INTRODUCTION:
Cardiovascular diseases represent a significant aetiology of mortality in chronic alcoholics worldwide. Cardiomyopathy (CM) means “disease of the heart muscle’’ that damages the muscle tone of the heart and reduces its ability to pump blood to the rest of the body. In CM the impairment ofmyocardial muscle occurs due to various causes, such as genetic and sporadic mutations of muscle proteins, as well as external factors such as hypertension, ischemia, and inflammation, usually leading to heart failure and sudden cardiac death1.CM is a major contributor to heart failure and the most frequent cause of heart transplantation. It is fatal because it often goes unrecognized and untreated2. Although it can be inherited, dilated cardiomyopathy (DCM) is primarily brought on by a number of different conditions, such as severe coronary artery disease, alcoholism, thyroid illness, diabetes, viral infections of the heart, abnormalities of the heart valves, and drug-induced CM. (e.g., doxorubicin)3. Alcohol-related toxicity causes non-ischemic dilated cardiomyopathy, which is marked by loss of contractile function and enlargement of the cardiac ventricles. Alcoholic cardiomyopathy (ACM) contributes to approximately one-fifth of all sudden cardiac death4. ACM necessitates a more prolonged exposure to alcohol intake and is seldom caused by short-term ethanol consumption5. In general, alcoholic patients who have consumed alcohol for more than 5 years at a rate of more than 90g/day (about seven to eight standard drinks) are at risk for developing asymptomatic ACM. The prevalence of ACM among patients of heart failure or DCM varies from 4 to 40 percent or more, depending upon the characteristics of population and the threshold alcohol consumption used to identify ACM6. ACM is associated with a number of adverse histological, cellular and structural changes within the myocardium. Different mechanisms have been hypothesized for ACM including oxidative stress via stimulation of stress signalling by increasing O2ˉ generation, aggregation of triglycerides, reformed fatty acid extraction, reduced myofilament Ca2+ sensitivity, increased cardiac lipid peroxidation and damaged protein synthesis7. Cardiac apoptosis contributing in destruction of cardiomyocytes plays a major role in the pathogenesis of ACM8. The major ethanol metabolite acetaldehyde is suspected to play a culprit role in the onset of this myopathic state9.
The PI3K/Akt Pathway is a signal transduction pathway that promotes cell survival, growth, migration, proliferation and metabolism (lipid and glucose); cell cycle progression; cardiomyocyte contractility; angiogenesis in response to extracellular signals10. Chronic ethanol intake has been documented to decrease the phosphorylation of PI3K/Akt survival kinase pathway11. Downregulation of PI3K/Akt may be a key factor for destruction of cardiomyocytes due to apoptosis and free radical generation, which ultimately results in ACM12. Additionally, it has been proven that chronic alcohol consumption activates protein tyrosine phosphatases (PTPases)13. PTPases (PTP1B) are a class of enzymes that control a variety of biological functions and has been identified as a negative regulator of PI3K/Akt signaling pathway, preventing the activation of the VEGF receptor. PTPases increase proapoptotic genes including Caspase, BIM, BAD, and BCL-2 and decrease the amounts of antioxidant enzymes like catalase, glutathione peroxidases, and super oxide dismutase by blocking the function of the survival pathway PI3K/Akt14. In addition, downregulation of the PI3K/Akt pathway results in higher levels of iNOS (inducible nitric oxide synthase), which increases nitric oxide (NO) production. Because of this rise in NO, the left ventricle's (LV) ability to contract properly is compromised, the LV stroke volume is decreased, and the cardiomyocytes are subjected to cytotoxic consequences15. These PTPase-mediated modulation may result in myocardial cell death and downregulation of endothelial function, which can impede ventricular function and may be the cause of alcohol-induced cardiomyopathy. Therefore, using SOV:Sodium orthovanadate (Protein tyrosine phosphatases inhibitor) and SMT:S-methyl isothiourea sulfate (iNOS inhibitor), the present study was designed to investigate the role of PTPases and possible downstream involvement of inducible nitric oxide synthase (iNOS) in experimental ACM.
MATERIALS AND METHODS:
Drugs and chemicals:
Sodium orthovanadate (SOV) and S-methyl isothiourea sulfate (SMT) were obtained from Sigma-Aldrich Ltd., Bangalore, India. All other chemicals and reagents used were of analytical grade.
Experimental animals:
Young male Wistar rats weighing 150g to 250g were procured from the Central Animal House facility of the ISF College of Pharmacy Moga and were acclimated to a lab environment before study. The experimental protocol was approved by the Institutional Animal Ethics Committee (CPCSEA Approval No: ISFCP/CPCSEA/M17/P294) and was carried out in accordance with the guidelines of the Indian National Science Academy (INSA) for the use and care of experimental animals.
