*Corresponding Author:
Mawen Lei
Department of Anesthesiology, Chenzhou No.1 People's Hospital, Chenzhou, Hunan Province 423000, China
E-mail:
leimawen@163.com
Date of Received 19 December 2022
Date of Revision 22 May 2023
Date of Acceptance 17 November 2023
Indian J Pharm Sci 2023;85(6):1745-1751  

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Abstract

To examine the effect and mechanism of dexmedetomidine pretreatment mediated adenylate adenosine monophosphate-activated protein kinase pathway on hypoxic reoxygenation injured rat cardiomyocytes. A total of sixty Sprague-Dawley rats were allocated into three groups, namely the sham surgery group, model group, and dexmedetomidine intervention group, utilizing a random number table. Each group consisted of twenty rats. The model group and the intervention group of dexmedetomidine were used to prepare a rat myocardial hypoxia reoxygenation injury model using an improved thread occlusion method. The sham surgery group only underwent thoracotomy without ligation, and the intervention group of dexmedetomidine was pretreated with dexmedetomidine before establishing the model. Western blot was used to detect adenosine monophosphate-activated protein kinase, uncoupling protein 2, and Kruppel-like factor 2 proteins; using flow cytometry to detect the average fluorescence intensity of reactive oxygen species; detection of cell apoptosis rate using terminal deoxynucleotidyl transferase dUTP nick end labeling method; using the enzyme-linked immunosorbent assay detection kit manual to detect serum interleukin-6 and tumor necrosis factor-alpha, interleukin-1 beta and the levels of various indicators such as superoxide dismutase and malondialdehyde. Adenosine monophosphate-activated protein kinase, Kruppel-like factor 2, and uncoupling protein 2 proteins in the myocardium of the model group rats were markedly lower than those of the sham operation group; adenosine monophosphate-activated protein kinase, Kruppel-like factor 2, and uncoupling protein 2 in the myocardium of rats in the dexmedetomidine intervention group were markedly higher than the model group. The superoxide dismutase level of the model group rats was markedly reduced than the sham operation group, with malondialdehyde, interleukin-6 and tumor necrosis factor-alpha, interleukin-1 beta was markedly higher than that of the sham surgery group; superoxide dismutase, malondialdehyde, interleukin-6 and tumor necrosis factor-alpha in the intervention group of dexmedetomidine were markedly higher than the model group, tumor necrosis factor-alpha, interleukin-1 beta was markedly reduced than that of the model group. Dexmedetomidine can activate adenosine monophosphate-activated protein kinase pathway, upregulation of uncoupling protein 2 expressions and the inhibition of mitochondrial reactive oxygen species production are observed, thereby restraining the oxidative irritability of myocardial tissue under hypoxia/reoxygenation conditions and playing a role in myocardial cytoprotection.

Keywords

Dexmedetomidine, titin/myosin heavy chain signal axis, myocardial ischemia-reperfusion, cardiac surgery

Myocardial Ischemia-Reperfusion Injury (IRI) is a common clinical pathophysiological phenomenon and a common postoperative adverse event in patients undergoing cardiac surgery[1,2]. At present, there is no specific means to effectively avoid the occurrence of myocardial injury after cardiac surgery, so it is of great significance to explore effective prevention and treatment measures. Studies have found that pretreatment with Dexmedetomidine (DEX) can play a protective role in myocardial function[3]. However, the specific molecular mechanism is still not fully elucidated, so the value of DEX in myocardial injury needs to be further elucidated. Titin and beta-Myosin Heavy Chain (β-MHC) are proteins closely related to myocardial function, and their expression levels are closely related to myocardial function[4]. Previous research has found that DEX can inhibit myocardial IRI through multiple signaling pathways. However, the mechanism of titin, β-MHC protein and related signaling pathway proteins in myocardial IRI, and the effect and mechanism of DEX on cardiomyocyte function remain unclear. The protective mechanism of DEX on cardiomyocytes will be further clarified by this work, and also provide new clues for clinical reduction of myocardial function injury after cardiac surgery.

Materials and Methods

General information:

Rat cardiogenic H9C2 cells were purchased as research subjects. They were divided into three groups; Blank Control Group (BCG), hypoxic incubation group (Hypoxic Group) ((HYG)) and DEX group. The BCG was the most basic culture environment, which was cultured in Dulbecco's Modified Eagle Medium (DMEM) low glucose medium of fetal bovine serum. The low oxygen concentration group was cultured under 5 % oxygen concentration environment. The DEX group was incubated in a culture environment in which the DEX concentration was 100 μm/ml.

