Melatonin protected against myocardial infarction injury in rats through a Sirt6 -dependent antioxidant pathway

Background. The Sirt6 , one of the members of the sirtuin family, has been regarded as a key factor in the pathogenesis of myocardial infarction (MI) through its antioxidant defense mechanisms. A previous study reported that melatonin is an antioxidant drug that can act as an agent for cardioprotection in cardiac ischemia-reperfusion (I/R) injury. However, whether melatonin could protect against cardiac remodeling after myocardial injury via the Sirt6 -dependent antioxidant pathway remains unknown. Objectives. To explore the protective effects and the potential mechanisms of melatonin on MI-induced injury in rats. Materials and methods. A cardiac remodeling model was established through left coronary artery ligation surgery. The dose of melatonin was 10 mg/kg body weight. Four weeks after the treatment for 7 successive days, the infarct size and hemodynamic parameters were evaluated. The relative mRNA level and protein level of Sirt6 were also determined. Finally, the levels of oxidative stress, including reactive oxygen species (ROS) and superoxide dismutase (SOD), were measured, and the expression of nitric oxide (NO), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and their corresponding phosphorylation were evaluated. Results. After the treatment with melatonin, infarct size, the left ventricular end-diastolic diameter (LVEDd), and left ventricular end-systolic diameter (LVEDd) and minimum first derivative of developed pressure (min dP/dt) decreased, while left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS) and maximum first derivative of developed pressure (max dP/dt) increased in the melatonin-MI (MM) group compared to the placebo-MI (PM) group. Furthermore, the expressions of Sirt6 , both in mRNA and protein level, were significantly increased in the MM group treated with melatonin, as compared to the mela-tonin-control (MC) group treated with melatonin. In addition, melatonin enhanced SOD activity and reduced ROS levels. At the same time, we observed that the eNOS/NO signaling pathways were activated. Conclusions. Melatonin improved cardiac function through the Sirt6 -dependent antioxidant pathway in MI rats.


Background
Acute myocardial infarction (MI) is generally caused by coronary artery disease (CAD), which remains a leading cause of morbidity and mortality and imposes a tremendous economic burden worldwide. 1 After MI injury, the heart undergoes a series of cardiac wound-healing responses, including cardiomyocyte necrosis and apoptosis, inflammation, and cardiac hypertrophy and fibrosis. As cardiac wound healing progresses, patients suffer from post-MI left ventricular (LV) remodeling, heart failure (HF) or even death. 2,3 Oxidative stress plays an important role in the pathophysiology of various cardiovascular disorders, including atherosclerosis, cardiac hypertrophy, cardiomyopathy, HF, ventricular remodeling, and myocardial injury after ischemia followed by reperfusion (I/R). During the development of cardiac remodeling, oxidative stress aggravates heart disease into HF. [4][5][6] Elevated oxidative stress may result in peroxidation of proteins, DNA and lipids, triggering mitochondria-induced cell death pathways and contributing to I/R injury. 7 The Sirt6 is one of the 7 mammalian sirtuin homologs and it contains a domain with NAD + -dependent deacetylase activity. 8 Among sirtuin family members, both Sirt1 and Sirt6 have demonstrated cardioprotective abilities. However, no direct evidence can verify the antioxidative role of Sirt1 in protecting heart function, while Sirt6 has been shown to play a vital protective role in cardiomyocytes in I/R through its antioxidative ability. 9 Melatonin, a hormone synthesized and secreted by the pineal gland, is a well-known antioxidant. 10,11 Extensive evidence suggests that melatonin has profound cardioprotective effects. 12,13 For example, Hu et al. demonstrated that melatonin alleviated post-infarction cardiac remodeling and dysfunction by inhibiting Mst1. 14 Zhou et al. demonstrated that melatonin protected against I/R in myocardial infarction by suppressing platelet activation and function. 15 However, whether melatonin could protect against cardiac remodeling after myocardial injury via a Sirt6-dependent antioxidant pathway, remains unknown.

Objectives
The aim of the study was to explore the protective effects and the potential mechanisms of melatonin on MI-induced injury in rats.

