Ischemia reperfusion myocardium injuries in type 2 diabetic rats: Effects of ketamine and insulin on LC3-II and mTOR expression

Abstract

Objectives: Myocardiopathy occurs in ischemia-induced injury caused by dysregulation of autophagy of cardiac tissues. The present report evaluates the protective effect of ketamine and insulin against myocardial injury in type 2 diabetic rats (T2DM).

Methods: The effects of ketamine and its combination with insulin on biochemical parameters and inflammatory cytokines in the serum of I/R-induced myocardial injury in T2DM rats were evaluated. The parameters of reactive oxygen species and the expression of autophagosome signaling pathway proteins were also determined. Using transmission electron microscopy, we investigated autophagosomes. Western blots were used to detect autophagy-associated signaling pathways. Myocardial function was determined by echocardiography and histopathological changes in myocardial tissues were also determined in I/R-induced myocardial injury in type 2 diabetic rats.

Results: There was a significant reduction in glucose, AST, LDH, and CK-MB levels and cytokines (IL-1β, IL-6, and TNF-α) in serum of the ketamine (p < .05) and ketamine + insulin (p < .01) groups than in the diabetic + I/R. MDA and ROS levels were reduced with a substantial (p < .05) increase in GSH levels through improved cardiac function in the ketamine (p < .05) and ketamine + insulin (p < .01) groups than the diabetic + I/R group. There was an increase in mature autophagosomes in diabetic+I/R+Kt+In compared to diabetic+I/R+Kt alone in infarction and marginal zones. It should be noted that the significant increase (p < .01) in protein levels of the autophagy-associated intracellular signaling pathways AMPK and mTOR, as well as an increase in LC3-II and BECLIN-1, suggests that ketamine combined with insulin-activated autophagy-associated intracellular signaling AMPK and mTOR.

Conclusion: The findings of the study suggest that ketamine combined with insulin administration remarkably protects I/R-induced myocardial injury in rats with T2DM by reducing the dysregulation of autophagy.

Keywords

ketamine insulin autophagy ischemia reperfusion

Introduction

The prevalence of type 2 diabetes (T2DM) increases every year and patients with it also report a higher risk of developing ischemic heart disease than non-diabetics.^1,2 Patient with diabetes shows poor recovery from ischemic heart disease and increased sensitivity to ischemia reperfusion injury to the heart.^3,4 Ischemia reperfusion injury and diabetes are closely related to autophagy in a study in rat models.^5,6 Survival, translation, autophagy, and cell growth regulated by the mammalian target of rapamycin (mTOR) and the cell autophagy process are reported to be controlled by the p-mTOR/mTOR signaling pathway.^7,8 Furthermore, autophagic activity is determined by evaluating the autophagy-related factor, that is, LC3-II. Dysregulation of autophagy contributes to the development of cardiovascular disorders such as myocardial infarction.⁹ The inflammatory reaction improves due to the activation of innate immune pathways that develop myocardial ischemic disease. In the infarcted area, inflammatory cell recruitment occurs due to the upregulation of inflammatory cytokines. Dead cells and their debris are digested by the phagocytosis process through inflammatory cells. Ischemia reperfusion injury has been reported to occur due to a combination of the inflammatory response, cell death, and autophagy.¹⁰ The need for surgery is also increasing with the increase in the number of cases of heart disease associated with clinical diabetes. Therefore, it is increasingly important to select and apply perioperative anesthetic drugs in patients with DM2 and coronary heart disease. It is important to select and apply the proper anesthetic drugs in patients with T2DM. Ketamine is an anesthetic commonly used clinically to produce anesthesia.¹¹ Ketamine has also been reported to possess an anti-inflammatory and neuroprotective effect. It inhibits apoptosis and regulates autophagy by regulating the NF-kB pathway.¹² Moreover, ketamine also protects liver injury by modulating the NF-kB pathway.¹³ The Nrf2/HO-1 pathway is targeted by S-ketamine (a dextrorotatory enantiomer of ketamine) to alleviate liver injury caused by carbon tetrachloride.¹⁴ Research has shown that ketamine greatly improves neuroprotection in PD (Parkinson’s disease) rats and reduces cognitive impairment after surgery when administered subanesthetic doses of ketamine.¹⁵ The PI3K/Akt/mTOR pathway could be inhibited by S-ketamine to alleviate neuropathic pain. It is clear that the findings provide a clear understanding of the molecular mechanisms that contribute to the protection of myocardial injury caused by S-ketamine.¹⁴ The literature suggests that guinea pigs were anesthetized with higher doses of ketamine and xylazine and had reduced myocardial infarction size and improved hemodynamic function after experimental ischemia reperfusion.¹⁶ Ketamine has been reported to provide antidepressant effects at a dose less than 10 mg/kg, but 25 mg/kg is needed for analgesia.¹⁷ However, ketamine has not been studied in diabetes mellitus associated with myocardial I/R injury. There has yet been any investigation on the effects of ketamine on the expression of LC3-II, mTOR, and pmTOR in diabetic rats with myocardial injury induced by I/R that undergoes ketamine treatment, to the best of our knowledge. Consequently, in the current study, a subanesthetic dose (<10 mg/kg) of ketamine was considered to determine its effects on improving autophagy dysregulation in I/R-induced myocardial injury in type 2 diabetes rats.

Insulin (IN) preparations have also been found to have a specific cardioprotective effect when used in animal models of ischemia reperfusion injury (I/R) as a means of treating diabetes.^18–20 Several favorable effects contributed to the development of the myocardial survival pathway, including an increase in endothelial nitric oxide synthase (eNOS) expression in the myocardium, coronary vasodilation, and activation.²¹ In a recent study, insulin-induced activation of the Akt protein was discovered to reduce cardiomyocyte apoptosis.²¹ A recent study has shown that insulin exerts cardioprotective effects following myocardial infarction, which can reduce the harmful effects on the heart.^22,23

Therefore, ketamine and insulin have shown potential to protect ischemia/reperfusion-induced injury and consequently protect ischemia reperfusion (I/R) myocardial injury in rats. A combination of ketamine and insulin has not yet been reported to alleviate autophagy dysregulation in type 2 diabetes rats after I/R-induced myocardial injury. Therefore, in the current study, ketamine was combined with insulin in a sub-anesthetic dose to evaluate its protective effects against myocardial injury in type 2 diabetic rats (DM2).

