|Year : 2018 | Volume
| Issue : 3 | Page : 92-100
Protective role of heartogen against myocardial infarction in rats
C Hamssika1, Q Sakeena1, G Ramakrishnan2, M Sugumar3, B Sathyanarayana4, MV Shetty5, AH Tousif6, N Chethan6, B Abid6, R Bipul6, Saravana Babu Chidambaram6
1 Department of Biotechnology, Jeppiaar Engineering College, Chennai, India
2 Department of Biochemistry, PRIST University, Thanjavur, India
3 Research and Development Centre, Bharathiar University, Coimbatore, Tamil Nadu, India
4 Muniyal Ayurveda Research Centre, Muniyal Institute of Ayurveda Medical Sciences, Manipal, India
5 Dr. Krshna Life Sciences Ltd., Manipal, India
6 Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Mysore, Karnataka, India
|Date of Web Publication||20-Jun-2018|
Saravana Babu Chidambaram
Department of Pharmacology, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Mysore, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Heartogen [HTN], an Ayurvedic medicine is a combination of herbs and bhasmas used in cardiac ailments. Objectives: The present study is aimed at investigating the cardio-protective effects of HTN against isoproterenol [ISP] induced myocardial infarction in rats. Methods: Male rats were pre-treated with HTN [50 and 100mg/kg/day, p.o] or vehicle [0.3% CMC] for 21 days. ISP [120mg/kg, s.c; 2 doses at 24 h interval] was administered on days 19 and 21. On day 22, plasma and heart tissues were collected for biochemical, molecular and histopathological analyses. Results: Pretreatment with HTN significantly reduced plasma CK-MB and cardiac LPO while increased SOD, GSH and GPx levels when compared vehicle treated rats. HTN improved NaKATPase, CaATPase and MgATPase activities in the hearts of ISP intoxicated rats. HTN down-regulated p53, caspase-3 and Bax and up-regulate Bcl2 gene expression. HTN treated rats showed minimal degree of histoarchitectural changes in heart when compared to positive control. Conclusion: Hence, in this manuscript we propose that HTN has the ability to protect against myocardial infarction via anti-oxidant and anti-apoptotic mechanisms.
Keywords: Anti-apoptosis, Ayurveda, cardioprotection, Heartogen, isoproterenol, myocardial infarction
|How to cite this article:|
Hamssika C, Sakeena Q, Ramakrishnan G, Sugumar M, Sathyanarayana B, Shetty M V, Tousif A H, Chethan N, Abid B, Bipul R, Chidambaram SB. Protective role of heartogen against myocardial infarction in rats. Int J Nutr Pharmacol Neurol Dis 2018;8:92-100
|How to cite this URL:|
Hamssika C, Sakeena Q, Ramakrishnan G, Sugumar M, Sathyanarayana B, Shetty M V, Tousif A H, Chethan N, Abid B, Bipul R, Chidambaram SB. Protective role of heartogen against myocardial infarction in rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2018 [cited 2018 Nov 18];8:92-100. Available from: http://www.ijnpnd.com/text.asp?2018/8/3/92/234813
| Introduction|| |
Changes in lifestyles, behavioral patterns, sociocultural, and technological advancements are leading to sharp increase in the prevalence of noncommunicable diseases such as diabetes, cardiovascular diseases, stroke, and cancer. Ischemic heart disease is a leading cause of morbidity and mortality in both men and women. Due to changing lifestyles in developing countries, such as India, and particularly in urban areas, myocardial infarction (MI) is making an increasingly important contribution to mortality statistics. It is predicted that cardiovascular diseases will be the most important cause of mortality in India by 2020.
MI occurs when the blood supply to a part of the heart is interrupted, resulting in complex phenomenon affecting the mechanical, electrical, structural, and biochemical properties, causing myocardial insult, and in turn death of heart tissues. Clinical trials with antiarrhythmic drugs (sodium-channel blockers, potassium-channel blockers, and calcium-channel blockers) failed to prevent cardiac death triggered by malignant arrhythmias in patients with MI. Moreover, at least Na + channel-blocking antiarrhythmia agents increases mortality., β-Adrenergic blockers are widely used in the clinic for the treatment of cardiovascular diseases such as hypertension, coronary heart disease, and hyperlipidemia, especially for preventing sudden cardiac death in patients suffering acute and chronic MI.,,, These agents are shown to produce wide varieties of effects, including the improvement of coronary circulation, reduction of infarct size, protection from cardiomyocyte apoptosis, prevention of Ca 2+ overload, scavenging of oxygen-free radicals, and amelioration of cellular metabolism.,, Although modern drugs are effective in preventing cardiovascular disorders, their use is often limited due to their side effects. This, in turn, has led the entire world population in turning toward natural drugs due to the widespread belief that “green medicines” are safer than synthetic ones. Medicinal plants have long been valued as sources of new compounds with cardioprotective activity.
