 5(a) and (b) indicates the activities of lysosomal enzymes in the myocardium of control and experimental animals. A significant elevation in the activities of β-glucosidase, β-glucuronidase, α- and β-galactosidase, cathepsin-B, and cathepsin-D was observed in the heart homogenate of ISO-induced MI rats. In rats pretreated with ARB (50 mg/kg bw), a significant decrease in the activities of these hydrolases was observed when compared to rats administered with ISO alone.

Figure 5.

(a and b) Effect of ARB on the activities of β-glucosidase, β-glucuronidase, α- and β-galactosidase, cathepsin-B, and cathepsin-D in the heart of control and ISO-induced rats. Values are given as means ± SD for six rats. Values not sharing a common superscript differ significantly at p ≤ 0.05 (DMRT). Units: mmol of p-nitrophenol liberated/h. ARB: arbutin; SD: standard deviation; ISO: isoproterenol; DMRT: Duncan’s multiple range test.

Evaluating DNA damage by comet assay

Figure 6(a) and (b) shows the levels of DNA damage (% head and tail DNA and tail length) in the myocytes of control and experimental rats. ISO-injected rats significantly increased the percentage of tail DNA and tail length and decreased the percentage of head DNA. ARB administration significantly reduced the levels of DNA damage.

Figure 6.

(a) Effect of ARB on DNA damage by comet assay in the myocytes (×20). (a) Untreated control cells, (b) 50 mg/kg bw of ARB, (c) 60 mg/kg bw of ISO, (d) 25 mg/kg bw of ARB + 60 mg/kg bw of ISO, (d) 50 mg/kg bw of ARB + 60 mg/kg bw of ISO shows DNA damage, as characterized by the % head DNA, tail DNA, tail moment, and live tail movement. (b) Comet assay analyzed by image analysis software. Values are given as means ± SD for six rats in each group. (a–e) Values not sharing a common superscript (a, b, c, d, e) differ significantly at p < 0.05. (DMRT). ARB: arbutin; SD: standard deviation; ISO: isoproterenol; DMRT: Duncan’s multiple range test; bw: body weight.

Histopathological changes on heart tissues by Masson’s trichrome staining

Figure 7 shows the Masson’s trichrome-stained heart tissue of control and treated rats. ISO-injected rats revealed interstitial collagen accumulation. Prior administration of ARB (50 mg/kg bw) to ISO-administered rats reduced the accumulation of interstitial collagen in the myocardial tissues. Control- and ARB alone-treated rats showed normal distribution of collagen.

Figure 7.

Histology of heart stained with Masson’s trichrome (×40). (a) Control rats show the normal distribution of collagen. (b) Rats with ARB alone administration show the normal distribution of collagen. (c) ISO-induced MI rats showed interstitial collagen accumulation (blue color). (d) ARB 25 mg/kg bw with ISO administration shows reduced interstitial collagen accumulation. (e) The heart of the rat that received 50 mg/kg bw of ARB showing smaller regions of fibrosis than the ISO alone administered rats. (f) The percentage of Masson’s trichrome-positive area per section was quantified. ARB: arbutin; SD: standard deviation; ISO: isoproterenol.

Discussion

A number of studies have reported that pathophysiological, morphological, and metabolic changes that occur in the heart of experimental animals following ISO administration are similar to those observed in humans.⁴⁷ ISO metabolism produces quinines, which react with oxygen to produce superoxide anion and hydrogen peroxide, leading to oxidative stress, thereby damaging the myocardial cells. Furthermore, free radicals could initiate the peroxidation of membrane-bound polyunsaturated fatty acids (PUFA), leading to the membrane damage, and phospholipids are a vital part of the biomembrane rich in PUFA, which are susceptible to free radicals.⁴⁸ ISO has been reported to produce stress and membrane degradation on the myocardium due to the generation of free radicals by its auto-oxidation.