Materials and methods

Materials

Solarbio Life Sciences (Beijing, China) provided us with ketamine (purity >98%). The LDH and CK assay kits were purchased from Sigma-Aldrich, USA. Streptozocin (STZ) and insulin were obtained from Sigma-Aldrich, USA.

Animals

Sprague-Dawley rats (SD; male) weighing approximately 220–250g, 8–9 weeks old, were obtained from Chuhu Hospital of Anhui Medical University, China. All experiments were approved by the Animal Ethics Committee (approval number 2022-AMU-0034, Wuhu, Anhui, China) according to the Animal Care and were using Guidelines (NIH) and conducted over a period of 3–4 months. SD rats were raised in a controlled environment with a humidity of 50%, as well as a 12-h day/night rhythm in the laboratory. Water and diet were administered to rats on a regular basis.

Experimental design

Diabetes induction and blood glucose measurement

Induction of type 2 diabetes mellitus was performed according to the following protocol.²⁴ The animals were subjected to a high-sugar, high-fat diet, and were given free access to drinking water at a temperature of 25⁰C, and a humidity of 40%. A single dose of 40 mg/kg body weight of freshly prepared streptozotocin (STZ, Sigma-Aldrich, USA) in citrate buffer (0.1 M, pH 4.8) was administered intravenously into the caudal vein of the group of animals after 8 weeks of high-fat diet treatment. Citrate buffer alone was administered to the control group via the same injection route as in the experimental group.^25,26

It was necessary to deprive the rats of food overnight prior to injection, but they had free access to water at all times. Several factors were considered when monitoring the severity of diabetes, including diet, weight gain, and blood sugar levels. We tested the results of a modified glucose dehydrogenase method in blood obtained from the tips of the tail of the rats. Rats were found to be found after 72 h (with 8 h fasting), fasting blood glucose levels greater than 16 mmol/L, consumed a greater amount of food and water and urinated more often. This way, the diabetic rat model was successful. The onset of diabetes and ischemic injury were separated by at least 2 weeks.²⁷

Establishment of the myocardial I/R model

Pentobarbital sodium was administered intraperitoneally at a dose of 40 mg/kg to male diabetic rats to induce anesthesia. Following tracheal intubation, the rat was hooked up to a ventilator that provided ventilation with a 3:1 inspiration-to-expiration ratio, a tidal volume of 2 mL, and a frequency of 50 breaths per minute. Using standard lead II electrocardiograms, all four limbs of the rats were fixed before an electrocardiogram of their hearts was recorded. An incision was made along the left side of the sternum approximately 3-4 ribs from the left atrium of the heart. A hemostatic forceps was used to dissect the main pectoralis muscle layer by layer. The left anterior descending branch (LAD) of the left coronary artery was located by cutting the pericardium with ocular forceps. A 6–0 suture was used to ligate the left anterior descending branch. Ischemia of the myocardium was caused by the presence of appropriate sizes of polyethylene plastic tubes that compressed and occluded the coronary arteries, causing myocardial ischemia. An ischemia of 30 min was followed by the removal of the plastic tube to restore blood flow to the rats. The thoracic cavity was clamped with a pair of clamps during reperfusion for 24 h to reduce interference from non-experimental factors during reperfusion. The study involved the collection of cardiac tissue and serum, which were to be used for further investigation.²⁸

Modeling Myocardial I/R: Criteria for Success

A recording of the ECG was made. To confirm that the ligation was successful, an increase in ST segment was observed for at least 15 min after the procedure. Cyanosis was observed at the distal end of the suture near the epicardium. There was a gradual return to baseline levels of the ST segment after the plastic tube was removed, and the color of the left ventricle returned to its original state after the plastic tube was removed.

Treatment Design

A total of eighty (n = 80, 16 rats in each group) diabetic rats were randomly assigned to five groups according to their verified diabetes levels, such as the control group; Diabetic group; Diabetic + I/R; Diabetic+I/R+Kt; and Diabetic+I/R+Kt+In. Ketamine was administered intravenously at 1 mg/kg and insulin subcutaneously at a dose of 1 IU/kg/day for 10 days after successful myocardial injury in T2DM rats. An equal amount of saline was administered to control rats, the diabetic group, and the diabetic group for I/R.

Measurement of reactive oxygen species, malondialdehyde (MDA), and glutathione (GSH) levels

Malondialdehyde levels in heart tissue were determined using an MDA assay kit. A commercially available kit was also used to measure GSH levels in heart tissues. We estimate cardiac ROS levels using MitoSOX red mitochondrial superoxide indicator. First, tissue homogenates were stained for 30 min at room temperature under dark conditions with 5 mM MitoSOX red. Intracellular ROS levels were calculated using a fluorescence plate reader. The excitation and emission wavelengths of the plate reader were 510 and 570 nm, respectively.

Measurement of Serum Aspartate Aminotransferase, Creatine Kinase MB Isoenzyme (CK-MB), Lactate Dehydrogenase, and Glucose Levels

The levels of myocardial injury markers aspartate aminotransferase (AST), CK isoenzyme MB (CK-MB), lactate dehydrogenase (LDH), and glucose level were determined in serum using a biochemical analyzer (DxC 800, Beckman Coulter, Shanghai, China) according to the direction of the manufacturer of commercial kit.

Determination of cardiac function and echocardiography

An ultrasound system with a 17.5 MHz imaging transducer (VisualSonics Inc, Canada) was used for transthoracic echocardiographic analysis. Cardiovascular structure and function were evaluated by echocardiography 24 h after reperfusion. A 10% (3 mL/kg, intraperitoneally) chloral hydrate anesthetic was used to anesthetise the rats and then placed them in the supine position.²⁹ During an M-mode echocardiography, chordae tendineae were closely examined, and LVEDD (left ventricular end-diastolic diameter), LVESD (left ventricular end-systolic diameter), the LVPWT (left ventricular posterior wall thickness), and the (left interventricular septal thickness) were measured; a computer algorithm was employed to calculate the LVEF (left ventricular ejection fraction) and the LVSF (left ventricular shortening fraction), respectively.