Isoproterenol (1- [3,4-dihydroxyphenyl]-2-isopropylaminoethanol hydrochloride) is a synthetic catecholamine and β-adrenergic agonist, which causes severe stress in the myocardium resulting in infarct-like necrosis. Isoproterenol (ISP)-induced myocardial damage causes many metabolic and morphologic aberrations in the heart of experimental animals similar to those seen in human. Administration of ISP depletes the energy reserve of cardiac muscle cells, and this leads to complex biochemical and structural changes that cause irreversible cellular damage, which is a prelude to necrosis; hence, isoproterenol model is used as a model for studying the effects of cardioprotective agents.
Heartogen (HTN), an Ayurvedic cardioprotective medicine, contains rare herbs and Bhasmas is used as a supportive remedy for various cardiac ailments such as mild hypertension, angina pectoris, cardiac arrthythmiasis, and dyspnea. The present study was undertaken to evaluate the effects of HTN against ISP-induced myocardial alteration and its role in myocardial membrane enzymes and anti-oxidant markers in ISP intoxicated rats' heart.
| Materials and Methods|| |
Male Sprague-Dawley rats 150–200 g body weight were used for the study. Animals were housed in colony cages in a well-ventilated room under an ambient temperature of 22°C ± 3°C and 40%–65% relative humidity, with artificial photoperiod (12-h light/12-h dark cycle). They were provided with standard rodent pellet diet (Nutrilab Rodent, Tetragon Chemie, India) and purified water ad libitum (RIOS, USA). Experimental animals were acclimatized for 7 days to the laboratory conditions before experimentation. Institutional Animal Ethical Committee (IAEC/XX/SRU/148/2010) approved the study protocol.
Chemicals and reagents
HTN was a kind gift from M/s. Muniyal Institute of Ayurveda Medical Sciences and Post-Graduate Centre, Manipal, Karnataka [Table 1]. Isoproterenol was purchased from Sigma Aldrich, USA. Creatinine kinase-MB (CK-MB) was purchased from Spinreact, Spain. Ammonium molybdate, tetrasodium pyrophosphate, and thiobarbituric acid (TBA) were purchased from Himedia laboratories, India and all other chemicals and reagents used were of analytical grade and were procured from SISCO Research Laboratories Pvt. Ltd., India.
Experimental groups and design
Rats were randomized into five groups with six in each. Animals were pretreated with vehicle or HTN (50 and 100 mg/kg, p.o) for 21 days. On day 19 and 21, ISP (120 mg/kg, s. c; 2 doses at 24 h interval) was administered 1 h after HTN administration. Carvedilol, a nonselective beta-blocker/alpha-1 blocker, was used as standard drug.
- Group I (normal control): 0.5% carboxymethylcellulose (CMC) + saline s.c.
- Group II (positive control): 0.5% CMC + ISP
- Group III (standard): Carvedilol (2 mg/kg/day, p.o) + ISP
- Group IV (low dose): HTN (50 mg/kg, p.o) + ISP
- Group V (high dose): HTN (100 mg/kg, p.o) + ISP.
At the end of the experiment, blood was collected for the determination of plasma CK-MB. Animals were euthanized under excessive ketamine dose; heart, kidney, and adrenals were dissected out and weighed. The left ventricle was separated, and used for biochemical and molecular investigation.
Assay for Creatinine Kinase-MB
CK-MB was measured using commercial kit Spinreact, Spain in a semi-automated biochemical analyzer (Star21Plus, India).
Determination of myocardial oxidative stress markers
The method involved heating of 0.5 ml of heart homogenate of experimental rats with 0.8 ml saline, 0.5 ml of butylated hydroxytoluene, and 3.5 ml TBA reagent for 11/2 min in a boiling water bath. After cooling, the solution was centrifuged at 2000 rpm for 10 min and the precipitate obtained was removed. The absorbance of the supernatant was determined at 532 nm using spectrophotometer against a blank that contained all the reagents minus the biological sample. The values were expressed in mg/g tissue.
Superoxide dimutase was assayed by taking 0.05 ml of heart homogenate followed by addition of 0.3 ml of sodium pyrophosphate buffer (0.025M, pH 8.3), 0.025 ml of PMS (186 Mm), and 0.075 ml of NBT (300 μM in buffer of pH 8.3). The reaction was started by addition of 0.075 ml of NADH (780 μM in buffer of pH 8.3). After incubation at 30°C for 90 s, the reaction was stopped by addition of 0.25 ml glacial acetic acid. Then, the reaction mixture was stirred vigorously and shaken with 2.0 ml of n-butanol. The mixture was allowed to stand for 10 min and centrifuged. 1.5 ml of n-butanol alone was served as blank. The color intensity of the chromogen was read at 560 nm.
