The efficacy and mechanism of dexmedetomidine in myocardial apoptosis via the renin–angiotensin–aldosterone system

Abstract

Introduction:

Pharmacological preconditioning limits myocardial infarct size after ischemia/reperfusion. Dexmedetomidine is an α₂-adrenergic receptor agonist used in anesthesia that may have cardioprotective properties against ischemia/reperfusion injury. We investigated whether dexmedetomidine induces cardioprotection against myocardial apoptosis injury.

Methods:

In order to assess the role of dexmedetomidine on myocardial apoptosis, we established a grave scalding rat model. Blood and myocardial tissue from the ventriculus sinister were harvested, then troponin, myocardial apoptosis, and expression of caspase-12, GRP78, and CHOP were assessed.

Results:

Dexmedetomidine significantly reduced myocardial apoptosis, improved functional recovery, and reversed myocardial injury induced by grave scalding. The heart rate in the five groups studied was significantly different (p < 0.05). The number of buffy-stained nucleoli in the myocardial cell was highest in the simple scald group. The expression of caspase-12 obviously increased in the simple scald group. The expression of GRP78 and CHOP increased in the simple scald and scald and 50 μg/kg dexmedetomidine groups (p < 0.05).

Conclusions:

The results show that dexmedetomidine (DEX) produces cardioprotection against myocardial apoptosis injury. DEX is not only a useful sedative, but also plays a pivotal role in anesthetic cardioprotection. The potential benefits of DEX protection in high risk cardiovascular patients undergoing surgery are enormous.

Introduction

Dexmedetomidine (DEX) is a potent and highly selective α₂-adrenergic receptor agonist used in anesthesia. Clinical evidence suggests that induced autonomic nervous system modulation during perioperative administration of DEX is associated with a trend towards improved cardiac outcomes following non-cardiac surgery.^1–4 Previous experiments on animals have shown the benefits of DEX administration in the ischemic heart.^5–7 Cardioprotective effects have also been reported in global ischemia of isolated rat hearts. ⁸ Dexmedetomidine is a new type of highly selective and specific α₂-adrenergic receptor agonist which possesses sedative, antianxiety, and analgesic effects that are dose dependent. It often is used during the process of operations on burn patients. Myocardial damage, which is also known as heart shock, is one of the grave complications in burn patients. Its major mechanism is ischemia–reperfusion injury, out of control inflammation, the dysbolismus of oxygen and energy, and myocardial apoptosis of different degrees. Some studies have demonstrated that DEX could protect the organs by lessening the inflammation and the damage caused by ischemia–reperfusion (I/R) injury.^9–11 However, perioperative myocardial ischemia and infarction are limited to a region of hypoperfused myocardium. Currently, whether DEX has preconditioning action against regional I/R injury remains to be further investigated, as well as whether DEX causes activation of signaling pathways associated with cardiac survival. However, determination of the efficacy and mechanism of DEX in myocardial apoptosis has yet to be reported using a grave scalding rat model. We hypothesize that DEX could exert direct protective effects against myocardial apoptosis and damage induced by grave scalding.

Materials and methods

Animals

This study conformed to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH, Publication No. 85-23, revised in 1996), and was approved by the Institutional Ethics Review Committee, Fujian Medical University. The SPF grade male Sprague Dawley (SD) rats (purchased from the Medical Laboratory Animal Center in Fujian Medical University, Fujian, China) weighed between 0.22 and 0.28 kg. The rats were quarantined and acclimatized one week before the experiments in the Animal Laboratory of Fujian Medical University. Throughout the study, the experiments were performed on the rats under the following standard conditions: room temperature 23±2°C, relative humidity 60%±10%, and alternating 12-hour light–dark cycles (8am to 8pm). All experimental procedures conformed to the Statement for the Use of Animals and were approved by the Medical Ethics Committee of Fujian Medical University.

Animal experiments procedure

The grave scalding rat model was established as follows: the rats were anesthetized by intraperitoneal injection of 40 mg/kg napental, the hair on the back shaved, and the back then immersed in 94°C hot water for 12 s until 30% of the body surface area had third-degree burns. The 30 SPF male SD rats were randomly divided into five groups (n = 6): normal control (group C), simple scald group (group B), scald and 50 μg/kg dexmedetomidine group (group D1), scald and 30 μg/kg dexmedetomidine group (group D2), and scald and 10 μg/kg dexmedetomidine group (group D3). In group C, the rats were fake burned by immersing them into 37°C warm water for 12 s. The 2 μg/ml dexmedetomidine was prepared by isotonic saline solution. In group D1, the rats were injected with 50 μg/kg dexmedetomidine intraperitoneally after the grave scalding was established, and in groups D2 and D3, respectively, 30 μg/kg and 10 μg/kg dexmedetomidine were separately injected by intraperitoneal injection after the grave scalding was established. The rats in groups D1, D2, and D3 were injected by fluid infusion of isotonic saline solution intraperitoneally according to the formula of Parkland. The rats in all the groups were bred in a single cage that enabled them to take food and drinking freely. Blood pressure and heart rate were atraumatic measured at 0, 3, 6, 9, and 12 h. Blood and myocardial tissue from the ventriculus sinister were harvested and were divided into three portions before being assayed for troponin, myocardial apoptosis, and the expression of caspase-12, GRP78, and CHOP by immunohistochemistry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and western blotting.