Methods:

Cell culture and passage: The frozen storage tube of the cell line was removed from the refrigerator at -80° and rapidly melted in a water bath at 37°. The frozen cells were absorbed into a clean centrifuge tube under aseptic conditions, and an appropriate amount of fresh medium was added to mix gently. After a series of treatments, the cells were cultured, and the fresh medium was changed for passage after the cells were completely adherent to the wall.

Cell grouping and DEX co-culture: Confluent adherent cells cultured under conventional conditions were divided into three groups; BCG, HYG and Differentially Expressed Gene (DEG). The BCG was the most basic culture environment, which was cultured in DMEM low glucose medium of fetal bovine serum. The low oxygen concentration group was cultured under 5 % oxygen concentration environment. The DEX group was incubated for 24 h in a culture environment perfused with DEX concentration of 100 μm/ml.

Observation indicators:

Cell Counting Kit–8 (CCK-8) was used to measure cell viability: A 100 l cell suspension is prepared in a 96-well plate and then pre-cultured. After 24, 48 or 72 h of incubation, 10 µl of CCK-8 is added to each sample and incubated for 4 h. Absorbance at 450 nm is measured by a spectrophotometer.

Detection of apoptosis rate: After 24 h of culture, trypsin was used to digest each group's cells without the use of Ethylenediaminetetraacetic Acid (EDTA), centrifuged, and then single-cell suspension was prepared by adding 500 μl binding buffer. The suspension was mixed and incubated for 10 min at room temperature. Finally, using flow cytometry, the apoptosis rate of the cells in each group was found.

Determination of cardiomyocyte activity (myocardial zymogram, telomerase activity): The 6-well plate was taken out of the incubator for observation, and the ice box and cell scraper for protein extraction were prepared in advance. The 6-well plate was placed on the ice box, and 2 ml Phosphate Buffered Saline (PBS) was taken from each well 3 times. Cells were lysed with Radio- Immunoprecipitation Assay (RIPA) protein lysate, and the activities of myocardial zymography (cardiac Troponin I (cTnI), Lactate Dehydrogenase (LDH), Creatine Kinase-Myocardial Band (CK-MB)) were measured by Bicinchoninic Acid (BCA) assay method. Telomerase in plasma and cardiac nucleus was detected by Enzyme-Linked Immunosorbent Assay (ELISA). The procedure was carried out exactly as the reagent's instructions required. A microplate reader was used to measure the Optical Density (OD) value of telomerase in each group within 5 min of the reaction's completion in order to assess each group's activity.

Cardiomyocyte contractile related proteins titin, β-MHC and signal pathway proteins: After 24 h of treatment, the cells were washed with PBS, and the total protein was extracted. The titin and β-MHC were determined by Western blot method. According to literature reports, the myocardial infarction is closely related to the effect on myocardial cells of signaling pathways including Extracellular Signal Regulated Kinases (ERK1/2) and Phosphatidylinositol-3 Kinase (PI3K) protein, Therefore, the levels of ERK1/2 and PI3K proteins were determined by Western blot.

Data processing:

The statistical software Statistical Package for the Social Sciences (SPSS) 20.0 was employed to conduct the analysis, specifically utilizing the Chi-square (χ2) test for count data, and was used for measurement data. The t-test was employed to conduct a comparison between two distinct groups. In comparison to the control group, statistical significance was observed (p<0.05).

Results and Discussion

Compared with the BCG, the survival rate of cardiomyocytes in HYG was decreased; Compared with HYG, the survival rate of cardiomyocytes in DEG was increased as shown in Table 1. The LDH, CTnI and CK-MB in myocardial cells of rats in HYG were higher than BCG, and the LDH, CTnI and CK-MB in myocardial cells of rats in DEG were reduced than HYG as shown in Table 2. There was no discernible difference between the hypoxic group and the control group in terms of the amount of telomerase present in the cardiomyocytes. The telomerase level of cardiomyocytes in the DEG was higher than the HYG as shown in Table 3. The apoptosis rate of cardiomyocytes in HYG was higher than BCG, and the apoptosis rate of cardiomyocytes in DEG was reduced than HYG as shown in Table 4. Compared with the BCG, the titin protein was decreased and β-MHC protein was increased in the HYG, while the expression of titin and β-MHC in the DEG was higher than the HYG as shown in Table 5.