The animals
All the experiments were performed with the approval of the Ethics Committee at Shandong University Qilu Hospital (approval No. KYLL-KS-2021180). Three-monthold male Wistar rats weighing 250-270 g were purchased from Charles River Laboratories (Beijing, China). The rats were randomly divided into 4 groups: placebo-control (PC), melatonin-control (MC), placebo-MI (PM), and melatonin-MI (MM). Each group included 10 rats. In the PM and MM groups, the rat MI model was established through left coronary artery ligation surgery. In addition, the PC group was regarded as a negative control group, and it was designed to show whether the model was established successfully and to eliminate the influence of the operation.

Rat MI model
The rats were placed in an induction chamber and 4% isoflurane was delivered until they reached a deep plane of anesthesia. After the intubation was finished, the chest was opened at the left 4 th intercostal space. The pericardial tissue was removed and the left anterior descending artery was visualized under a light microscope (model YAN-6A; Yuyan Instruments Co., Ltd., Shanghai, China) and ligated by a 6-0 silk suture (Yuyan Instruments), 1 mm below the tip of the left atrial appendage. The PC and MC groups underwent surgery without the left anterior descending artery ligation. After ligation, the left artery presented a pale appearance. Echocardiography was used to confirm the successful ligation, which would appear as ST segment and Q wave changes.

Melatonin treatment
For 7 days following the surgical procedures, melatonin (Sigma-Aldrich, St. Louis, USA) dissolved in dimethyl sulfoxide was administered to the MC and MM groups by daily intraperitoneal injection, at a dose of 10 mg/kg body weight. In the placebo groups, an equivalent volume of dimethyl sulfoxide (DMSO) was injected.

Hemodynamic measurement and determination of infarct size
A microtip catheter transducer (Millar Instruments, Houston, USA) was inserted into the right carotid artery and advanced into the left ventricle to measure the following parameters: the left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular ejection fraction (LVEF) and fractional shortening (FS). For the hemodynamic analysis, LV maximum first derivative of developed pressure (max dP/dt), and LV minimum first derivative of developed pressure (min dP/dt) were analyzed. Four weeks after the treatment, the animals were anesthetized again for hemodynamic measurement as described in another study. 16 Infarct size was determined with Masson's trichrome staining as described in an earlier publication. 17 After the animals were sacrificed, the LV myocardium was dissected, fixed in 4% paraformaldehyde, embedded in paraffin, and then cut into 7-μm sections. The sections were stained with Accustian Trichrome Stain (Masson) kit (Sigma-Aldrich). Infarct size was determined using the Microsoft Image Composite Editor v. 14.4 (Microsoft Corp., Redmond, USA).

Measurement of Sirt6 in mRNA and protein levels
The primary rat cardiomyocytes were isolated and cultured from the animals in the 4 groups as described in a previous study. 2 When the cells were prepared, the expression of Sirt6 in cardiomyocytes was measured using western blot and quantitative real-time polymerase chain reaction (qRT-PCR). The anti-Sirt6 (Cell Signaling Technology, Danvers, USA) and tubulin (T5168; Sigma-Aldrich) were used. The following primers were used: Sirt6: forward 5′-TCTTCCAGTGTGGT-GTTCCA, reverse 5′-CCTCCATGGTCCAGACTCC; β-actin: forward 5′-CCAACCGCGAGAAGATGA, reverse 5′-CCAGAGGCGTACAGGGATAG.

Analysis of oxidative stress (ROS and SOD)
Intracellular reactive oxygen species (ROS) production in cardiomyocytes was evaluated with a fluorometric assay using 2',7'-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene, USA), according to the manufacturer's protocols. The cells were incubated with DCFH-DA for 1 h, after which the fluorescence intensity was measured at 488 nm and 525 nm emission wavelengths. Lysate and cardiomyocytes from fresh rat hearts were collected before total superoxide dismutase (SOD) activity was determined, using a commercial SOD Kit (Beyotime Biotech Inc., Jiangsu, China). The 50% inhibition activity of SOD (IC50) values was determined under an optic density (OD) of 560 nm, using a Model 550 Microplate Reader (Bio-Rad Laboratories Inc., Hercules, USA).