Hemodynamic measurements

We performed invasive hemodynamic measurements in rats under anesthesia with intraperitoneal injection of sodium pentobarbital (40 mg/kg) as previously described.³⁰ The procedure consisted of inserting a microtip catheter with a pressure transducer into the right carotid artery and advancing it into the LV cavity using a pressure transducer as a guide (Blood Pressure analysis module attached to a computerized data acquisition system with Lab-Chart 7 pro-software (Power Lab, AD Instruments, USA) and SPR-407 rat pressure catheter with PCU-2000 pressure control unit (ADI and Miller Instruments, USA).³¹ The LV pressure, positive and negative LV dP/dt were monitored continuously and measurements were recorded at a speed of 1 mm/s with a sampling rate of 1000 Hz. The HR, MAP, LV dp/dtmax, LV dp/dtmin, LVESP, and LVEDP were monitored and recorded.

Masson’s trichrome staining

Tissue processing

To process tissue specimens in labeled cassettes or tubes, we used a tissue processor (Tissue-Tek®, Sakura Finetek Inc., CA) according to the protocol for the processing of animal tissues that our laboratory follows: 80% ethanol for 30 min xylene for 30 min, and paraffin wax at a temperature of 50° C for 30 min.

Paraffin embedding

After the tissue had been processed, each component was assembled in a mold containing molten paraffin. As a standard procedure, the paraffin block of tissue was sectioned at a thickness of 4 μm. The sections were mounted on microscope slides and heated at 60° C for 1 hour to attach the sections to the microscope slide. A graded concentration of ethanol was used to rehydrate the sections before staining was performed because they had been deparaffinized in xylene and rehydrated prior to staining. The Masson trichrome stain was the histological stain used for the samples. The Olympus BX-51 microscope detected pathological changes in myocardial tissue based on the extent of the necrotic area. Angiographic measurements of the ischemic myocardium were calculated as a percentage of the total area at risk of the left ventricle. The infarcted area in the myocardial tissue was considered as collagen and the blue appearance on the TS of the tissue shows the presence of collagen fibers.

Measurement of myocardial infarct area

As soon as the rats' hearts were excised after cardiac function measurement, Evans blue (1.2%, 3.5 mL) was injected into the vena cava to track the perfused region and the area at risk (AAR) as previously reported.³² The hearts were cut into 1mm thick so that they could be cut longitudinally along the ligation point. We then placed them in 1.2% TTC (2, 3, 5-triphenyltetrazolium chloride) in PBS and incubated them for 10 min at 37° C in an incubator to identify the infarct area. A digital camera was used to record the ischemic regions (area at risk, AAR) and the infarct area (stained with Evans blue; non-ischemic area), while a digital imaging system was used to analyze the blue area (not stained by TTC, but stained by Evans blue; non-ischemic area). The myocardial AAR area was identified as the region lacking blue staining. The weights of the infarct area (white), AAR (red), and non-ischemic zones of the LV (blue) were measured. Infarct size was calculated taking into consideration the percentage of the AAR mass. A ratio was calculated based on the area at risk (AAR) and the entire section, as well as a ratio based on the size of the infarct to the overall area-at-risk, was calculated.

Estimation of cytokine activity

We used ELISA to determine blood serum levels of the inflammatory mediators IL-1β, IL-6, and tumor necrosis factor (TNF)-α. We used commercial kits according to the manufacturer's protocol (R&D Systems) to determine the levels of inflammatory mediators at an optical density of 450 nm, and these levels were compared with standard curves.

Western blot analysis

We used Western blot to evaluate the level of protein expression of p-mTOR, m-TOR, p-AMPK, AMPK, ATG5, BECLIN-1, LC3, and GAPDH in myocardial tissue homogenates. A BCA protein assay kit (ThermoFisher Scientific, USA) was used to determine the amount of total protein in myocardial tissues (area-at-risk) as a function of homogenization in RIPA buffer containing protease inhibitors, followed by quantitative analysis of protein content. After centrifuging the tissue protein lysate at 13,000 rpm for 20 min at 45°C, the protein supernatant was collected and stored at −20°C.

A 10% SDS-PAGE was used for protein separation after loading 20 μg of protein into it, then electroblotting them onto nitrocellulose membranes. The blocking solution, which contained 5% non-fat milk, was then applied for 2 h to the membranes. A total of three washes were performed in TBST followed by overnight incubation with primary antibodies at 4° C in blocking buffer: p-mTOR (cat.no. # 2971, Cell Signaling Technology, USA; 1:500, dilution), m-TOR (cat.no. # 2983, Cell Signaling Technology, 1:800, dilution), p-AMPK (cat.no # PA5-17831, Invitrogen, USA; 1:500, dilution), AMPK (cat.no. # PV4674, Invitrogen, USA; 1:2000, dilution), ATG5 (cat.no. # A0203; Abclonal, USA; 1:2000, dilution) BECLIN-1 (cat.no. # A7353, Abclonal, USA; 1:2000, dilution), LC3 (cat.no. # CBA-5116. 96, Cell Biolabs, USA; 1:2000, dilution), and GAPDH (cat.no. # SRE0024, Sigma-Aldrich, USA; 1:8000, dilution). The membranes were then thoroughly washed, followed by incubation of the membranes with a goat secondary antibody conjugated with horseradish peroxidase (1:2000) added to the blocking buffer for 2 h at room temperature the following day. As soon as the samples had been washed three times with TBST, we detected and visualized them with the Amersham 600 (General Electric Company, USA) using Enhanced Chemiluminescent. Using ImageJ software, a quantification of the amount of expression of the target band was carried out compared to the GAPDH loading control.

Transmission electron microscopy

Myocardial samples were processed according to the procedure described previously [21] to study the myocardium with transmission electron microscopy. A cacodylate buffer (100 mM) containing glutaraldehyde (2.5%) was used to fix the hearts overnight at 4⁰C. A cacodylate buffer was used to wash the hearts the next day, and afterwards, the hearts were kept in a freezer at 4°C for future processing the next day. An acetone series was used to dehydrate the hearts after being fixed with 1% tannic acid and embedded in Embed-812 resin after cleaning with 1% osmium tetroxide. The electron microscope was equipped with a Tecnai G2 Spirit BioTWIN electron microscope to image ultrathin sections of 100 nm thickness on copper or nickel grids that were set up using single-slot or 200-mesh slots.