Immunopathology and immunohistochemistry

Myocardial tissue blocks from the ventriculus sinister were harvested and studied using standard histologic and immunohistochemical analysis. Six millimeter serial paraffin sections were prepared for immunohistological study by the avidin–biotin–peroxidase complex method and for hematoxylin–eosin (H&E) staining. Myocardial tissue blocks were fixed in 4% paraformaldehyde and then embedded in paraffin. Serial sections were cut and stained with hematoxylin–eosin. The sections were analyzed under a light microscope. The myonecrosis, inflammatory cell infiltration, and edema were evaluated in the section.

Serial paraffin sections were prepared, deparaffined, and dehydrated, incubated in 3% H₂O₂ for 20 min, put in a 92–98°C microwave oven for 4 min, sealed off by goat serum, labeled by mouse polyclonal antibody (caspase-12 polyclonal antibody, 1:50 dilution) and by biotin-conjugated goat anti-rabbit immunoglobulin-G (American Qualtex, CA), which was depleted of cross-react anti-rat immunoglobulin-G. The photos were collected using an Olympus BX51 automated image acquisition system (R&D Corporation).

TUNEL assay

The TUNEL assay was operated as follows: after sacrifice, the myocardial tissue blocks were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated through ethanol and xylene, and embedded in paraffin. Five-micron thick sagittal sections through the myocardial tissue blocks were obtained and mounted on poly-l-lysine-coated glass slides. The ApopTag peroxidase kit (Oncor, Gaithersburg, MD) was applied to paraffin sections according to the manufacturer’s instructions. Briefly, residues of digoxigenin nucleotide were catalytically added by TdT to the 3′-OH ends of double- or single-stranded DNA. The labeled product was visualized using diaminobenzidine (DAB), which yielded brown granules mainly localized on apoptotic cells. Following this, the sections were counterstained with 1% methyl green. Omission of TdT or digoxigenin nucleotide gave completely negative results (not shown). Comparisons of levels of neuronal cell death were determined from the number of morphologically intact cells and the number of deep brown-stained, TUNEL-positive, damaged myocardial cells in the ventriculus sinister. Apoptotic cells were identified by the distinctive condensed or fragmented nuclear structure within cells stained with the TUNEL assay (KeyGEN). The number of TUNEL-positive cells was presented as a percentage of the total cardiomyocytes. Preparations were imaged using a bright field microscope (Nikon Microscope Eclipse E600W, Tokyo, Japan) and were photographed with a microscope digital camera (Nikon DP70, Tokyo, Japan).

Transmission electron microscopy

The myocardial tissue blocks, which were fixed by 5% glutaraldehyde in 4°C for 3 h, rinsed using 0.1 mol/L PBS with 6% cane sugar, fixed by 2% OSO₄ for 1 h, gradiently dehydrated by acetone, blocked dyed by 1% uranyl acetate (dispensed by dehydrated alcohol) for 1 h, embedded in epoxide resin, located by serial section (1–2 μm), were ultramicrocut (50–70 nm), double stained by uranyl acetate and medlar hydrochloric acid lead, and observed and photographed using a Hitachi H-600 transmission electron microscope.

Western blotting analysis

Western blot analysis was performed using a previous method. ¹² In brief, the cytoplasm and nuclear protein samples (30 mg per lane) were prepared with Nuclear Extract Kit (Active Motif, Carlsbad, CA) and measured by a BCA protein assay kit (Pierce, Rockford, IL), were mixed with 6×SDS reducing sample buffer and boiled for 5 min before loading. Proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE gel) and transferred electronically onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% non-fat milk in TTBS (50mM Tris (pH 7.5), 0.9% NaCl, and 0.1% Tween-20) for 1 h at room temperature, incubated with primary antibodies against GRP or CHOP (1:1000) overnight at 4°C (Cell Signaling, USA), and then with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The secondary antibody was biotin-conjugated goat anti-mouse immunoglobulin-G (American Qualtex, CA), which was depleted of cross-react anti-rat immunoglobulin-G. All primary antibodies were used at a dilution of 1:1000. Subsequently, membranes were incubated with secondary antibodies at a 1:5000 dilution at 37°C for 30min. The blots were visualized with ECL-Plus reagent (Santa Cruz, USA) and analyzed with Quantity One System image analysis software (Bio-Rad, USA) and then the protein amount of target protein and β-actin protein was analyzed by Western Blot.