Group n Survival rate of cells
BCG 10 98.65±0.21
HYG 10 46.38±4.27a
DEG 10 75.39±5.62ab
F   26.544
p   <0.001

Table 1: Comparison of Cardiomyocyte Survival Rate

Group n LDH (KU/L) CTnI (ng/ml) CK-MB (U/ml)
BCG 10 0.25±0.03 36.87±6.52 15.46±2.35
HYG 10 1.46±0.13a 1365.48±21.57a 104.68±7.11a
DEG 10 0.77±0.11ab 349.65±21.54ab 83.97±6.47ab
F   14.683 365.574 25.621
p   <0.001 <0.001 <0.001

Table 2: Comparison of Myocardial Zymogram Levels of Cardiomyocytes (x͞ ±s)

Group n Telomerase (ng/mg)
BCG 10 56.38±3.65
HYG 10 60.64±4.82a
DEG 10 83.27±3.68ab
F   14.624
p   <0.001

Table 3: Comparison of Myocardial Zymogram Levels of Cardiomyocytes (x͞ ±s)

Group n Apoptosis
BCG 10 2.83±0.23
HYG 10 37.95±3.54a
DEG 10 18.23±2.11ab
F   16.582
p <0.001

Table 4: Comparison of Cell Apoptosis in Each Group (x͞ ±s)

Group n Titin β-MHC
BCG 10 1.03±0.05 1.03±0.03
HYG 10 0.68±0.04a 1.13±0.03a
DEG 10 0.83±0.07ab 1.05±0.04b
F   5.642 0.357
p   <0.01 0.751

Table 5: Comparison of Contractile Protein Expression in Cardiomyocytes of Rats (x͞ ±s)

Compared with the BCG, the p-ERK protein in cardiomyocytes of rats in the HYG and DEG was decreased, and the Nuclear Factor Kappa B (NF- κB) protein was increased, while the p-ERK in cardiomyocytes of rats in DEG was higher than the HYG, and the NF-κB was reduced than the HYG as shown in Table 6. The p-PI3K and phospho-Protein kinase B (p-Akt) in myocardial cells of rats in HYG and DEG were higher than those in BCG, and the p-PI3K and p-Akt in DEG were higher than HYG as shown in Table 7.

Group n p-ERK ERK NF-kB
BCG 10 1.03±0.06 1.02±0.08 1.03±0.04
HYG 10 0.57±0.05a 0.98±0.02a 1.68±0.21a
DEG 10 0.79±0.06ab 0.97±0.03ab 1.23±0.12ab
F   6.572 0.462 7.675
p   <0.01 0.658 <0.01

Table 6: Comparison of Protein Expression Levels of ERK Signaling Pathway in Myocardium of Rats (x͞ ±s)

Group n p-PI3K p-AKT
BCG 10 0.26±0.04 0.23±0.03
HYG 10 0.36±0.06a 0.37±0.04a
DEG 10 0.63±0.05ab 0.59±0.05ab
F   6.543 5.567
p   <0.01 <0.01

Table 7: Comparison of PI3K/AKT Pathway Protein Expression Levels in Cardiomyocytes of Rats (x͞ ±s)

DEX hydrochloride is a novel, highly selective and highly effective Alpha (α) 2 adrenergic receptor agonist with sedative, anti-sympathetic excitability, analgesic and mononuclear properties[5,6]. DEX, a clinically highly selective α2 adrenergic receptor agonist, has been used in cardiac surgery to attenuate cardiovascular responses. DEX attenuates hemodynamic response to endotracheal intubation in patients undergoing cardiac surgery. Intravenous administration of DEX before cardiopulmonary bypass can attenuate the cardiovascular response to skin excisions and sternal splits[7]. DEX has been reported to stabilize hemodynamics during cardiac surgery in both adults and children. Although DEX has side effects of causing bradycardia and hypotension, it has a significant effect on reducing catecholamine secretion and maintaining hemodynamic stability during cardiac surgery[8]. Pasero et al.[9] found that intra-coronary infusion of DEX could significantly improve the myocardial contractility and reduce the plasma concentration of norepinephrine after ischemia-reperfusion in the innervated area of the left anterior descending coronary artery of porcine, which may be related to stimulation of cardiac presynaptic α2-adrenoceptor and decrease the concentration of norepinephrine in the ischemic area[10]. Previous research has found that ketamine combined with DEX has a myocardial protective impact compared with sevoflurane with sufentanil. DEX can reduce myocardial damage after cardiac surgery, however, the specific mechanism of the myocardial effect of DEX is still unclear[11].