We differentiated autophagosomes, lysosomes, and autolysosomes in our experimental conditions based on the following criteria to verify the accuracy of TEM quantitation. An autophagosome is a double membrane-enclosed vesicle that engulfs cytoplasmic material with its double membrane. Lysosomes are monolayer organelles that have a high electron density compared to the rest of the cell. Autolysosomes are defined as a monolayer structure that contains abundant undegraded materials (autophagosomes and other membrane components) and has a significantly enlarged volume.

Calculation and justification of sample size

We considered the following factors when determining the sample size: power to evaluate the variability of the samples, standard deviation to evaluate the variability of the samples, type-1 error at p < .05, and 80% study power with a two-tailed test. The sample size estimate for the study was based on the G power to determine the number of animals.

Statistical analysis

The data was analyzed using Graphpad Prism (Version 9.2, USA) as statistical software. A Shapiro–Wilk test was performed on all parametric data in order to determine the normality of the distribution. The mean +standard error of mean (SEM) is calculated if the data are normally distributed and have homogeneous variance. An analysis of variance (ANOVA) was used for comparisons between groups in intergroup comparisons. For comparisons within groups, repeated measures ANOVA was used as the statistical method of choice. Post hoc comparisons were conducted using Tukey's multiple comparison tests. Kruskal–Wallis and Mann–Whitney U tests were used for inhomogeneous variance or non-normal distributions. p-values less than 0.05 were considered statistically significant.

Results

Mortality rates among the different groups and their comparison

The death rates in each group were analyzed and the statistical data did not show significant differences (Control: 5.34%, Diabetic: 5.31%, Diabetic+IR: 6.45%, Diabetic+IR+Kt: 6.14%, Diabetic+IR+Kt+In: 6.91%). In all deaths, bleeding was the cause of death during improper cannulation of the carotid artery or improper ligation of the LAD coronary arteries, respectively.

Evaluation of the effects of ketamine and insulin on biochemical parameters

Biochemical parameters in serum of I/R-induced myocardial injury in diabetic rats were estimated as presented in Table 1. Significant increases (p < .01) in blood glucose were observed (p < .01) in the diabetic and diabetic+I/R group compared to the control group of rats. Treatment with ketamine alone did not show a significant reduction (p = .325) in the level of blood glucose in diabetic rats. However, glucose levels were significantly reduced (p < .01) in the Kt+In the treated group compared to the diabetic and diabetic+I/R+kt group. AST activity of AST (p = .235), CK-MB activity (p < .01), and LDH activity (p < .01) was significantly higher in the serum of the diabetic group compared to the control group. Furthermore, the activity of AST, CK-MB, and LDH increased considerably (p < .01) in the serum of the diabetic + I/R group compared to diabetic rats. However, treatment with Kt and Kt+In significantly (p < .01) reduced AST, CK-MB, and LDH in serum compared with the diabetic+I/R group of rats. AST (p = .361), CK-MB (p = .371), and LDH (p = .216) activities decreased considerably (p < .05) in the serum of the diabetic + I/R + Kt treated group compared to diabetic+IR+Kt+In rats.

Table 1.

Assessment of ketamine, insulin, and its combined effects on the biochemical parameters in the serum of I/R-induced myocardial injury in diabetic rats.

S. No.	Groups	Glucose (mmol/L)	AST (U/L)	CK-MB (U/L)	LDH (U/L)
1	Control	4.7 ± 0.42	110.8 ± 5.2	23.6 ± 1.8	105 ± 3.9
2	Diabetic	25.2 ± 1.25^$$	187.3 ± 5.7^$	124.2 ± 2.1^$$	874 ± 3.2^$$
3	Diabetic+I/R	24.9 ± 0.96^$$	265.5 ± 12.6^@@	197.3 ± 13.8^@@	1425 ± 35.2^@@
4	Diabetic+I/R+kt	23.4 ± 0.85^$$	183 ± 9.4^**	59.8 ± 4.2^**	903 ± 19.4^**
5	Diabetic+I/R+Kt+In	5.1 ± 0.38^**,##	159.6 ± 8.3^**,#	51.5 ± 2.8^**,#	648 ± 15.2^**,#

Mean ± SEM (n = 16).

^$p < .05; ^$$p < .01 compared to control group; ^@@p < .01 compared to diabetic group; **p < .01 compared to Diabetic+I/R; ^#p < .05 and ^##p < .01 to Diabetic+I/R+kt group.

Evaluation of the effects of ketamine and insulin on electrocardiogram parameters

There was almost no difference in the changes of all cardiac parameters between the durations of time before ischemia and after reperfusion, so we considered only the average changes of the first 2 min before ischemia and the first 10 min of the reperfusion period. In the diabetic+I/R group, there was a significant reduction in cardiac function factors as well as coronary flow compared to the control group during the ischemic period compared to the diabetic+I/R group (p < .001). Furthermore, to avoid additional chaos in the figures, a comparison was made between the highest performing group and the diabetic + I/R group. In the first 2 min after reperfusion, the heart rate in the groups of diabetic+I/R+kt and diabetic+I/R+kt+In showed a significant increase compared to the diabetic+I/R group (p < .01); however, this increase trend continued slowly by the end of the reperfusion period (p < .05). Compared with the diabetic+I/R group, heart rate and mean arterial pressure increased significantly in the first 2 min after reperfusion in the diabetic+I/R+kt+In and diabetic+I/R+kt+In groups. However, this trend continued to increase slowly at the end of the reperfusion period (p < 0,1).

Electrocardiography was used to determine the effects of ketamine and insulin on cardiovascular function in I/R-induced myocardial injury in diabetic rats (Figure 1). No significant differences were found in LVEDD (p = .762) in the diabetic group compared to the control rats. However, LVESD (p = .271) was considerably higher in diabetic rats compared to the control group. Furthermore, LVEDD (p < .01; p < .001) and LVESD (p = .113; p < .001) were significantly higher in the diabetic+I/R group than in the control and diabetic groups, respectively. Furthermore, LVEDD (p = .0361; p = 217) and LVESD (p = .0412; p = .317) were markedly reduced in the diabetic + I/R + Kt and diabetic + I/R + Kt + In groups, respectively, compared to diabetic+I/R rats. A substantial decrease in LVEDD (p = .0391) and LVESD (p = .0378) was also observed in the group treated with ketamine (diabetic + I/R + Kt) was also observed compared to the combined treatment group (diabetic + I/R + Kt + In).