Glutathione peroxidase was assayed by taking 200 μl of tris HCl buffer (0.4 M), 0.4 mM ethylenediaminetetraacetic acid (EDTA) along with 100 μl of sodium azide and 200 μl of tissue homogenate and mixed well. Thereafter, 200 μl of reduced glutathione solution (2 mM) followed by 0.1 ml H2O2 were added. The overall reaction was arrested by adding 0.5 ml of 10% trichloroacetic acid (TCA). The precipitate was removed by centrifugation at 4000 rpm for 10 min. The absorbance was read at 412 nm using spectrophotometer. The nonenzymatic reaction rate was correspondingly assessed by replacing the enzyme sample by buffer. The results are expressed as mg of reduced glutathione (GSH) consumed/min/mg protein.
Glutathione content was estimated as described earlier. A volume of 0.25 ml of tissue homogenate was added to equal volume of ice cold 5% TCA. The precipitate was removed by centrifugation at 4000 rpm for 10 min. To 1 ml aliquot of the supernatant, 0.25 ml of 0.2M phosphate buffer, pH 8.0 and 0.5 ml of DTNB (0.6 mM in 0.2M phosphate buffer, pH 8.0) was added and mixed well. The absorbance was read at 412 nm using spectrophotometer. The values were expressed in mg/g tissue.
Determination of myocardial membrane-bound enzymes
Na + K + ATPase
Na + K + ATPase was assayed by taking 250 μl of tris HCl buffer followed by the addition of 50 μl of 600 mM NaCl, 50 μl of 50 mM KCl, along with 50 μl of 1 mM Na.EDTA, and 50 μl of 80 mM ATP. The reaction mixture was preincubated at 37°C for 10 min. Then, 25 μl of 10% homogenate was added to the test alone and further incubated at 37°C for 1 h. The reaction was immediately arrested by the addition of 10% TCA. The control reaction rate was correspondingly assessed by adding 25 μl of 10% homogenate only after arresting the reaction. The precipitate was removed by centrifugation at 3500 rpm for 10 min. To 50 μl of the supernatant, 1075 μl of distilled water, 125 μl of ammonium molybdate, and 50 μl of ANSA were added and incubated for 10 min at 37°C. The intensity of blue color was read at 640 nm using spectrophotometer against a blank that contained all the reagents minus the supernatant. The results are expressed in μg of inorganic phosphate (Pi) liberated/min/mg of protein.
Ca 2+ ATPase
Ca 2+ ATPase was assayed by taking 0.75 ml of tris HCl buffer followed by the addition of 50 μl of 100 mM KCl, 50 μl of 100 mM CaCl2, and 50 μl of 80 mM ATP. The reaction mixture was pre-incubated at 37°C for 2 min. Then, 50 μl of 10% homogenate was added to the test alone and further incubated at 37°C for 20 min. The reaction was immediately arrested by the addition of 500 μl of 10% TCA. Control reaction rate was correspondingly assessed by adding 50 μl of 10% homogenate only after arresting the reaction. The precipitate was removed by centrifugation at 3500 rpm for 10 min. To 50 μl of the supernatant, 1075 μl of distilled water, 125 μl of ammonium molybdate, and 50 μl of ANSA were added and incubated for 10 min at 37°C. The intensity of blue color was read at 640 nm using spectrophotometer against a blank that contained all the reagents minus the supernatant. The results are expressed in μg of Pi liberated/min/mg of protein.
Mg 2+ ATPase
Mg + ATPase was assayed by taking 0.75 ml of tris HCl buffer followed by the addition of 50 μl of 100 mM KCl, along with 50 μl of 100 mM MgCl2, and 50 μl of 80 mM ATP. The reaction mixture was preincubated at 37°C for 2 min. Then, 50 μl of 10% homogenate was added to the test alone and further incubated at 37°C for 20 min. The reaction was immediately arrested by the addition of 500 μl of 10% TCA. Control reaction rate was correspondingly assessed by adding 50 μl of 10% homogenate only after arresting the reaction. The precipitate was removed by centrifugation at 3500 rpm for 10 min. To 50 μl of the supernatant, 1075 μl of distilled water, 125 μl of ammonium molybdate, and 50 μl of ANSA were added and incubated for 10 min at 37°C. The intensity of blue color was read at 640 nm using spectrophotometer against a blank that contained all the reagents minus the supernatant. The results are expressed in μg of Pi liberated/min/mg of protein.
Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to determine the level of mRNA expression of caspase-3, Bax, Bcl2, and p53. In brief, total RNA was extracted from left ventricle using TRIzol Reagent (Sigma, USA). After homogenization, the tubes were incubated for 10 min and centrifuged at 1000 rpm for 5 min. A volume of200 μl of chloroform was added to the supernatant, allowed to incubate for 5 min at room temperature and centrifuged at 12,000 rpm for 20 min. Then, 500 μl of isopropyl alcohol was added to the supernatant to precipitate the total RNA and centrifuged at 12,000 rpm for 15 min following the incubation period of 10 min. The supernatant was decanted carefully; the pellet was washed three times with 75% ethanol, centrifuged at 12,000 rpm for 15 min and the pellet was allowed to air dry. The pellet was resuspended in 20 μl of RNase-free water and stored in −80°C until use. The isolated RNA was allowed to undergo reverse transcription and polymerization reaction to get cDNA using PCR master cycler gradient. The formed cDNA was loaded on an agarose gel, allowed to run the electrophoresis at 80V for 30 min and the gene expression was analyzed using the bands formed. A total of 200 nanograms of RNA were used for reverse transcription polymerase chain reaction RT-PCR according to the manufacturer's instructions (Genet Bio, Korea). Primers used in the study are given in [Table 2].
Heart tissues were washed in saline immediately following necropsy and then fixed in 10% neutral buffered formalin solution. After fixation, the heart tissue was processed and embedded in paraffin. Then, the heart tissue was sectioned (5 μm) longitudinally and stained with hematoxylin and eosin. These sections were then examined under a light microscope for the histoarchitectural changes in treatment-blinded manner. Photomicrographs were taken at ×20 magnification for further analysis.
Statistical analysis was performed using GraphPad Prism, 4.03 San Diego, USA. Data were expressed as a mean ± standard error of the mean. Mean difference was analyzed using one-way ANOVA with Tukey's multiple comparison as the post hoc test. P ≤ 0.05 was fixed as the statistical significance criterion.
| Results|| |
Effect of Heartogen on body weight and heart weight
Measurement of body weight in the study gives an idea about the use of the drug. The effect of the HTN on body weight was recorded weekly for 21 days. When compared to normal group, a significant body weight loss was observed in ISP control rats, whereas pretreatment with HTN at (50 and100 mg/kg) followed by ISP administration showed a significant alteration in the body on day 21 [Figure 1]. There was no difference in body weight between the experiment animals from day 0 to day 14 which can be corroborated by normal age-related growth.
|Figure 1: The effect of Heartogen on the body weight of the animals during the course of administration. Results values were expressed in mean ± standard error mean (n = 6); ##P < 0.01 (comparison between normal and positive control); *P < 0.05 (comparison between positive control and other treatment groups)|
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Effects of ISP and influence of HTN on the heart, kidneys (paired), and adrenals (paired) weights were determined at the end of the study. Vehicle-treated ISP administered rats exhibited increased (24%) heart weight when compared to normal control rats. HTN (100 mg/kg) decreased the increase in heart weight by 12.3% in ISP intoxicated rats [Table 1]. However, there was no significant change in the kidneys and adrenals weights between the experimental groups (data not shown).
Heartogen decreased plasma creatinine kinase-MB in isoproterenol administered rats
ISP administration significantly (P< 0.01) increased plasma CK-MB when compared to the normal control rats. Pretreatment with HTN decreased (P< 0.01) CK-MB activity in comparison to vehicle-treated ISP rats [Figure 2].
|Figure 2: The effect of Heartogen on creatinine kinase MB levels of the experimental animals. Results were expressed in mean ± standard error mean (n = 6); ##P < 0.01 respectively (comparison between normal and positive control); **P < 0.01 (comparison between positive control and other treatment groups)|
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Heartogen decreased lipid peroxidation and increased superoxide dimutase, glutathione peroxidase and reduced glutathione levels
Vehicle-treated ISP rats showed significant increase in lipid peroxidation (LPO) (P< 0.01) and decrease in superoxide dimutase (SOD) (P< 0.01), GSH (P< 0.01), and glutathione peroxidase (GPx) (P< 0.01) when compared to control group rats. HTN significantly decreased LPO and improved SOD, GPX, and GSH levels in the left ventricle of ISP injected rats. Effect of HTN is comparable to carvedilol [Figure 3], [Figure 4], [Figure 5], [Figure 6].