Animal Model and experimental protocol:
ACM was induced by regular administration of 5ml of 20% alcohol per 100g (7.9g/kg) for 60 days by intragastric intubation (po). The animals were divided into six groups containing six rats in each group. Normal control rats received glucose solution equivalent to the calorific value of ethanol (15g of glucose/kg, (p.o) i.e., 5ml of 30% glucose solution/100g).
Figure 1: Experimental design of study
After 45 days of alcohol administration drug treatment was started i.e., from 46th day to 60th day, treatment groups 3,4, and 5 were given three different doses of sodium orthovanadate (SOV) i.e. 2.5mg/kg (very low dose), 5 mg/kg (medium dose) and 10mg/kg (high dose) respectively and Treatment group 6 was administered with SOV (10mg/kg. P.o) + SMT (S-methylisothiourea) (5mg/kg; i.p)16 (Table 1). Rats were used for pharmacological evaluation after 60th day of study. (Figure 1).
Table 1: Experimental Protocol (In each group n=6)
|
Group |
Name/Treatment Schedule |
|
1 |
Normal Control Glucose solution (NC) (15g/kg p.o) |
|
2 |
Alcoholic cardiomyopathy (ACM) Ethanol (7.9g/kg., p.o) |
|
3 |
ACM + SOV (SOV: Sodium orthovanadate) (2.5mg/kg. p.o) |
|
4 |
ACM + SOV (5mg/kg. p.o) |
|
5 |
ACM + SOV (10mg/kg. p.o) |
|
6 |
ACM + SOV (10mg/kg. p.o) + SMT (S-methylisothiourea) (5 mg/kg; i.p) |
Hemodynamic and Morphological assessments:
After 60 days of the study period, the rats were sacrificed by spinal dislocation, the thorax was opened and the heart was excised after intraperitoneal heparinization (500U/kg body weight) and placed into chilled, heparinized to decrease work load of the heart. The heart was immediately mounted on digital Ludendorff’s apparatus (RADNOTI, Monrovia, CA, USA) [27] and perfused with Kreb’s-Henseleit solution (NaCl 118Mm; KCl 4.7 Mm; CaCl2 2.5Mm; MgSO4.7H2O 1.2Mm; NaHCO3 25Mm; KH2PO4 Mm; C6H12O6 11Mm), gassed with 95% O2-5% CO2, pH7.4, maintained at 37˚C. For the measurement of cardiac functions, a double distilled water-filled latex balloon was inserted through the mitral valve into the left ventricle, and left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP) (mmHg), rate of left ventricular pressure increase (dp/dtmax) andrate of left ventricular pressure decrease (dp/dtmin) were assessed using pressure transducer (BIOPAC MP100 System, California, USA). The coronary effluent volume was measured at the various time intervals for a total of 120 min. Coronary blood flow, in ml/min /g was defined as the total volume collected during the perfusion interval divided by the time, normalized by the heart wet weight (g), which was measured at the beginning of the experiment17. Further MABP (mmHg) was measured using tail cuff method (BIOPAC System, California, USA)18. After sacrificing animals LVW/BW (Left ventricular weight: Body weight) was estimated by measuring the weight of Left ventricle with the interventricular septum and was expressed as mg/g of body weight for determining cardiomyopathy in respective groups of animals19.
Biochemical Assessments:
a) Protein content:
The left ventricle was separated and stored at -80˚C refrigerator for quantitative estimation of biochemical parameters. The protein composition of the left ventricle was ascertained using Lowry's method. Briefly 0.2 M potassium phosphate buffer containing 1 mM EDTA and mercaptoethanol was used to homogenize left ventricular tissue in a glass-Teflon homogenizer at 4˚C. Five test tubes were filled with 0.2ml of the Bovine Serum Albumin (BSA) working standard, and 1ml of it was made with distilled water. The test tube with 1ml distilled water served as blank. 4.5ml of the reagent I was added, and the mixture was incubated for 10 minutes. Next, 0.5ml of reagent ΙI was added, and the mixture was incubated for 30minutes. Using a standard Bovine serum albumin graph as a reference, the absorbance of the resulting sample was measured at 750 nm. Calculated total left ventricular protein content was represented as mg/g of left ventricular weight20.