Figure 1.

Effects of ketamine and its combination with insulin on cardiovascular function in I/R-induced myocardial injury in diabetic rats. Left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), left ventricular ejection fraction (LVEF) and left ventricular systolic function (LVSF), LV maximum positive dP/dt (mmHg) [+LV dp/dt(max)], LV maximum negative dP/dt [−LV dp/dt(max)]. Mean ± SEM (n = 16). *p < .05, **p < .01, ***p < .001 versus control group; #p < .05 versus diabetic+I/R; ^$p < .05 versus Diabetic+I/R+kt group.

Furthermore, LVEF (p = .0412), LVSF (p = .315), +LV dp/dt max (p = .327), and −LV dp/dt max (p = .0182) were significantly lower in the diabetic group compared to control rats. Furthermore, LVEF, LVSF, +LV dp/dt max, and −LV dp/dt max were considerably (p < .001) reduced in the diabetic+I/R group compared to diabetic rats. Furthermore, there was a substantial increase in LVEF (p = .0412; 0.0347), LVSF (p = .0361; 0.0278), +LV dp/dt max (p = .268; 0.0184), and −LV dp/dt max (p = .0267; 0.0242) when treated with ketamine and its combination with insulin compared to diabetic+I/R rats. Furthermore, a significant increase in LVEF (p =.0273), LVSF (p=.0382), +LV dp/dt max (p = .0223), and −LV dp/dt max (p = .243) was observed in the diabetic group + I/R + Kt + In compared to the ketamine alone. As a result, our findings indicated that ketamine and insulin improved cardiac function and reduced myocardial dilation in diabetic rats with I/R injury (Figures 1(a)–(f)). Illustrative LV M-mode echocardiograms are shown in Figure 2. Compared to the control group, diabetic and diabetic+I/R rats showed a significant increase in the thickness of the interventricular septum (IVS) wall with greater aortic and left atrial diameters showing a substantial increase in LVEDD and LVESD (left ventricular end-diastolic diameter and left ventricular end-systolic diameter, respectively) with a higher LV mass and posterior wall (PW). However, after treatment with ketamine and its combination with insulin, there was a considerable decrease in the thickness of the interventricular septum (IVS) wall thickness with a decrease in the aortic and left atrial diameters showing a reduction in LVEDD and LVESDD with a lower LV mass and posterior wall (Figure 2).

Figure 2.

An illustration of an M-mode echocardiogram of the left ventricle. Treatment with ketamine and its combination with insulin showed considerable decrease in the interventricular septal (IVS) wall thickness with decreased aortic and left atrial diameters showing reduced LVEDD and LVESDD with comparatively less LV mass and posterior wall thickness. LVEDD and LVESD: left ventricular (LV) diastolic and systolic diameters, respectively; PW: LV posterior wall; IVS: interventricular septum.

Effect of ketamine and insulin on cardiac function and hemodynamic parameters

The cardiac function of rats was significantly decreased in diabetic and diabetic+I/R groups after 24 h of reperfusion. Data showed that heart rate (HR, 381 ± 7.5 vs 309 ± 8.1, p < .05), systolic blood pressure (SBP, 127 ± 4 vs 121 ± 2.1; p < .05), diastolic blood pressure (DBP, 79 ± 2 vs 74 ± 3, p < .05), mean arterial pressure (MAP, 85.2 ± 3.3 vs 70.3 ± 1.6, p < .05), the left ventricular end-systolic pressure (LVESP, 143.2 ± 9.5 vs 131.6 ± 2.7, p < .05), rates of maximum positive left ventricular pressure (±dp/dtmax, 4873 ± 241.2 vs 4231 ± 121.4, p < .05), and rates of maximum negative left ventricular pressure (-dp/dtmax, − 4511 ± 218 vs −4121 ± 203, p < .05) were markedly decreased, while the left ventricular end-diastolic pressure (LVDSP, 3.41 ± 1.2 vs 6.23 ± 0.9, p < .05) were markedly increased in diabetic group compared with control group. Similar significant (p < .05) reductions in heart rate, SBP, DBP, MAP, LVESP, dp/dtmax and –dp/dtmax and substantial increase in LVDSP were observed in diabetic+I/R group when compared to diabetic group. However, after treatment with ketamine, insulin and its combination, there was significant (p < .05) improvements in the cardiac function and hemodynamic parameters. The data suggests that the combination of ketamine and insulin improved cardiac function induced by I/R injury (Table 2).

Table 2.

Effects of ketamine, insulin, and its combination on cardiac function and hemodynamic parameters.

S.No	Parameters	Control	Diabetic	Diabetic+I/R	Diabetic+I/R+Kt	Diabetic+I/R+Kt+In
1	Heart rate, bpm	381 ± 7.5	309 ± 8.1^$	243 ± 9.4^@@	321 ± 7.8^*	347 ± 8.1^#
2	SBP, mmHg	127 ± 4	121 ± 2.1^$	109 ± 3^@@	115 ± 2.7^*	121 ± 3^#
3	DBP, mmHg	79 ± 2	74 ± 3^$	69 ± 1^@@	74 ± 2^*	76 ± 3^#
4	MAP, mmHg	85.2 ± 3.3	70.3 ± 1.6^$	43.5 ± 2.7^@@	67.1 ± 4.5^*	75.6 ± 3.1^#
5	LVESP, mmHg	143.2 ± 9.5	131.6 ± 2.7^$	121.4 ± 6.7^@	132.8 ± 1.9^*	138.2 ± 5.5^#
6	LVEDP, mmHg	3.41 ± 1.2	6.23 ± 0.9^$	7.71 ± 1.1^@	6.05 ± 0.8^*	4.51 ± 0.5^#
7	+dP/dtmax, mmHg/s	4873 ± 241.2	4231 ± 121.4^$	2013 ± 212.5^@@	3431 ± 145.1^*	4056 ± 211.7^#
8	−dP/dtmin, mmHg/s	−4511 ± 218	−4121 ± 203^$	−2421 ± 113.7^@@	−3237 ± 189.2^*	-4189 ± 156.8^#

Mean ± SEM (n = 16); Cardiac function parameters. SAP (Systolic arterial pressure), DAP (Diastolic arterial pressure), MAP (Mean arterial pressure), LVESP (left ventricular end-systolic pressure), LVEDP (left ventricular end-diastolic pressure), +dp/dtmax (rates of maximum positive left ventricular pressure development), −dp/dtmax (rates of maximum negative left ventricular pressure development). $p < .05 compared to control group; @p < .05 and @@p < .01 compared to diabetic group; *p < .05 and **p <.01 compared to Diabetic+I/R; ^#p <.05 to Diabetic+I/R+kt group.