|Figure 3: The effect of Heartogen on LPO levels in Heart tissue. Results were expressed in Mean±SEM [n=6]; #, ## - denotes P<0.05 and 0.01 respectively [Comparison between Normal and Positive control]; *, ** - denotes P<0.05 and 0.01 respectively [Comparison between Positive control and other treatment groups]|
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|Figure 4: The effect of Heartogen on SOD levels in Heart tissue. Results were expressed in Mean±SEM [n=6]; #, ##- denotes P<0.05 and 0.01 respectively [Comparison between Normal and Positive control]; *, ** - denotes P<0.05 and 0.01 respectively [Comparison between Positive control and other treatment groups]|
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|Figure 5: The effect of Heartogen on GSH levels in Heart tissue. Results were expressed in Mean±SEM [n=6]; #, ##- denotes P<0.05 and 0.01 respectively [Comparison between Normal and Positive control]; *, ** - denotes P<0.05 and 0.01 respectively [Comparison between Positive control and other treatment groups]|
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|Figure 6: The effect of Heartogen on GPx levels in Heart tissue. Results were expressed in Mean±SEM [n=6]; #, ##- denotes P<0.05 and 0.01 respectively [Comparison between Normal and Positive control]; *, ** - denotes P<0.05 and 0.01 respectively [Comparison between Positive control and other treatment groups]|
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Effect of Heartogen on membrane stabilizing enzyme levels in heart tissue
Ca 2+ ATPase (P< 0.01) was significantly increased while Na + K + ATPase and Mg 2+ ATPase (P< 0.01) were significantly decreased in vehicle-treated ISP rats when compared to the control group. Pretreatment with HTN exhibited significantly decreased Ca 2+ ATPase and increased Na + K + ATPase and Mg 2+ ATPase (P< 0.01) activities when compared to ISP alone treated rats. Effect of HTN was comparable with carvedilol [Figure 7], [Figure 8], [Figure 9].
|Figure 7: The effect of Heartogen on Ca2+ATPase levels in heart tissue. Results were expressed in Mean±SEM [n=6]; #, ## - denotes P<0.05 and 0.01 respectively [Comparison between Normal and Positive control]; *, ** - denotes P<0.05 and 0.01 respectively [Comparison between Positive control and other treatment groups]|
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|Figure 8: The effect of Heartogen on Na+K+ATPase levels in heart tissue. Results were expressed in Mean±SEM [n=6]; #, ## - denotes P<0.05 and 0.01 respectively [Comparison between Normal and Positive control]; *, ** - denotes P<0.05 and 0.01 respectively [Comparison between Positive control and other treatment groups]|
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|Figure 9: The effect of Heartogen on Mg2+ATPase levels in heart tissue. Results were expressed in Mean±SEM [n=6]; #, ##- denotes P<0.05 and 0.01 respectively [Comparison between Normal and Positive control]; *, ** - denotes P<0.05 and 0.01 respectively [Comparison between Positive control and other treatment groups]|
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Effect of Heartogen on apoptosis marker
In the present study, induction of MI with ISP up-regulated the mRNA expression of caspase 3, p53 and Bax and down-regulated Bcl2 expression. Treatment with HTN markedly up-regulated the expression of Bcl2 and down-regulated caspase-3, p53 and Bax expression when compared to ISP rats [Figure 10].
|Figure 10: The effect of Heartogen on apoptotic markers. (a) on p53 expression: Line: L1: Normal control, L2: Positive control, L3: Carvedilol (2 mg/kg), L4: Heartogen (50 mg/kg), L5L Heartogen (100 mg/kg) (b) on caspase 3: Line: L1: Normal control, L2: Positive control, L3: Carvedilol (2 mg/kg), L4: Heartogen (50 mg/kg), L5L Heartogen (100 mg/kg) (c) Bax expression: Line: L1: Normal control, L2: Positive control, L3: Carvedilol (2 mg/kg), L4: Heartogen (50 mg/kg), L5L Heartogen (d) Bcl2 expression: Line: L1: Normal control, L2: Positive control, L3: Carvedilol (2 mg/kg), L4: Heartogen (50 mg/kg), L5L Heartogen|
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Myocardium in all control rats exhibited normal histology, cross striations, and intact basement membrane. ISP treated vehicle control group showed lesions of maximum severity. There were multifocal confluent areas of necrosis and myocardial fragmentation along with interstitial edema and mononuclear cells (macrophages and lymphocytes) infiltration. In carvedilol treated group, minimal myocardial damage characterized by mild interstitial edema and focal degeneration and necrosis of myofibers. Occasionally, confluent necrotic areas with focal mononuclear cells infiltration were observed. In HTN (50 and 100 mg/kg) treated group, the lesions were minimal as similar to that of carvedilol group. In the HTN (100 mg/kg) group, only interstitial edema was evident, but necrotic lesions were infrequent and focal [Figure 11].
