Zhentao Jiang, Xiaofeng Ma, Shengquan Lin, Jiankai Tan and Zhaobing Li*

Department of Cardiology, Nanhua Hospital Affiliated to Nanhua University, Hengyang, Hunan Province 421002, China

*Corresponding Author:
Zhaobing Li
Department of Cardiology, Nanhua Hospital Affiliated to Nanhua University, Hengyang, Hunan Province 421002, China
E-mail: 17026340352@163.com
Date of Received 16 June 2021
Date of Revision 08 February 2022
Date of Acceptance 28 September 2022
Indian J Pharm Sci 2022;84(5):1303-1308  

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DOI: 10.36468/pharmaceutical-sciences.1027

Abstract

To research the effects of melatonin on cardiac function, myocardial cell metabolic stress and apoptosis in heart failure after myocardial infarction rats. Thirty specific-pathogen-free male Sprague Dawley rats were randomly divided into sham operation group (n=10), model group (n=10) and melatonin group (n=10). The model group and the melatonin group were constructed by ligating the left anterior descending coronary artery, while not in the sham operation group. Melatonin group (10 mg/kg) was intraperitoneally injected with melatonin (Sigma, United States of America) 30 min before operation. The sham operation group and the model group were injected intraperitoneally with the same amount of normal saline. The cardiac function of rats was measured by left ventricular ejection fraction, left ventricular fractional shortening, left ventricular end diastolic diameter and left ventricular end-systolic diameter. The myocardial cell apoptosis in ischemic areas of each group was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. The expression of related proteins, measure myocardial fibrosis and determine the oxidative stress parameters (nicotinamide adenine dinucleotide phosphate oxidases activity, superoxide anion, malondialdehyde and superoxide dismutase) were detached via Western blot. Left ventricular fractional shortening % and left ventricular ejection fraction % in the melatonin group were significantly increased than those in the model group (p<0.05), while left ventricular end diastolic diameter and left ventricular end-systolic diameter in the melatonin group were significantly decreased (p<0.05). The apoptotic cardiomyocytes number in melatonin group was significantly less in model group (p<0.05). The levels of B-cell lymphoma 2 associated X-protein and caspase-3 protein in rat cardiomyocytes in melatonin group were significantly lower than those in model group (p<0.05). The proportion of sirius red staining in the melatonin group was significantly less than that in model group (p<0.05). Collagen I/III and transforming growth factor of rat cardiomyocytes in melatonin group-beta was significantly down-regulated than that in the model group (p<0.05). Nicotinamide adenine dinucleotide phosphate oxidase activity, superoxide anion and malondialdehyde level of rats in melatonin group were significantly decreased than those in model group, while superoxide dismutase activity was significantly increased than that in model group (p<0.05). Melatonin can improve heart failure and cardiac fibrosis by restraining oxidative stress in heart failure rats. Nicotinamide adenine dinucleotide phosphate oxidases 1 play an important role in regulating the role of melatonin in reducing cardiac fibroblasts fibrosis.

Keywords

Melatonin, heart failure, myocardial infarction, cardiac function, metabolic stress, apoptosis

Heart Failure (HF) is caused by the structural or functional cardiac disease and it is featured as the cardiac fibrosis[1]. Cardiovascular fibrosis is a significant driving factor for Chronic Heart Failure (CHF) and the excessive cardiac fibrosis can lead to the large infarct scar, cardiac insufficiency and heart dilation[2]. The Cardiac Fibroblasts (CF) are currently considered to be the main source of cardiac fibrosis after ischemic injury[3]. CF has important role in the remodeling after infarction, which can eventually lead to pathological cardiac fibrosis and HF[4]. CF is a sign of HF and there is no efficient therapeutic drug. Melatonin is one kind of neurohormones and maintains the Circadian Rhythm (CR)[5]. Abundant researches indicate that it has the antioxidant activity[6]. Clinical evidence showed that melatonin plays a protective role in regulating hypertension, pulmonary hypertension, HF, atherosclerosis, arrhythmia and Ischaemia- Reperfusion Injury (IRI)[7]. Melatonin is known to protect the heart by activating the G protein coupled membrane receptor, tumor necrosis factor receptor, toll like receptor, riboretinoic acid receptor and others[8]. Oxidative stress is considered to play a key role in pathological cardiac remodeling and the transformation of HF. Collagen production induced by the Angiotensin II (Ang II) is caused by the production of the Reactive Oxygen Species (ROS) in the adult rat CF. Ang II activates ROS-sensitive kinase, a key protein in cardiac fibrosis remodeling. However, it is not clear whether the melatonin administration inhibits the oxidative stress to alleviate the HF mice’s cardiac fibrosis induced by Myocardial Infarction (MI). This research aims to discuss the anti-fibrosis effect of melatonin on HF rats induced by MI and whether oxidative stress participated in melatonin in reducing HF.