Evaluation of the effects of ketamine and insulin on inflammatory mediators

The cytokine level was estimated in the serum of ketamine and insulin in diabetic rats. There was a significant increase in the levels of IL-1β (p = .0116), IL-6 (p = .0183), and TNF-α (p = .0135) levels in the serum of diabetic rats compared to the control group. A similar trend was observed in the significant increase (p < .001) increase was noticed in the levels of IL-1β, IL-6, and TNF-α in diabetic+I/R rats compared to diabetic rats alone. However, ketamine treatment and its combination with insulin significantly (p < .01) reduced the level of cytokines [IL-1β (p = .0131; p = .0213), IL-6 (p = .0371; 0.0121) and TNF-α (0.0312; 0.0271)] compared to I/R-induced myocardial injury in diabetic rats. Furthermore, a synergistic effect was observed with a significant reduction in cytokine levels [IL-1β (p = .0257), IL-6 (p = .0313), and TNF-α (0.0279) in the Kt + In treated group compared to the Kt treated group (Figure 3).

Figure 3.

Effects of ketamine and its combination with insulin on the level of cytokines in the serum of I/R-induced myocardial injury in diabetic rats. Mean ± SEM (n = 16). *p < .05, ***p < .001 versus control group; #p < .05, ##p < .01 versus diabetic+I/R; ^$p < .05 versus Diabetic+I/R+kt group.

Evaluation of the effect of ketamine and insulin on oxidative stress

Parameters of oxidative stress, such as the level of MDA and ROS and activity of SOD were determined in the myocardial tissue of I/R-induced myocardial injury in diabetic rats. MDA (p = .0281) and ROS (p = .0327) levels were significantly enhanced in the myocardial tissue of the diabetic group than in the control rats. Furthermore, GSH activity (p = .0278) was considerably reduced in the myocardial tissue of the diabetic group compared to control rats. In a similar trend, MDA and ROS increased significantly (p < .01) in the myocardial tissue of the diabetic + I/R group compared to diabetic rats. Furthermore, GSH activity was also reduced considerably (p < .001) in the myocardial tissue of the diabetic + I/R group compared to diabetic rats. However, treatment with ketamine and its combination with insulin significantly improved the altered levels of oxidative stress parameters such as MDA, level of MDA (p = .0141; p < .01), ROS (p = .0244; 0.0366) and GSH activity (p = .0155; p < .01) in the myocardial tissue from I/R-induced myocardial injury in diabetic rats compared to diabetic+IR+Kt rats and diabetic+IR+Kt+In treated rats, respectively (Figure 4). However, there was a significant improvement in altered levels of MDA (p = .279), ROS (p = .388), and GSH (p = .0188) in Kt+In treated with Kt + In compared to ketamine alone.

Figure 4.

Effects of ketamine and its combination with insulin on the parameters of oxidative stress in the cardiac tissues of I/R-induced myocardial injury in diabetic rats. Mean ± SEM (n = 16). *p < .05, **p < .01, ***p < .001 versus control group; #p < .05, ##p < .01 versus diabetic+I/R; $p < .05 versus diabetic+I/R+kt group.

Insulin and ketamine significantly reduced myocardial collagen deposition in diabetic rats

Masson's stain was used to further evaluate the effect of ketamine and insulin on collagen deposition in cardiac tissues (Figure 5). According to our findings, collagen fibers were predominantly found in blood vessels and only a small amount was present in the control group. However, in the diabetic+I/R group, there was a substantial increase (p < .01) increase in collagen fibers scattered throughout the myocardium compared to control and diabetic rats. Collagen fibers were considerably reduced after ketamine (p < .01) and insulin (p < .001) intervention in the diabetic + IR groups, respectively. Furthermore, compared with the diabetic+IR+Kt treated group, the collagen amount decreased significantly (p = .0367) after insulin treatment and restored to normal in the diabetic + IR + Kt + treated group.

Figure 5.

Effects of ketamine and its combination with insulin on the histopathology and size of myocardial infracted tissues of I/R-induced myocardial injury in diabetic rats. (a) Masson’s staining was used for pathological verification of myocardial infarction. Muscle fibers were reddish in color. The infarcted area in the myocardial tissue was considered as deposition of collagen and the blue appearance of the tissue shows the presence of collagen fiber. Scale bar – 100 µm. (b) Quantitative results presented as the percentage of myocardial fibrosis area and collagen content. Mean ± SEM (n = 16). **p < .01, ***p < .001 versus control group; #p < .05, ##p < .01 versus diabetic+I/R; $p < .05 versus Diabetic+I/R+kt group.

The effects of ketamine and insulin on the size of the myocardial infarct after I/R injury in diabetic rats

The infarct size is a good indicator of possible cardiomyocyte injury in myocardial infarction. Compared to a control group, it is evident that I/R significantly increased cardiomyocyte injury as a result of an increased ratio between infarct size and area at risk, nevertheless, in agreement with cardiac function, the ratio between infarct size to area-at-risk in the ketamine and insulin groups was lower than in the I/R group (p < .05). Based on these data, we were able to demonstrate that ketamine and insulin could protect diabetic rats from I/R-induced cardiomyocyte injury (Figure 6).

Figure 6.

The effects of ketamine and insulin on the size of myocardial infarctions in diabetic rats + I/R. (a) Representative images of left ventricle slices following myocardial I/R injury in diabetic rats of each group. (b) TTC was used to measure the ratio of area at risk plus the infarct size to the whole section. (c) The ratio between the infarct size to the area at risk plus the infarct size. Mean ± SEM (n = 16). ***p < .001, versus control group; *p < .05, **p < .01, versus Diabetic+I/R group.