|Figure 11: (a-e) Photomicrographs of heart tissue of normal and ISO treated rats (H and E, ×20). (a) Normal histological appearance of myocardium. (b) ISO-treated positive control shows confluent myocardial necrosis, fragmentation and infiltration with macrophages. (c) Carvedilol treated group (2 mg/kg) shows mild interstitial edema and focal degeneration of myofibres. (d) Heartogen (50 mg/kg)-treated group shows focal myocardial necrosis and fragmentation, interstitial edema, macrophage infiltration. (e) Heartogen (100 mg/kg)-treated group shows mild interstitial edema and focal excess contraction of myofibres|
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| Discussion|| |
Isoproterenol is a synthetic catechol compound, and nonselective beta-blocker produces MI at high doses in experimental rats. Continuous and severe stimulation of adrenergic receptors causes cardiotoxicity through the the destruction of myocardial cells. As a result of intense tachycardia, cytosolic enzymes such as creatine phosphokinase, lactate dehydrogenase, and transaminases (alanine transaminase and aspartate transaminase) were released into bloodstream and serve as the diagnostic markers of myocardial tissue damage. The amount of these cellular enzymes in blood reflects the alterations in myocardial membrane integrity and/or permeability.
CK-MB is one of the most frequently used cardiac markers in the diagnosis of MI. In the present study, administration of ISP increased plasma CK-MB levels revealing the induction of MI in rats. This increase in CK-MB is accompanied with increase in wet weight of heart confirming cardiac hypertrophy and edema. The extent of cardioprotective action offered by a drug is associated with the significant attenuation of CK-MB activity. Pretreatment with HTN significantly decreased CK-MB elevation which shows its cardioprotective effect.
Oxidative stress increases cAMP levels by exhausting ATP and decreases sarcolemmal Ca 2+ transport, resulting in intracellular calcium overload, which leads to ventricular dysfunction and contractile failure in rat heart., Isoproterenol administration is known to produce free radical moieties through its quinine metabolites that react with oxygen ultimately resulting in the enhanced generation of reactive oxygen species (ROS). ROS, the highly toxic by-products of aerobic metabolism are known to react extensively with cell membranes and macromolecules enhancing formation of lipid peroxides, thus leading to tissue damage. Lipid peroxidation is an important pathogenic event in myocardial necrosis and accumulation of lipid hydroperoxides reflects damage of the cardiac constituents. The increased levels of MDA, a lipid peroxidation end-product, observed in the present study following isoproterenol administration might be due to free radicals mediated membrane damage. HTN decreased lipid peroxidation which might be due to its ability to neutralize the formation free radicals through the high content of flavonoids and total phenolic-based ingredients. Among the free radical scavenging antioxidants, SOD was considered to be the prime cellular defense factor. The results envisaged that the decreased levels of SOD following ISP administration were ameliorated to a greater extent by pre-treatment with HTN. HTN might have the ability to up-regulate SOD protein expression which requires further studies.
Glutathione is known to protect the myocardium against the free radicals-mediated injury by the reduction of hydrogen peroxide. On the contrary, decreased glutathione levels is shown to aggravate myocardial necrosis. ISP administration was found to reduce the level of GSH in plasma and cardiac tissue. Inactivation of endogenous anti-oxidant enzyme GPX in the heart leads to accumulation of oxidized glutathione  and inhibits cellular protein synthesis. In the present study, increased GSH and GPX with HTN treatment reveals its antioxidant potential and protection of myocytes against free radicals [Figure 5] and [Figure 6].
Membrane-bound ATPases of cardiac cells play a significant role in contraction and relaxation cycles by maintaining normal ion levels. Changes in the properties of ion pumps affect cardiac function. Inhibition of Ca 2+ ATPase causes perturbation in cellular calcium homeostasis, and this is associated with an increase Ca 2+ permeability. ISP reported to decrease membrane-bound ATPases activities. Loss of ATPase activity in the ischemic state may be responsible for causing not only functional damage but also reversible necrotic changes in myocardial cells. Oxidation of membrane lipids inactivates Na + K + ATPase due to the degradation of “SH” groups present in the active site leading to the conformational alterations in the enzymes.
Hence, the study further extended to investigate the role of HTN on membrane-bound enzymes activity in ISP administered rats. Pretreatment with HTN showed noticeable elevation in Na + K + ATPase, Ca 2+ ATPase, and Mg 2+ ATPase activity. Na + K + ATPase, a membrane-bound enzyme is responsible for sodium ion influx and potassium ion efflux during muscle contraction. Intracellular calcium (Ca 2+) and Mg 2+ levels are maintained and regulated by Ca 2+ ATPase and Mg 2+ ATPase, respectively. The restoration of membrane-bound enzymes Na + K + ATPase, Ca 2+ ATPase, and Mg 2+ ATPase by HTN indicates its membrane stabilizing effect which may be due to the anti-oxidant properties.