Nitric oxide measurement
The level of nitric oxide (NO) in MI cardiac tissue was detected using a diagnostic assay kit (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China) as described in a previous study. 18

Measurement of iNOS and eNOS with western blot
Total proteins were extracted from LV tissues. Protein concentrations were detected using a Pierce BCA Assay Kit (Thermo Fisher Scientific Inc., Waltham, USA). Then, samples containing 20 μg protein were resolved with electrophoresis on 10% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. Rabbit anti-endothelial nitric oxide synthase (eNOS), anti-inducible nitric oxide synthase (iNOS), polyclonal antibodies, rabbit anti-P1177 eNOS, and rabbit anti-GAPDH monoclonal antibody (all from Abcam, Cambridge, USA) were used to detect the levels of iNOS, eNOS and phosphorylated eNOS (p-eNOS) in this assay.

Statistical analyses
Data were shown as median and interquartile range (IQR) due to a small sample size. Statistically significant differences among the groups were analyzed using the Kruskal-Wallis test. Pairwise multiple comparisons post hoc tests were carried out using Dunn's test with a Bonferroni correction. Two-tailed, unpaired Mann-Whitney tests were used to analyze the comparisons between 2 groups. The value of p < 0.05 was taken to indicate statistical significance.

Melatonin decreased the myocardial infarct size in the rat MI model
As shown in Fig. 1A, in which the fibrotic scar appears blue and the viable myocardium is red, melatonin did not affect the cardiac structure in the MC group, while in the MM group, melatonin treatment decreased the myocardial infarct size (U = 0; p < 0.0001). The quantitative analysis confirmed this finding (Fig. 1B).

Melatonin improved hemodynamic parameters
Hemodynamic analyses were performed in all 4 groups. In the MM group, LVEDd, LVESd and min dP/dt decreased, while LVEF, LVFS and max dP/dt increased, but there was no significant difference after Dunn's Bonferroni adjustment (Table 1). No differences in these parameters were observed in the MC group, as shown in Fig. 2.

Melatonin increased Sirt6 expression both in mRNA and protein levels
After treatment with melatonin, the expressions of Sirt6 both in mRNA (p < 0.0001 after adjustment) and protein levels (p < 0.0001 after adjustment) were increased in the MM group compared to the PM group, as shown in Fig. 3 (Table 1).

Melatonin ameliorated MI-induced oxidative stress
Because of the fact that increased oxidative stress deteriorated cardiac remodeling, 2 oxidative stress-related molecules were detected. The results indicated that when treated with melatonin, the ROS level from primary cardiomyocytes was lowered in the MM group compared to the PM group, yet it was nonsignificant after Dunn's Bonferroni adjustment (p = 0.3343; Table 1). There was no significant difference among the PC and MC groups, as shown in Fig. 4A. The SOD activity was enhanced in the MM group compared to the PM group, but it was nonsignificant after Dunn's Bonferroni adjustment (p = 0.3336; Table 1), as shown in Fig. 4B.

Melatonin increased eNOS and NO production and inhibited iNOS production
In order to demonstrate whether the important role of melatonin as an antioxidant was involved in NO signaling, we performed assays to measure the production of NO-related molecules. The results showed that the levels of NO (p = 0.3346 after adjustment when compared to the PM group), eNOS (p < 0.0001 after adjustment) and p-eNOS (p = 0.3343 after adjustment when compared to the PM group) were increased in the MM group, but not in the PC and MC group, as shown in Fig. 5. This indicated that melatonin might promote cardioprotection by activating the eNOS/NO signaling pathway. At the same time, we found that the levels of iNOS (p = 0.0008 after adjustment when compared to the PM group) and phosphorylated iNOS (p-iNOS) (p = 0.0136 after adjustment when compared to the PM group) were significantly decreased when treated with melatonin in the hearts of the MM rats but not in the PC and MC groups, as shown in Fig. 5C. This indicated that melatonin might promote cardioprotection by inhibiting iNOS (Table 1).