Evaluation of the effects of ketamine and insulin on the signaling pathway of autophagosomes

The protein expression of the autophagosome signaling pathway was determined in the myocardial tissues of ketamine- and insulin-treated I/R-induced myocardial injury in diabetic rats, as shown in Figure 7. Relative expression of the proteins LC3-II (p = .0281), p-AMPK/AMPK (p = .0297), BECLIN-1 (p = .0273) and ATG5 (p = .384) increased significantly and reduced the expression of p-mTOR/mTOR (p = .0163) in the myocardial tissue of diabetic rats than in the control group. In a similar trend, there was a significant increase in the relative expressions of LC3-II (p = .0267), p-mTOR/mTOR of (p = .0387), BECLIN-1 (p = .0257) and ATG5 (p = .0193) were significantly increased and reduced in the expression of p-AMPK/AMPK (p = .0348) in the myocardial tissue of diabetic+I/R rats compared to diabetic.

Figure 7.

Effects of ketamine on the protein expression of signaling pathway of autophagosomes in the cardiac tissues of I/R-induced myocardial injury in diabetic rats. Analysis of the protein levels of LC3-I, LC3-II, BECLIN-1, and ATG5, and the ratios of p-mTOR to mTOR and p-AMPK to AMPK. Mean ± SEM (n = 16). *p < .05, **p < .01, ***p < .001 versus control group; #p < .05, ##p < .01 versus diabetic+I/R; $p < .05 versus Diabetic+I/R+kt group.

After treatment with ketamine and its combination with insulin, ketamine significantly reduced the expression of LC3-II (p = .0487; p < .01), p-AMPK/AMPK (p = .0213; p < .001), BECLIN-1 (p = .0347; p < .01) and ATG5 (p = .0284; p < .01) protein and increased the expression of p-mTOR/mTOR (p = .0273; p < .01) protein p-mTOR/mTOR (p = .0273; p <.01) in myocardial tissue compared to the diabetic+I/R group. Furthermore, there was also a significant (p < .05) increase in the expression of LC3-II, p-AMPK/AMPK, BECLIN-1, and a ATG5 protein and decrease in the expression of the p-mTOR/mTOR protein in the myocardial tissue of the ketamine + insulin-treated group compared to the diabetic group + I/R + Kt.

Evaluation of the effect of ketamine and insulin on autophagy

Effects of ketamine on autophagic flux in the cardiac tissues of I/R-induced myocardial injury in diabetic rats were observed by transmission electron microscopy as shown in Figures 8 and 9. There was a significantly lower number of autophagosomes (p < .001), cellular lysis, mitochondrial swelling, and myofibril disorganization in the diabetic + I/R group than in the control and diabetic group of rats. However, treatment with ketamine (p < .01) and its combination with insulin (p < .05) significantly improved autophagosome count in infracted (Figure 7) and marginal (Figure 8) myocardial tissue than the diabetic + I/R group.

Figure 8.

Effects of ketamine and its combination with insulin on the activated autophagy of marginal and infracted zone in the cardiac tissues of I/R-induced myocardial injury in diabetic rats. The distribution of autophagosomes in infarction zones. Infarction zone profiles and autophagosome analysis of rats treated with ketamine and insulin in combination with myocardial injury induced by I/R in diabetic rats. Scale bar – 2.0 µm. It is evident that autophagosomes are indicated by red arrows, which were counted across a minimum of 10 different fields. Mean ± SEM (n = 16). ***p < .001 versus control group; #p < .05, ##p < .01 versus diabetic+I/R; $p < .05 versus Diabetic+I/R+kt group.

Figure 9.

Effects of ketamine and its combination with insulin on the activated autophagy of marginal and infracted zone in the cardiac tissues of I/R-induced myocardial injury in diabetic rats. The distribution of autophagosomes in marginal zones. Marginal zone profiles and autophagosome analysis of rats treated with ketamine and insulin in combination with myocardial injury induced by I/R in diabetic rats. Scale bar – 2.0 µm. It is evident that autophagosomes are indicated by red arrows, which were counted across a minimum of 10 different fields. Mean ± SEM (n = 16). ***p < .001 versus control group; #p < .05 versus diabetic+I/R; $p < .05 versus Diabetic+I/R+kt group.

Discussion

Diabetes is one of the modifiable factors that contribute to the development of atherosclerosis, leading to several complications associated with ischemia-induced injury, such as myocardiopathy.³³ Myocardiopathy occurs in ischemia-induced injury due to dysregulation of autophagy of cardiac tissues.³⁴ The present report evaluates the protective effect of ketamine and insulin against myocardial injury in rats with DM2. Despite the lack of clinically effective treatment for I/R injury, several preclinical studies have shown that ketamine and insulin reduce the size of the infarct, improve coronary blood flow, reduce inflammation, and improve myocardial oxygen consumption.^16,35 However, so far, reports have reported the combined synergistic effect of ketamine and insulin on I/R injury in rats with DM2.

Several biochemical parameters such as CK-MB, LDH, and AST have been reported to be improved in patients suffering from myocardial injury.³⁶ Ketamine and the combination of ketamine and insulin were found to reduce the incidence of I/R in rats with DM2, as indicated by lower levels of AST, LDH, and CK-MB in the blood (Table 1). Combined with insulin, ketamine reduced ROS and MDA production after I/R injury in rats with Type 2 diabetes and promoted GSH synthesis. Proteins and nucleic acids are crosslinked by MDA in cardiac myocytes, the most important metabolite of lipid peroxidation. Myocardial cell injury leads to increased MDA levels during mutation, senescence, denaturation, and cardiomyocyte death. As a result, myocardial injury is often evaluated by the detection of MDA.³⁷ According to our findings reported in this study, both ketamine and its combined effects with insulin were found to have antioxidant effects, which may be the mechanism through which they prevented I/R damage.¹⁸ The findings of these experiments support the hypothesis that ketamine and insulin effectively mitigate myocardial injury and maintain cardiac function in an I/R model of myocardial infarction. However, more research is needed to understand the mechanisms underpinning the protective effects of their combined effects.