Lipids play a key role in CVDs by developing the cholesterol deposition and modifying the composition, structure, and stability of cellular membranes. Elevated levels of blood cholesterol and its buildup in cardiac tissue are well connected with cardiovascular damage.⁴⁹ In the present study, ISO-administered rats showed a significant increase in the levels of TC, TG, and FFA in myocardium and plasma while the levels of PLs increased in the plasma and decreased in heart, which is in line with the previous studies.¹⁵ ISO has been reported to produce stress in the myocardium due to the production of free radicals by its auto-oxidation, which leads to membrane degradation.⁵⁰ Membrane PLs are important factors of maintaining cell membrane integrity and functions of the cells, and the observed decrease in the levels of PLs in ISO-injected rats myocardium might be due to the acceleration of membrane degradation of PLs by phospholipase. The lipolytic action of ISO on fat cells is mediated by the cAMP cascade and ISO activate membrane-bound adenylate cyclase by activating protein kinase, thereby increasing the formation of the cAMP. cAMP promotes lipolytic activity of hormone-sensitive lipase by activating cyclic adenosine monophosphate (cAMP) dependent protein kinase, which phosphorylates hormone-sensitive lipase (HSL)⁵¹ and increases the mobilization of FFAs from the fat depots, thereby causing hyperlipidemia. ARB pretreatment significantly prevented these alterations in the levels of lipids, thereby maintaining the normal membrane fluidity and functions of the myocardial cells. ARB is a selective inhibitor of the catalytic activity of cAMP-dependent protein kinase,⁵² which may decrease the HSL activity, thereby reducing the hydrolysis of stored TG.

An increase in plasma TC, LDL-C, and VLDL-C with a decrease in plasma HDL-C was also observed in ISO-induced rats. Elevated levels of LDL-C correlated with a higher risk of MI, while high levels of HDL-C correlated with a lower risk of MI. The increased LDL-C concentration and decreased HDL-C concentration by ISO in the blood lead to the buildup of harmful deposits in the arteries, thus favoring MI. Changes in the level of lipids are because of enhanced lipid biosynthesis by increased cAMP produced by ISO. HDL-C facilitates the transport of excess cholesterol from peripheral tissue to the liver for the excretion.¹² The non-HDL-C to HDL-C ratio is beneficial in determining the risk of CVDs and it indicates the deposition of plaque of lipids and foam cells in coronaries and heart.⁵³ ARB administration significantly decreased the levels of all atherogenic cholesterols (TC, LDL-C, and VLDL-C) and increased the level of antiatherogenic cholesterol (HDL-C).

About 45% of the myocardial cell volume is occupied by the mitochondria. Mitochondria are important subcellular organelles involved in the ATP production and are susceptible to oxidative stress. Mitochondrial dysfunction plays a significant role in the pathogenesis of CVDs.⁵⁴ ISO induces lipid peroxidation in mitochondrial membrane and accelerated lipid peroxidation damages the heart mitochondrial membrane in ISO-treated rats.⁵⁵ Literature suggest that ISO damages the myocardium by decreasing the antioxidant capacity and increases the production of mitochondrial ROS and alterations of mitochondrial respiratory chain function through downregulation of the activity of the mitochondrial respiratory chain enzymes.¹⁵ In the present study, the catalytic activity of the mitochondrial respiratory chain enzymes, such as SDH, MDH, ICDH, and α-KGDH, was significantly decreased in the mitochondria of ISO-administered group of rats while compared to control group of rats. The decreased activities of TCA cycle enzymes, which are located in the outer membrane, could be due to the membrane destabilization caused by ISO. Decreased activities of these enzymes affect the mitochondrial substrate oxidation, resulting in decreased oxidation of substrates and decreased rate of transfer of reducing equivalents to molecular oxygen, thereby depleting cellular energy.⁵⁶ ARB pretreatment (50 mg/kg bw) restored the activities of these enzymes to near normal in the cardiac mitochondria.

Lysosomes have phospholipid enriched membranes, which are a potential site for free-radical attack consequently causing loss of membrane stability that leads to the release of hydrolytic enzymes from the lysosome. Lysosomes are essential for controlled intracellular digestion of cellular components by different pathways, such as autophagy, heterophagy, and endocytosis, and release of the lysosomal enzymes to the cytosol, leading to myocardial cellular injury and death.⁵⁷ Previous reports demonstrated that the administration of the ISO to the rats causes a significant elevation in the activities of the lysosomal enzymes in the myocardium and it might be due to the membrane damage. Intracellular release of the lysosomal enzymes causes the cell injury by directly activating the complement pathway.⁵⁸ Hence, considerable attention has been focused on lysosomal enzymes that might accompany myocardial damage. In our study, a significant increase in the activities of the lysosomal enzymes was observed in the cardiac tissues of ISO-administered rats. The previous study reported that the increased lipid peroxidation was observed in ISO-administered rats and it might be the reason for lysosomal membrane damage.⁵⁹ The stabilization of myocardial cell membranes, mainly the lysosomal membranes, may extend the viability of myocardial cells and prevent MI. Lysosomal membrane destabilization may be prevented either by prevention of free-radical attack or by inhibition of peroxidation of cellular membranes, which inhibits the leakage of lysosomal content. ARB is an antioxidant and has the potential to inhibit lipid peroxidation, ultimately resulting in decreased lipid peroxidative products.²⁴ In the present study, we observed that the oral administration of ARB to the ISO-induced MI rats significantly prevented the increased activities of lysosomal enzymes mediated by ISO in the myocardium, which might be due to decreased lysosomal membrane damage, thereby inhibiting the release of lysosomal enzymes.