Materials and Methods

Experimental animals and grouping:

Thirty Specific-Pathogen-Free (SPF) male Sprague Dawley (SD) rats were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine, weighing 250-300 g, adaptive feeding for 7 d under SPF environment with humidity (45 %±5 %) and temperature (20°±2°). Regular CR was provided, as well as feeding and drinking freely. The rats were fasted for 12 h and they were divided into sham-operated group (n=10), model group (n=10) and melatonin group (n=10). The model group and melatonin group established rat MI model by ligating the left anterior descending coronary artery, while it was not in the sham-operated group. Melatonin (Sigma, United States of America (USA)) was injected into the abdominal cavity for the melatonin group (10 mg/kg) 30 min before the operation. An equal amount of saline was injected intraperitoneally in other two groups.

Establishment of HF rat model:

The experimenters adopted the pentobarbital sodium to anesthetize the rats through the intraperitoneal injection and it was fixed in the supine position, connected the limbs to the Electrocardiogram (ECG), cut the neck skin, separated the muscle layer, exposed the trachea and connects the small animal ventilator (Chengdu Teichmann). The thorax was opened along the left 3rd and 4th intercostal spaces. Then, the 0 gauge guide wire was used to ligate the coronary artery along the left artery at 1 mm below the left atrial appendage. Heart beat weakened, myocardium turned white, myocardial tissue appeared cyanosis with ST elevation. It meant successful occlusion of the anterior coronary descending artery. Stitch the chest and anti-infection treatment was performed via intramuscular injection of penicillin within 12 h after surgery to record the 7 d survival of rats.

Cardiac function test:

The rat cardiac function was determined by M-type echocardiography with the small animal ultrasound instrument. Left Ventricular Ejection Fraction (LVEF), Left Ventricular Fractional Shortening (LVFS), Left Ventricular End Diastolic Diameter (LVEDD) and Left Ventricular End-Systolic Diameter (LVESD) were measured via BL-420E+.

Determination of myocardial apoptosis:

The experimenters killed the rats immediately after the experiment, removed their hearts separated the left ventricle. Myocardial apoptosis in the ischemic area was detected with the Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) kit by strictly following the instructions strictly. The experimenters observed and photographed the sections under a fluorescence microscope. TUNEL positive cells, which were apoptotic cells, showed the yellow green fluorescence, whereas TUNEL negative cells were normal without the fluorescence. The apoptosis level in myocardial cell was calculated for each group.

Detection of related protein expression by Western blotting:

The rats were killed and the left ventricle was isolated. According to the instructions of TUNEL staining kit (Beyotime Biotechnology), the number of cardiomyocyte apoptosis in ischemic area was detected. The sections were observed and photographed under a fluorescence microscope. The level of cardiomyocyte apoptosis in each group was calculated.

Sirius red staining:

The heart slice (5 μm) was inspected through the sirius red staining, to measure the fibrosis of the myocardial cell. 3-5 slices were selected for each animal (30-50 cells at each vision) to observe under the optical microscope. The Image-Pro Plus software was adopted to analyze the image.

Parametric measurement of oxidative stress:

We detached the Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidases (NOX) activity through enhanced chemiluminescence of lucigenin in CF. For the measurement of the superoxide anion, the superoxide anion level in CF was determined by chemiluminescence derived from glossin. An Enzyme-Linked Immunosorbent Assay (ELISA) kit was adopted to measure the level of Malondialdehyde (MDA). The micro plate reader was used to measure Superoxide Dismutase (SOD) according to the description.