The structural protein titin gene and the contractile protein β-MHC gene of cardiomyocytes are important functional protein genes regulating the contractile function of cardiomyocytes. During the development of cardiomyocytes, titin is expressed earlier than myosin and cTn[12,13]. Related experiments have also shown that Bone Marrow-Derived Mesenchymal Stem Cells (BMMSCs) can form connections with cardiomyocytes in mixed culture with cardiomyocytes, and the expression of titin is significantly enhanced. Some cells have sarcomele- like structures, indicating that titin plays an essential role in the early differentiation of mesenchymal stem cells into cardiomyocytes[14]. Titin is intimately associated with several different heart functions, including coagulation, myocardial contraction, myocardial fiber development, myocardial hypertrophy, myocardial tissue morphogenesis and other biological processes. Previous studies have shown that titin fragment in plasma is upregulated when it dissociates from cardiomyocytes during myocardial infarction caused by long-term ischemia, and when myocardial tissue dies, the contraction of striated myocardial tissue changes. This process is probably due to the degradation of actin by Matrix Metalloproteinase (MMP)-12[15]. Titin, especially exon N2B, can be used as a new biomarker specifically associated with cardiac injury in serum[16]. In this study, it was found that the level of titin in rat cardiomyocytes in DEG was higher than that in HYG, revealing that DEX could reduce the degradation of titin and play an essential role in cardiomyocytes. However, the specific changes in actin phosphorylation and actin splicing require further investigation to elucidate the specific role leading to actin modification[17].

Myosin is a characteristic protein of cardiomyocytes and a major component of thick myofilaments[18]. Before induction, MSCS showed weak expression of β-MHC gene, and the β-MHC gene was enhanced 1 d, 4 d, 7 d and 14 d after induction[19]. Studies have shown that when vitamin C induces Embryonic stem cells (Es) to differentiate into cardiomyocytes, β-MHC gene expression is also present before induction, and the expression gradually increases after induction, reaching the peak at 10 d, and then gradually decreases[20]. After the interference of titin gene expression, the distribution of β-MHC was dispersed, and the expression of the β-MHC gene did not alter considerably from before the interference, and the arrangement of myofibrils was disordered[21].

Myocardial zymography is an index for differentiating myocardium, skeletal muscle and brain injury. CK-MB is a dimer composed of brain-type subunits and myotype subunits. When patients have myocardial injury, the level of CK-MB increases significantly, which is one of the sensitive indicators of myocardial tissue injury[22]. CTnI is a regulatory protein of myocardial muscle contraction. Under normal circumstances, the level of CTnI is very low, but when myocardial injury, the level of CTnI is significantly increased. CTnI is an important index to evaluate myocardial injury. LDH exists in almost all tissues, most abundant in heart, skeletal muscle and kidney, and is a reducing oxidase of pyruvate, the final product of glucose colysis[23]. In this study, all indicators of myocardial enzymes after hypoxic culture showed serious injury of cardiomyocytes, but all indicators tended to be improved after DEX addition. One of the traditional Mitogen- Activated Protein Kinases (MAPKs) signaling pathways, the ERK signaling pathway is essential for the growth and differentiation of cardiomyocytes. Abnormal ERK signaling pathway can lead to coronary atherosclerosis, myocardial infarction, myocardial hypertrophy and other cardiovascular diseases[24,25]. Stress in and out of cardiomyocytes can affect the activation process of ERK signaling pathway. Activation of ERK signaling pathway can significantly increase the phosphorylation level of ERK protein, and then affect the expression of downstream target protein NF-kB, and regulate cell proliferation and apoptosis[26,27]. An essential channel for membrane receptor signal transmission into cells, the PI3K/Akt signaling pathway controls various cellular processes, including cell proliferation, death, metastasis, and other cellular processes[28]. Recent studies have found that DEX can alleviate LPS-induced lung cell apoptosis and mitochondrial apoptosis signal activation by activating PI3K/Akt signaling pathway, and then improve acute lung injury[29]. In this research, it was discovered that the p-ERK, p-PI3K and p-Akt in rat cardiomyocytes in DEG were higher than HYG, indicating that DEX could activate ERK1/2 and PI3K signaling pathways to protect cardiomyocytes after ischemia-reperfusion.

In conclusion, DEX’s ability to prevent cardiac IRI may be due to its ability to activate the ERK1/2 and PI3K signaling pathways, which prevents cardiomyocyte apoptosis and titin degradation.

Authors’ contributions:

This study was supported by scientific research program from Hunan province health and wellness committee (No. 202204113585).

Conflicts of interests:

The authors declared no conflict of interests.

References