Apoptosis is a tightly regulated, cell deletion process that plays an important role in various cardiovascular diseases, such as MI, reperfusion injury, and heart failure. Caspase, a critical enzyme in the induction and execution of apoptosis, has been the main potential target for achieving anti-apoptotic therapy. Caspases are members of a family of cysteine proteases that play a crucial role in the apoptotic pathway. Further apoptosis may result from abnormal expression of Bcl-2 and Bax. In the present study, ISP up-regulated the mRNA expression of apoptotic markers such as caspase 3, p53, and Bax and down-regulated the expression of anti-apoptotic marker Bcl2. In the apoptotic cascade, increased expression of Caspase 3 results in the phosphorylation of other caspases; on the other hand, p53 inhibits Bcl2 expression and conversely enhances Bax expression. Bax, in turn, inhibits the Cytochrome C, thereby augmenting the reactive oxygen species generation. Bcl2 and Bax proteins are known to modulate the cell survival signals of various apoptotic stimuli. Treatment with HTN markedly up-regulated the expression of Bcl2, whereas down-regulated the expression of caspase-3, p53, and Bax revealing that HTN exerts its cardioprotective action through the anti-apoptotic pathway.
| Conclusion|| |
HTN can stabilize the myocardial membrane and associated enzymes which might be due to its anti-oxidant and anti-apoptotic properties. Further, studies are in progress in the laboratory to identify the active bio principles in HTN.
Financial support and sponsorship
This study was financially supported by M/s. Muniyal trust for the kind gift of heartogen medicine.
Conflicts of interest
There are no conflicts of interest.
| References|| |
World Health Organisation. Statistical Annex 2: Death by Cause, Sex and Mortality Stratum in WHO Regions, Estimates for 2002. World Health Report; 2004. p. 121-5.
Levy RI, Feinleib M. Risk factors for coronary artery disease and their management. In: Brawnwald E, editor. Heart Disease: A Textbook of Cardiovascular Medicine. Philadelphia: W.B. Saunders; 1984. p. 1205-34.
Rajadurai M, Stanely Mainzen Prince P. Preventive effect of naringin on cardiac markers, electrocardiographic patterns and lysosomal hydrolases in normal and isoproterenol-induced myocardial infarction in wistar rats. Toxicology 2007;230:178-88.
Petrich ER, Schanne OF, Zumino AP. Electrophysiological responses to ischemia and reperfusion. In: Karmazyn M, editor. Myocardial Ischemia: Mechanisms, Reperfusion, Protection. Basel, Switzerland: Birkhauser; 1996. p. 115-33.
Waldo AL, Camm AJ, deRuyter H, Friedman PL, MacNeil DJ, Pauls JF, et al.
Effect of d-sotalol on mortality in patients with left ventricular dysfunction after recent and remote myocardial infarction. The SWORD investigators. Survival with oral d-sotalol. Lancet 1996;348:7-12.
Larsen JA, Kadish AH, Schwartz JB. Proper use of antiarrhythmic therapy for reduction of mortality after myocardial infarction. Drugs Aging 2000;16:341-50.
Cardiac Arrhythmia Suppression Trial (CAST) Investigators. Preliminary report: Effect of encainide and flecainide on mortality in a randomized trial of arrhythmia suppression after myocardial infarction. N
Engl J Med 1989;321:406-12.
Hjalmarson A, Herlitz J, Holmberg S, Rydén L, Swedberg K, Vedin A, et al.
The göteborg metoprolol trial. Effects on mortality and morbidity in acute myocardial infarction. Circulation 1983;67:I26-32.
Olsson G, Rehnqvist N, Sjögren A, Erhardt L, Lundman T. Long-term treatment with metoprolol after myocardial infarction: Effect on 3 year mortality and morbidity. J Am Coll Cardiol 1985;5:1428-37.
Lohse MJ, Engelhardt S, Eschenhagen T. What is the role of beta-adrenergic signaling in heart failure? Circ Res 2003;93:896-906.
Ruffolo RR Jr., Feuerstein GZ. Neurohormonal activation, oxygen free radicals, and apoptosis in the pathogenesis of congestive heart failure. J Cardiovasc Pharmacol 1998;32 Suppl 1:S22-30.
Reiken S, Wehrens XH, Vest JA, Barbone A, Klotz S, Mancini D, et al.
Beta-blockers restore calcium release channel function and improve cardiac muscle performance in human heart failure. Circulation 2003;107:2459-66.
Rajadurai M, Prince PS. Comparative effects of aegle marmelos extract and alpha-tocopherol on serum lipids, lipid peroxides and cardiac enzyme levels in rats with isoproterenol-induced myocardial infarction. Singapore Med J 2005;46:78-81.
Trivedi PC. Herbal Drugs and Biotechnology. India: Pointer Publishers; 2004.