Statistical method:

The data of the present study were analyzed by Statistical Package for Social Sciences (SPSS) 22.0. All data with normal distribution were compared and represented by (x?±s). One-way Analysis of Variance (ANOVA) was adopted for comparisons among multiple groups and the Student–Newman–Keuls (SNK)-q test for pairwise comparisons. The enumeration data was expressed in percent (%) and the comparison between the groups was made by Chi-square (χ2)-test. The statistical results were statistically significant if p<0.05.

Results and Discussion

After 7 d modelling, both LVEF % and LVFS % in the model group were lower than those of the sham operated group. However, LVEDD and LVESD increased significantly in the model group (p<0.01). LVEF % and LVFS % in the melatonin group were higher than those in the model group (p<0.05), while LVEDD and LVESD in the melatonin group were lower (p<0.05) as shown in Table 1.

Grouping n LVEF (%) LVFS (%) LVEDD (cm) LVESD (cm)
Sham operated group 10 84.85±4.36 46.65±3.65 0.72±0.06 0.37±0.05
Model group 10 45.48±3.29a 20.53±2.18a 0.94±0.05 0.76±0.08
Melatonin group 10 63.64±3.42ab 28.82±23.25ab 0.83±0.06 0.49±0.06
t   13.482 12.658 5.625 6.352
p   0.001 0.001 0.001 0.001

Table 1: Cardiac Function Indicators of RATS (x?±s)

Compared to the model group, the number of apoptotic cardiomyocytes in the sham operated group was decreased (p<0.05); meanwhile it was less in the melatonin group (p<0.05) as shown in Table 2.

Grouping n Number of TUNEL apoptotic cells (n/mm2)
Sham operated group 10 15.65±1.14
Model group 10 326.84±39.85a
Melatonin group 10 107.55±8.62ab
t   365.71
p   0.001

Table 2: Comparison of the Number of Tunel-Stained Apoptotic Cardiomyocytes of RATS among Groups

The B-Cell Lymphoma-2 (BCL-2) protein of rat cardiomyocytes in the model group was obviously down-regulated than that in the sham operated group (p<0.05). The BCL-2 protein of rat cardiomyocytes in the melatonin group was significantly up-regulated than that in the model group (p<0.05). BAX and caspase-3 protein of rats in the model group were significantly higher than those in the sham operated group (p<0.05); BAX and caspase-3 protein in the melatonin group were decreased compared with model group (p<0.05) as shown in Table 3.

Grouping n BCL-2 BAX Caspase-3
Sham operated group 10 0.13±0.06 0.17±0.03 0.42±0.08
Model group 10 0.06±0.03a 0.52±0.16a 1.09±0.13
Melatonin group 10 0.09±0.04ab 0.33±0.14ab 0.69±0.09
t   1.65 4.58 5.47
p   0.045 0.001 0.001

Table 3: Expression Comparison OF BCL-2, BAX and CASPASE-3 Protein Tables in RAT Cardiomyocytes (x?±s)

Compared to model group, the proportion of sirius red staining was increased in the sham operated group and model group (p<0.05) as shown in Table 4.

Grouping n Proportion of sirius red staining (%)
Sham operated group 10 6.57±1.05
Model group 10 38.72±6.84a
Melatonin group 10 18.26±3.24ab
t   17.65
p   0.001

Table 4: Cardiac Fibrosis of Each Group of the MICE

Collagen I/III and the Transforming Growth Factor Beta (TGF-β) protein of the rat cardiomyocyte in the model group were significantly raised from the sham-operated group (p<0.05); collagen I, collagen III and TGF-β protein of rat cardiomyocyte in the melatonin group were significantly reduced than those in the model group (p<0.05) as shown in Table 5.

Grouping n Collagen I Collagen III TGF-β
Sham-operated group 10 0.48±0.06 0.39±0.05 0.29±0.05
Model group 10 1.08±0.11a 0.89±0.23a 0.84±0.10
Melatonin group 10 0.78±0.10ab 0.56±0.17ab 0.56±0.09
t   6.58 5.64 5.18
p   0.001 0.001 0.001

Table 5: Expression of Collagen I/III And TGF-? of RAT Cardiomyocytes in each Group (x?±s)

The NOX activity, superoxide anion and MDA level of the rat’s cardiomyocyte in the model group were raised than that of the sham-operated group and the SOD activity was significantly lower than the operation group (p<0.05); NOX activity, superoxide anion and MDA level of the rats in the melatonin group were reduced than that of the model group and the SOD activity was up-regulated than that of the model group (p<0.05) as shown in Table 6.