b) Collagen content:
The LV collagen content was estimated by measuring hydroxyproline concentration. In brief the LV tissue was homogenized in 4ml of 6N HCl and then hydrolysed at 110˚C for 16 hrs. After dissolving the dried hydrolysate in 1000µl of 50% isopropanol, 1.2ml of 50% isopropanol was added to 30µl of mixture, and it was incubated for 10minutes at room temperature with 0.2 ml of 0.84% chloramine-T in acetate citrate buffer (pH 6.0; citric acid -5.5g, sodium acetate -57g, tri-sodium citrate.2H2O-37.5g, and n-propanol -385ml to bring the volume up to 1000ml with distilled water). Then Ehrlich’s reagent was added and the mixture was incubated at 60˚C for 25 min. The absorbance of the sample solution was measured at 560nm wavelength. The hydroxyproline content in 200mg of LV tissue was calculated using standard curve of 4-hydroxy-L- proline 21.
Collagen content was calculated as
· Hydroxyproline/gm of LV = Concentration of hydroxyproline/ml of homogenate × 33.3 × 5
· Collagen content (mg/g of LV) = Concentration of hydroxyproline (per gm of LV) × 6.94
c) Estimation of total LV cholesterol:
The total LV cholesterol was estimated by cholesterol oxidase peroxidase (CHOD-PAP) method using commercially available kit (Coral clinical system, Goa, India). 1000µl of cholesterol reagent was added to 50µl of re-dissolved lipid extract in dioxane/isopropanol mixture, 50µl of standard cholesterol (200mg/dl) and 50 µl of purified water to prepare test, standard and blank, respectively. All test tubes were incubated at room temperature for 15min. The absorbance of test and standard samples were noted against blank at 505 nm spectrophotometrically. The intensity of colour formed is directly proportional to the amount of cholesterol present in the sample.
The serum total cholesterol was calculated as:
Absorbance of Test
Total cholesterol (mg/dl) = --------------------------- × 200
Absorbance of Standard
d)Estimation of nitrite concentration:
The nitrite concentration was measured by Griess reaction. The accumulation of nitrite in the supernatant, an indicator of the production of nitrite oxide was determined by a colorimetric assay with Griess reagent (0.1% N-(1-napthyl) ethylene diaminedilhydrochloride, 1% sulphanilamide, and 5% Phosphoric acid). Equal volumes of the supernatant and the Griess reagent were mixed and the mixture was incubated for 10 min at room temperature in the dark. The absorbance was measured at 540nm using spectrophotometer. The concentration of the nitrite in the supernatant was determined from the sodium nitrite standard curve and express as in μ mole per gram tissue22.
Molecular Assays:
a) Estimation of tumor necrosis factor –alpha (TNF-α) levels:
TNF-α level was estimated by using rat TNF- αkit (Ray Bio, Rat TNF-alpha ELISA kit protocol) which uses a microtiter plate reader (450nm). Concentrations of TNF-α were calculated from plotted standard curve. The reagent and standard dilutions were prepared as suggested by the manufacturer’s instructions. Absorbance of each well was read in an ELISA reader set to 450nm23.
b) Estimation of iNOS expression:
Estimation of iNOS expression was done using PCR technique, briefly 100mg of tissue homogenae was taken in 1ml of trizol reagent and incubated for 5 min at 20˚C. 0.2ml of chloroform was added to the homogenate, incubated at 20˚C for 3min. The mixture was centrifuged at 10,000g for 15min at 4˚C, the upper aqueous phase was isolated and 0.5 ml isopropyl alcohol was added to precipitate RNA. The sample was centrifuged at 10,000 g for 15 min at 4˚C to form the gel-like pellet of RNA in the tube. The supernatant was removed, RNA pellet was washed with 75% ethanol, mixed, and centrifuged at 7,500g for 5 min at 4˚C and RNA pellet were briefly vacuum dried for 5-10min. The
RNA was quantified by ultraviolet absorbance spectrophotometry to ascertain A260/A280 ratio < 1.6 and dissolved in RNA free water. The 5μl reverse primer
was added to crude RNA, 29μl reverse transcriptase buffer incubated for 10 min at 65˚C and cooled on ice. 16 U AMV transcriptase (10U/μL) and 5μl 10nM dTNB mixture were added, incubated at 42˚C for 1hr and 100 mM Tri’s buffer (pH- 7.5) was added to synthesized single stranded cDNA. 5μl forward primer, backward primer, 10 X amplification buffer, 0.9μl of Taq DNA polymerase enzyme (3U/μl) and 70.1μl RNA free water in PCR tube and overlaid with 100μl mineral oil. 24 PCR cycles of GADPH (94˚C for 1 min, 62˚C for 1min, 72˚C for 1 min) for 30 PCR cycle of iNOS (94˚C for 1 min, 62˚C for 1min, 72˚C for 1 min) followed by 1 cycle at 57˚C for 2 min and 72˚C for 7 min were performed using 1 half of the reverse transcription mixture (Biorad, MJ Mini Thermal cycler), sense and antisense primers for iNOS: (5’–CGAAACGCTTCACTTCCAA -3’ and 5’ TGAGCCTATATTGCTGTGGCT-3’ resp.) and for GADPH (5’-TCCCTCAAGATTGTCAG CAA-3’ and 5’-GATCCACAACGGATACATT-3’ resp.) were used. The PCR products so obtained were analyzed on ethidium bromide-stained agarose (1.5%) gel on Gel electrophoresis (Biorad). The iNOS and GADPH products were quantified using the image (Gel Doc EZ image, Biorad) and the amount of iNOS was normalized with respect to amount of GADPH product24,25.