Nivethetha M, Jayasri J, Brindha P. Effects of Muntingia calabura
L. on isoproterenol-induced myocardial infarction. Singapore Med J 2009;50:300-2.
Wexler BC. Myocardial infarction in young vs. old male rats: Pathophysiologic changes. Am Heart J 1978;96:70-80.
Ravichandran LV, Puvanakrishnan R, Joseph KT. Alterations in the heart lysosomal stability in isoproterenol induced myocardial infarction in rats. Biochem Int 1990;22:387-96.
Rona G, Chappel CI, Balazs T, Gaudry R. An infarct-like myocardial lesion and other toxic manifestations produced by isoproterenol in the rat. AMA Arch Pathol 1959;67:443-55.
Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351-8.
Kakkar P, Das B, Viswanathan PN. A modified spectrophotometric assay of superoxide dismutase. Indian J Biochem Biophys 1984;21:130-2.
Rotruck JT, Pope AL, Ganther HE, Swaason AB, Hafeman DG, Hoekstra WG. Selenium biochemical role as a component of glutathione peroxide. Science 1973;179:588-90.
Moron MS, Depierre JW, Mannervik B. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim Biophys Acta 1979;582:67-78.
Cortas N, Walser M. (Na+-K+)-activated ATPase in isolated mucosal cells of toad bladder. Biochim Biophys Acta 1971;249:181-7.
Amende LM, Chock SP, Albers RW. Characterization of the ca2+- and mg2+-dependent ATPases in electrophorus electroplax microsomes. J Neurochem 1983;40:1040-7.
Zheng Y, Zhou M, Ye A, Li Q, Bai Y, Zhang Q, et al.
The conformation change of bcl-2 is involved in arsenic trioxide-induced apoptosis and inhibition of proliferation in SGC7901 human gastric cancer cells. World J Surg Oncol 2010;8:31.
Thippeswamy BS, Thakker SP, Tubachi S, Kalyani GA, Netra MK, Patil U, et al
. Cardioprotective effect of Cucumis trigonus
Roxb on isoproterenol-induced myocardial infarction in rat. Am J Pharmacol Toxicol 2009;4:29-37.
Noronha-Dutra AA, Steen EM, Woolf N. The correlation between catecholamine and lipid peroxidation induced damage in heart cells. In: Adult Heart Muscle Cells. Heidelberg: Steinkopff; 1984. p. 133-36.
Bhagat B, Sullivan JM, Fischer VW, Nadel EM, Dhalla NS. CAMP activity and isoproterenol-induced myocardial injury in rats. Recent Adv Stud Cardiac Struct Metab 1976;12:465-70.
Tappia PS, Hata T, Hozaima L, Sandhu MS, Panagia V, Dhalla NS, et al.
Role of oxidative stress in catecholamine-induced changes in cardiac sarcolemmal ca2+ transport. Arch Biochem Biophys 2001;387:85-92.
Koçak H, Yekeler I, Başoğlu A, Paç M, Senocak H, Yüksek MS, et al.
The effect of superoxide dismutase and reduced glutathione on cardiac performance after coronary occlusion and reperfusion – An experimental study in dogs. Thorac Cardiovasc Surg 1992;40:140-3.
Ji LL, Stratman FW, Lardy HA. Antioxidant enzyme systems in rat liver and skeletal muscle. Influences of selenium deficiency, chronic training, and acute exercise. Arch Biochem Biophys 1988;263:150-60.
Ferrari R, Ceconi C, Curello S, Guarnieri C, Caldarera CM, Albertini A, et al.
Oxygen-mediated myocardial damage during ischaemia and reperfusion: Role of the cellular defences against oxygen toxicity. J Mol Cell Cardiol 1985;17:937-45.
Parthasarathy R, Joseph J. Study on the changes in the levels of membrane-bound ATPases activity and some mineral status in cyhalothrin-induced hepatotoxicity in fresh water tilapia Oreochromis mossambicus
. Afr J Environ Sci Tech 2011;5:98-103.
Chernysheva GV, Stoĭda LV, Amarantova GG, Kuz'mina IL. Effect of disseminated myocardial necrosis on ATPase activity, ca2+ transport, and lipid peroxidation in cardiac mitochondrial and microsomal membranes. Biull Eksp Biol Med 1980;89:563-5.
Kako K, Kato M, Matsuoka T, Mustapha A. Depression of membrane-bound Na+-K+-ATPase activity induced by free radicals and by ischemia of kidney. Am J Physiol 1988;254:C330-7.
Kerr JF, Wyllie AH, Currie AR. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972;26:239-57.
Sutton VR, Vaux DL, Trapani JA. Bcl-2 prevents apoptosis induced by perforin and granzyme B, but not that mediated by whole cytotoxic lymphocytes. J Immunol 1997;158:5783-90.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11]
[Table 1], [Table 2]