Discussion
Melatonin can counteract many pathological conditions, such as cardiovascular disorders, carcinogenesis, neurological diseases, and aging. 19 Recently, its cardioprotective function has gained a great deal of attention. [20][21][22][23] More and more evidence suggests that the cardioprotective functions of melatonin are due to its direct free radical scavenger properties, antioxidant activities and anti-inflammatory effects. In this study, we confirmed the cardioprotective role of melatonin in an animal model of MI, and for the first time we found that melatonin improved cardiac remodeling through a Sirt6-dependent antioxidant pathway.  Pharmacological data have clearly demonstrated the powerful antioxidant properties of melatonin. [24][25][26] For example, melatonin can protect the brain as a potent antioxidant, anti-inflammatory and antiapoptotic agent. 27 Melatonin has been shown to prevent granulosa cells from oxidative damage by targeting JNK-mediated autophagy, which may hold promise for patients with ovulation failure-related disorders. 28 In order to investigate the cardioprotective role of melatonin on MI, we established a MI rat model by ligating the left anterior descending coronary artery and verifying ST elevation using eletrocardiography (ECG). Four weeks after the treatment with melatonin, infarct size and hemodynamic parameters were measured. Infarct size was reduced significantly after the treatment with melatonin, which indicated that melatonin could protect the infarcted myocardium. Similarly, the results of hemodynamic studies indicated that post-MI cardiac remodeling had improved significantly following the treatment with melatonin.
Accumulated evidence indicates that oxidative stress is responsible for the progression of cardiac remodeling. 29,30 In order to investigate whether the cardioprotective effect of melatonin functions through its antioxidant role, ROS and SOD levels were measured as the indicators of oxidative stress. Excessive ROS accumulation leads to cellular oxidative stress and elicits an increased proinflammatory response, which is associated with cardiac remodeling. 7,31 Not surprisingly, after the treatment with melatonin, we found that ROS activity reduced significantly and SOD level increased significantly in the MM group. The evidence suggested that melatonin could prevent cardiac remodeling by decreasing oxidative stress.
The NO is a widely known ventricular dilator produced from L-arginine by a family of NOS, including eNOS and iNOS. 32 It has been shown that NO plays a role in reducing oxidative stress 33 and in blocking the progression of MI. 34 In our study, the expressions of NO, iNOS, eNOS, and their corresponding phosphorylations were determined. The results indicated that the expressions of NO and eNOS were upregulated and the expression of iNOS was lowered in the MM group compared to the PM group. This evidence suggests that melatonin might suppress the processes of oxidative stress by activating the eNOS/NO signaling pathway and inhibiting iNOS signaling pathway.
Interestingly, we found that the expressions of Sirt6, both in mRNA and protein levels, were significantly increased in the MM group compared to the PM group. This indicates that Sirt6 might be the target through which melatonin improves oxidative stress by activating the eNOS/NO signaling pathway in the heart.
In spite of the fact that a dose of 10 mg/kg of melatonin administered via intraperitoneal injection works cardioprotectively in the MM group, there is still no evidence to demonstrate whether this dose works for humans. Actually, it was reported that a total dose of 50 mg has been used during a major vascular surgery. 35 Regretfully, those researchers concluded that intracoronary and intravenous administration of melatonin did not improve the myocardial salvage index in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention. Hopefully, if the dose of melatonin was increased to 10 mg/kg, it would demonstrate some beneficial effects for the heart, considering its obvious Sirt6-dependent antioxidative ability in I/R. However, this speculation still needs to be verified.

Limitations
Firstly, this study proved the effect of melatonin on mouse MI model, but there is still no evidence to demonstrate whether this works for humans. Secondly, the study had no designed not designed dose gradient, so the optimal dose of melatonin for MI could not be obtained. Thirdly, we only analyzed the changes of Sirt6 expression and did not verify whether the knockout of Sirt6 would eliminate the cardioprotective effect of melatonin.

Conclusions
The current study demonstrated for the first time that intraperitoneal injection of melatonin could improve the cardiac function via a Sirt6-dependent antioxidant pathway in MI rats.