Statistical Analysis:
All results comprise Values that are expressed as mean ± SD; aP <0.05 vs. normal control; bP <0.05 vs. ACM; cP <0.05 vs. SOV (2.5mg/kg); dP <0.05 vs. ACM + SOV (10mg/kg) + SMT (5mg/kg). Various Hemodynamic parameters i.e., LVDP, LVEDP, dp/dtmax, dp/dtmin, LVW/BW, MABP, CFR and biochemical parameters i.e., LV protein, LV collagen, LV cholesterol and nitrite levels and molecular assays i.e., TNF-α levels and iNOS/ GAPDH ratio were statistically analyzed using one- way ANOVA followed by Tukey’s multiple comparison tests. The p values of less than 0.05 were considered to be statistically significant.
RESULTS:
Hemodynamic and morphological interventions:
Alcohol administered to rats for 8 weeks significantly decreases LVDP, dp/dtmax, dp/dtmin, and Coronary blood flowas compared to normal control rats. However, treatment with SOV, a PTPase inhibitor (2.5mg/kg, 5mg/kg, 10mg/kg., p.o) significantly increased LVDP, dp/dtmax, dp/dtmin, and Coronary blood flow in alcoholic cardiomyopathic rats. Furthermore, administration of SMT, an iNOS inhibitor (5mg/kg., i.p) with SOV (10mg/kg., p.o) significantly increased the ameliorative effect of SOV (Figure 2-5). Beside it LVEDP, MABP and LVW/BWwas significantly increased in alcohol administered rats as compared to normal control rats. Treatment with SOV (2.5mg/kg, 5mg/kg, 10mg/kg. P.o) significantly decreases LVEDP, MABP and LVW/BW in alcoholic cardiomyopathic rats. Moreover, administration of SMT (5mg/kg., i.p) with SOV (10mg/kg., p.o) significantly increased the protective effect of SOV (Figure 6-8).
Figure 2. Effect of pharmacological interventions on LVDP
Figure 3. Effect of pharmacological interventions on dp/dtmax
Figure 4. Effect of pharmacological interventions on dp/dtmin Figure
5. Effect of pharmacological interventions on Coronary blood flow
Figure 6. Effect of pharmacological interventions on LVEDP
Figure 7. Effect of pharmacological interventions on MABP.
Figure 8. Effect of pharmacological interventions on LVW/BW.
Biochemical interventions:
8 weeks of alcohol administration leads to significant decrease in LV protein content as compared to normal control rats. Treatment with SOV (2.5mg/kg, 5mg/kg, 10mg/kg., p.o) significantly improved the LV protein content in alcoholic cardiomyopathic rats. Furthermore, administration of SMT (5mg/kg., i.p) with SOV (10mg/kg., p.o) significantly potentiate the ameliorative effect of SOV (10mg/kg., p.o) (Figure 9). Further LV collagen content, LV cholesterol content and nitrite levels were significantly increased after alcohol administered of 8 weeks as compared to normal control rats. However, treatment with SOV (2.5mg/kg, 5mg/kg, 10mg/kg., p.o) significantly attenuated the increased LV collagen content in alcoholic cardiomyopathic rats. Further, administration of SMT (5mg/kg., i.p) with SOV (10mg/kg., p.o) significantly increased the ameliorative effect of SOV (Figure 10-12).