Earlier reports demonstrated that oxidative stress induced by ISO provokes DNA damage.⁶⁰ Auto-oxidation of ISO forms quinonoid compounds and generates free radicals, which damage the DNA by causing oxidative stress. In this study, we observed an increase in DNA damage in the myocytes of ISO-induced MI rats. ARB administration decreased the DNA damage caused by ISO. ARB has a strong inhibitory activity against superoxide formation and reduces both intracellular ROS levels and rate of H₂O₂-induced apoptotic cell death through the DNA damage. Thus, the antioxidant effect of ARB might be responsible for its protective effect against ISO-induced DNA damage.⁶¹

Collagen is a major fibrous protein of the normal extracellular matrix of the myocardium, linking the myriad of myocardial tissue components into a cohesive whole and ensuring efficient function as a biological pump.⁶² Collagen fibers play an important role in maintaining the structural integrity and tissue functions of the myocardium. Collagen deprivation after MI is associated with infarct expansion and functional decline of the myocardium.⁶³ The normal architecture of muscle fibers primarily confined to the intramuscular fascicule and it was observed in cardiac tissues of normal rats. ISO-injected rats showed muscle cell necrosis with disruption in the arrangement of collagen fibers. ARB-treated ISO rats showed minimally damaged collagen fibers exhibiting near the normal structure of heart tissue. ARB alone-administered rats showed normal distribution of collagen. In this study, we observed that the pretreatment with ARB (50 mg/kg bw) maintained the normal level of mitochondrial and lysosomal enzymes and prevented the DNA damage in ISO-induced MI rats. The schematic representation of the proposed mechanism of action of ISO is shown in Figure 8.

Figure 8.

The schematic representation of the proposed mechanism of action of ISO. ISO: isoproterenol.

Conclusion

Our study reveals that ARB exhibited protective effects against ISO-induced MI in rats by maintaining the lipid profile normal, preserving the integrity of the mitochondrial and lysosomal membranes, thereby restoring the activities of the enzymes to near normal and preventing the DNA damage in ISO-induced rats. Therefore, this study provides the basis for the development of ARB as an effective and safe treatment of MI.

Footnotes

Acknowledgment

The Department of Biochemistry and Biotechnology, Annamalai University, is supported by the University Grants Commission, Special Assistance Programme (UGC-SAP). Financial support from the University Grants Commission, New Delhi, India, in the form of RGNF (research fellow) to Ms SS is gratefully acknowledged.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

L Vennila

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Arbutin prevents alterations in mitochondrial and lysosomal enzymes in isoproterenol-induced myocardial infarction: An in vivo study

Abstract

Keywords

Introduction

Materials and methods

Chemicals

Experimental animals

Experimental induction of MI

Experimental design

Analysis of plasma and heart lipid profile

Isolation of mitochondria from the heart tissue and the assessment of mitochondrial enzymes activities

Isolation of lysosomal fraction from the heart tissue and the assessment of activities of lysosomal enzymes

Alkaline single-cell gel electrophoresis (comet assay)

Histopathology

Statistical analysis

Results

Plasma lipid profile

Myocardial and hepatic tissue lipids

Effect of ARB on the activities of mitochondrial TCA cycle enzymes in the heart of control and ISO rats

Effect of ARB on the activities of lysosomal enzymes in the heart of control and ISO rats

Evaluating DNA damage by comet assay

Histopathological changes on heart tissues by Masson’s trichrome staining

Discussion

Conclusion

Footnotes

Acknowledgment

Declaration of conflicting interests

Funding

ORCID iD

References