Grouping n NOX activity (MLU/min/mg) Superoxide anion (MLU/min/mg) MDA (nmol/mg) SOD (U/mg)
Sham-operated group 10 8.97±0.86 8.62±1.20 76.54±8.21 375.65±40.61
Model group 10 17.65±1.95a 17.55±2.01a 213.28±32.69 176.53±18.64
Melatonin group 10 12.43±1.34ab 11.27±1.32ab 131.54±14.12 234.73±26.23
t   9.546 9.875 18.273 16.584
p   0.001 0.001 0.001 0.001

Table 6: Comparison of Oxidative Stress Levels in RAT Cardiomyocytes

Melatonin is a hormone. It not only maintains CR, but protects the heart from the continuous ischemia-reperfusion injury[9]. It is observed from clinical research that the reduction in serum melatonin level is related to the increase of the heart attack and other acute cardiovascular events[10]. The acute MI is related to the lack of serum melatonin at night and the increase of oxidative stress, which indicates that melatonin, clears free radicals formed during acute MI at night. The patients with MI have the high-level oxidized Low-Density Lipoprotein (LDL), but the serum melatonin level is relatively low. It also shows that the heart attack is related to the level of melatonin in the serum[11]. Before operation, increased melatonin level leads to IRI and the decrease of inflammatory markers intercellular adhesion molecule 1, Interleukin 8 (IL-8), Troponin I (TnI). Wherein, the 20 d melatonin treatment may produce significant anti-ischemic and anti-angina effects and normalize oxidative stress in individuals with Cardiovascular Disease (CVD)[12]. It is also reported that people with the vascular diseases have a significantly reduced level of melatonin at night and the reduced melatonin secretion is associated with a raised risk of high Body Mass Index (BMI) in women[13]. In addition, supplemented melatonin could improve the ejection fraction and reduce cardiac TnI and IL-1 in plasma β and caspase-3 enzyme, which significantly inhibits IRI in the myocardium IRI. It indicates that the melatonin could alleviate the IRI related to the cardiac surgery[14]. In addition, compared with the control group, the circulating melatonin levels are also low in patients with acute MI and dilated cardiomyopathy, which may be associated with the myocardial injury and cardiac output of the patients with the dilated cardiomyopathy[15]. The HF impairs the ability of the ventricle filling or ejection to resulting in progressive loss of contractility and ejection fraction, ventricular wall thinning, humoral homeostasis, activation of cytokines and neurohumors[16]. In this research, the LVEF % and LVFS % of the mice in the modeling group after modeling for 7 d are significantly lower than that of the sham operated group, LVEDD and LVESD increase significantly in the model group, while melatonin administration inhibits the increase. These results indicate that melatonin could alleviate the HF caused by MI and improve cardiac insufficiency of HF, for the formation of CF in HF, it could develop to HF[17]. At present, it is still unclear for the relationship between the melatonin and cardiac fibrosis of HF. In the current research, the result indicates that collagen I/III and TGF-β level increase in the MI rats’ heart, while the melatonin alleviates the increase. These results indicate that the CF of MI mice increases and the melatonin improves the cardiac fibrosis of HF rat.

As an important molecule in the organism, ROS is involved in lots of signal pathways. As a key factor of the organic damage, the oxidative stress is associated with various diseases[18], including CF[19]. Increased apoptosis and mitochondrial ROS production in hypoxic and deoxygenated cells reduce the exposure to short N-terminal gelatin fragments 2-15[20]. The antioxidant has been shown to improve cardiac function and produce the antifibrosis effect[21,22]. Currently, it is found that the NOX activity, superoxide anion and MDA levels increase in the hearts of MI rats and CFs and the SOD level decreases, while melatonin reverses these changes. Melatonin could reduce oxidative stress to inhibit the cardiac fibrosis in HF. To sum up, the melatonin could inhibit the HF rat’s oxidative stress, to improve the HF and cardiac fibrosis. NOX1 plays an important role in melatonin of CF fibrosis.

Conflict of interests:

The authors have no conflict of interest to report.

References