The effect of ketamine and insulin on inflammatory cytokines in serum of I/R-induced myocardial injury in type 2 diabetes. Our data showed that ketamine, in addition to the combined effects of ketamine and insulin, significantly inhibited the release of proinflammatory cytokines such as IL-1β, IL-6 and TNF-β. Previous studies have found similar results, where the results showed that low-dose ketamine inhibited TLR4/MAPK/NF-κB by activating the α7nAChR-mediated cholinergic anti-inflammatory pathway, reducing the levels of IL-6, IL-1β and TNF-α, thereby producing the protective effect on neuronal apoptosis and neuroinflammation.^38,18

Cardiac function was determined by echocardiography and histopathological changes in myocardial tissues were determined in I/R-induced myocardial injury in type 2 diabetes. The results suggest that treatment with ketamine alone and in combination with insulin significantly improved cardiac function by echocardiography in I/R-induced myocardial injury in DM2. It has been found that the combination of ketamine and insulin significantly ameliorates cardiac hypertrophy in rats with DM2 and improves cardiac contractility in the face of I/R-induced myocardial injury. According to Chen et al., our findings showed that after ketamine and insulin, LVEDD and LVESD decreased significantly. Furthermore, the LVEF and LVEF increased considerably. We also discovered that when ketamine is combined with insulin, it has the effect of attenuating myocardial dilation and improving cardiac function, supporting the conclusions of our study.³⁹

There was a marked reduction in the deposition of collagen fibers in Masson stained cardiac tissues, indicating that ketamine and its combination with insulin improved myocardial fibrosis based on histopathological findings. The results of our study are in agreement with those of a study published in literature,³⁵ our study showed that ketamine, in combination with insulin, significantly reduced the number of fibrous tissues in the heart, including Col I and Col III, and therefore decreased the level of myocardial fibrosis, cardiac hypertrophy, and interstitial fibrosis (Figure 4). There is a correlation between the increase in collagen synthesis and the decrease in collagen degradation that results in the formation of myocardial fibrosis.

The literature reveals that I/R promotes the dysregulation of autophagy due to oxidative stress and inflammatory cytokines. There are several proteins, such as Beclin-1, Bcl 2, involved in the regulation of autophagy, that are dysregulated in cardiac diseases. Autophagy has been reported to be induced in cardiac diseases and the expression of ATG5 involved in it.⁴⁰ Furthermore, the expression of AMPK, BECLIN-1, and ATG5 proteins enhances and inhibits the mTOR protein involved in the induction of autophagy in cardiovascular diseases.^41–43 The study suggests that ketamine treatment and its combination with insulin ameliorated the altered expression of mTOR, AMPK, BECLIN-1, and ATG5 protein in myocardial tissues of I/R-induced myocardial injury in DM2. Our findings were further supported by the study report,⁴² where autophagy-associated signaling pathways such as AMPK/mTOR were suggested to play a crucial role in protecting against myocardial ischemia-reperfusion injury in diabetes mellitus (DM). Furthermore, our findings were supported by results from another study⁴³ in which the findings indicated that icariin attenuated cardiomyocyte hypertrophy and prevented cell injury mediated by the AMPK/mTOR pathway to improve autophagy and reduce apoptosis related to autophagy.

An autophagy-associated gene (Atg) is responsible for regulating autophagy. It can be concluded that mTOR (LC3) and Atg6 (BECLIN-1) are some of the key protein complexes involved in regulating autophagy in cell.⁴⁴ Among them, mTOR inhibits autophagy through its negative regulation. Atg5 and LC3, a marker protein for autophagy expression, contribute to the synthesis of the autophagosome. It is also possible to determine whether autophagy expression changes with LC3 levels. Despite the fact that Beclin-1 is now independent of BCL2, there is a possibility that it can induce autophagy in cell⁴⁵ and the expression of BECLIN-1 and the protein moderator of LC3 are often used to detect autophagy in cell.⁴⁶ Therefore, in this experiment, we used LC3-II, BECLIN-1, and mTOR as variables to assess autophagy expression and associated signaling pathways.

There have been numerous studies on LC3-II, a factor related to autophagy, a protein involved in the development and maturation of autophagosomes, and a protein responsible for autophagy monitoring.⁹ Furthermore, LC3-II to LC3-I is reported to be a biomarker of autophagosomes, which alters I/R-induced myocardial injury. There was a significant increase in the expression of the LC3-II protein in myocardial tissue of the ketamine and ketamine + insulin-treated groups compared to the diabetic group + I/R rats. Our findings showed a significant improvement in autophagosome count in myocardial tissue of ketamine and combined insulin-treated I/R-induced myocardial injury in type 2 diabetes promoted autophagy to alleviate myocardial injury. These findings provide an understatement of the molecular mechanism underlying the protective effects of ketamine and insulin on myocardial injury. As a consequence of the I/R injury induced in the diabetic myocardium, these results indicate that there is an obvious increase in autophagy after the I/R-induced myocardial injury. Our findings are further supported by studies where ketamine showed induced autophagy to alleviate neuropathic pain by inhibiting the PI3K/Akt/mTOR pathway, and insulin inhibits osteogenesis by inhibiting autophagy through the TGF-β1 pathway.^47,48 It is consistent with our findings that previous studies reported that cardiomyocytes could activate autophagy by increasing mTOR expression and LC3-II expression under conditions of ischemia and hypoxia.^49,39 The study includes the following limitations: (1) The diabetic+I/R+Insulin group were not included in the study. (2) Additional biomarkers such as troponin levels have not been studied. (3) No additional biochemical or enzyme studies have been studied. (4) In vitro and in vivo myocardial insulin sensitivity in the presence or absence of ketamine has not been studied. (5) The additional molecular signaling cascades and bioenergetic pathway studies are needed in diabetic rats with myocardial I/R injury.

Conclusion

Based on the findings of the present study, it can be concluded that ketamine and its combination with insulin significantly increased the expression of p-mTOR of p-mTOR and decreased mTOR and LC3-II in the myocardium after I/R-induced myocardial injury was induced by I/R in rats with DM2. Furthermore, this treatment reduced myocardial ischemia/reperfusion damage by inhibiting autophagy and oxidative stress.

Footnotes

Declaration of conflicting interests

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

Funding

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

Ethics approval

Animal Ethics Committee of the Chuhu Hospital of Anhui Medical University - Approval number 2022–AMU-0034, Wuhu, Anhui, China.

Animal welfare

Animal Care and Use Guidelines (NIH) - Animal Ethics Guidelines and Care of the Chuhu Hospital of Anhui Medical University.

ORCID iD

Yuanhai Li

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