Figure 9. Effect of pharmacological interventions on LV Protein content
Figure 10. Effect of pharmacological interventions on LV Collagen content
Figure 11. LV Cholesterol content
Figure 12. Nitrite levels
Effect of pharmacological interventions onTNFα levels and mRNA expression of iNOS:
Continuous administration of alcohol for 8 weeks caused significant increase in TNFα levels and expression ratio of iNOS/GAPDH as compared to normal control rats. Treatment with sodium orthovanadate (2.5mg/kg, 5mg/kg, 10mg/kg., p.o) significantly attenuated the increased TNFα levels and expression ratio of iNOS/GAPDH in alcoholic cardiomyopathic rats. Further, administration of SMT (5mg/kg., i.p) with SOV (10mg/kg., p.o) significantly increased the beneficial effect of SOV (Figure 13-14).
Figure 13. TNF-α levels
Figure 14. mRNA expression of iNOS levels
DISCUSSION:
Alcoholic cardiomyopathy (ACM) is
characterized by cardiac hypertrophy, impaired ventricular contractility, and
decreased cardiac output26,27. This work demonstrates the
pharmacological and therapeutic potential of sodium orthovanadate (SOV), a
PTPases inhibitor, in treating alcohol-induced cardiomyopathy in Wistar rats.
The study used an isolated rat heart preparation that was retrogradely perfused
on lagendorff's equipment since functioning heart preparation is unaffected by
changes in system circulation. Furthermore, dilated cardiomyopathies are
associated with both systolic and diastolic dysfunction28,29.
Cardiovascular illness, hypertension, angina, irregular cardiac rhythm, and
alcoholism are the primary causes of the reduction in systolic function which
eventually rises end-diastolic and end-systolic volumes30,31. Early
compensation for systolic dysfunction and decreased cardiac output is
accomplished by increasing the stroke volume, heart rate, or both (cardiac
output = stroke volume 
heart rate), which is also accompanied by
an increase in peripheral vascular tone. Coronary blood flow(ml /min/g)
defined as the total volume collected during the reperfusion interval divided
by the time, indicated by the heart wet weight (g), which was measured at the
beginning of the experiment was found to be significantly reduced in alcoholic
cardiomyopathic rats as compared with normal rats. Dilator reserve of the
coronary microvasculature was also diminished in rats with DCM32,33.
Chronic ethanol consumption has been shown to reduce the phosphorylation of
PI3K/Akt survival kinase, which eventually results in ACM34,35.
However present study indicated the possible role of Protein tyrosine
Phosphatases (PTPases) in dephosphorylation of PI3K/Akt survival kinase pathway
that removes phosphate groups from phosphorylated tyrosine residues on proteins
and consequently leading to ACM by various mechanisms as shown in Figure 15.
Next to it, the myocardial vasculature's endothelium and vascular smooth muscle
cells contain the inducible nitric oxide synthase (iNOS), whose expression is
generally linked to ACM and eventually heart failure. The iNOS generates a
prolonged release of large amounts of NO which may be cytotoxic, inhibits
myocyte contractility and causes the reduction in LV stroke volume36,37.
However, data obtained by combining the dose of SOV and SMT (group 6), which
demonstrate a considerable reduction in ACM as compared to alone SOV dose
(group 5), confirmed the participation of iNOS and NO in PTPase-induced ACM.
Additionally, TNF-α can influence the production of nitric oxide synthase
by macrophages, cardiac myocytes, and other cells, which can directly and
indirectly lower heart performance38,39,40. The reduction in
elevated TNF-α in ACM rats caused by SOV and its combination with SMT
indicates the role of PTPases in the cytokine-induced activation of iNOS in the
myocardium.In summary, the research suggests that PTPases have a role in
Alcoholic Dilated Cardiomyopathy by means of multiple downstream pathways, such
as AKT/Pi3k, TNF-α, iNOS production, and NO-induced inflammation.
Figure 15. Proposed role PTPases, iNOS and inhibitors in ACM
CONCLUSION:
The current study provides evidence for potential involvement of Protein Tyrosine Phosphatase (PTPase) in alcoholic cardiomyopathy. In Wistar rats, alcoholic cardiomyopathy is lessened by sodium orthovanadate, a PTPases inhibitor, and SMT, an iNOS inhibitor. This effect may be attributed to activation of the PI3K/Akt survival pathway and a reduction in iNOS levels. PTPase may therefore be a promising molecular target for reducing ACM.
CONFLICT OF INTEREST:
The authors have no conflicts of interest regarding this investigation.
ACKNOWLEDGMENTS:
The authors would like to thank ISF college of Pharmacy, Moga and Chandigarh College of Pharmacy, Landran Punjab for providing facility for conducting this research.
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Received on 25.11.2023 Modified on 22.03.2024
Accepted on 27.05.2024 © RJPT All right reserved
Research J. Pharm. and Tech 2024; 17(8):3691-3699.
DOI: 10.52711/0974-360X.2024.00575