Statistical analysis

All values are presented as the mean ± standard deviation (SD). All statistical analyses were performed with SPSS software, version 13.0. The number of cells stained positively by immunohistochemistry for each first antibody was compared between the control and the treated groups using the same nonparametric test. The other data were compared by one-way analysis of variance, and a probability value of 0.05 was considered statistically significant.

Results

The assay of the heart rate of the rat

The heart rate (HR), which varied between the five groups, was significantly different (p < 0.05). The HR at different times also varied and was significantly different (p < 0.05). The HR in the simple scald group was obviously higher than that in the four other groups, which could be due to the stress-accelerated heart beat caused by scald. The assay of blood pressure in the five groups was within the range of normal values (Table 1).

Table 1.

The effect of dexmedetomidine on the heart rate (HR) of the rats (n = 10).

Group	3 hours	HR	9 hours	12 hours	p
		6 hours
B	363.67±5.74	363.33±5.89	367.65±5.25	369.33±4.99	<0.05
C	304.67±7.62	306.5±5.11	305.33±9.06	307.5±7.11	<0.05
D1	326.0±6.45	326.83±5.59	328.17±8.11	345.5±8.91	<0.05
D2	333.33±7.98	331.17±9.05	331.17±6.50	331.83±5.08	<0.05
D3	343.0±6.38	341.17±4.05	342.83±8.37	341.0±6.64	<0.05
p	<0.05	<0.05	<0.05	<0.05	<0.05

The results of immunopathology

Myocardial tissue blocks from the ventriculus sinister were harvested and studied using standard histologic analysis. It was shown that the structure of myocardium was integrated and clear; fusiform myocardia were almost the same size with the clarity of nuclear structure. The cardiac myocyte from the ventriculus sinister was observed clearly to be normal cardiac myocyte and cardiac muscle fiber, without edema and degeneration in the normal control group (group C). The cardiac myocyte in the simple scald group (group B) was obviously edema, with mesenchymal hyperemia and myofibrosis cordis; the transverse striation even vanished and exhibited cloudiness. In the scald and 50 μg/kg dexmedetomidine group (group D1) and the scald and 10 μg/kg dexmedetomidine group (group D3), the cardiac myocyte was slightly less edema with mesenchymal hyperemia and myofibrosis cordis than that in the B group. In the scald and 30 μg/kg dexmedetomidine group (group D2), the cardiac myocyte was less edema with mesenchymal hyperemia and myofibrosis cordis than that in the group D1 and D3. The rat pathology and morphology of myocardium in the scald and 30 μg/kg dexmedetomidine group (group D2) were less deteriorative than that in group D1 and D3. This indicated that the efficacy of 30 μg/kg dexmedetomidine on the myocardial cellular apoptosis may be optimal, compared with 50 μg/kg and 10 μg/kg dexmedetomidine (Figure 1(a)).

Figure 1.

The results of (a) of immunopathology, (b) TUNEL, and (c) TEM assays.

The results of TUNEL assay

Myocardial tissue blocks from the ventriculus sinister were harvested and studied using TUNEL assay. The normal nucleolus of myocardial cell should be blue-stained. In the simple scald group, the nucleolus of a myocardial cell was obviously stained buffy, with chromatin agglutination and condensation, which indicated that the apoptosis of myocardial cell was stained buffy. The number of buffy-stained nucleoli of myocardial cell was highest in the simple scald group. The number of buffy-stained nucleoli of myocardial cells in the D1 and D3 groups was more than that in the normal control group. The number of buffy-stained nucleoli of myocardial cells in the D2 group was less than that in the D1 and D3 groups, which was nearly the same as that of the normal control group. This indicated that the efficacy of 50μg/kg dexmedetomidine on the myocardial cellular apoptosis may be optimal, compared with 30 μg/kg and 10 μg/kg dexmedetomidine (Figure 1(b)).

Transmission electron microscopy

Myocardial tissue blocks from the ventriculus sinister were harvested and studied using TEM. The microstructure of myocardial tissue blocks was clear and integral, including undamaged nucleolus, regular assay of endoplasmic reticulum, and amount of mitochondria in the normal control group. In the simple scald group, the microstructure of myocardial tissue blocks was unclear and incomplete, including damaged nucleolus and swell mitochondria. It was observed that the edema and discreteness of rough endoplasmic reticulum, the chaotic and swell structure of mitochondria, and their number decreased. In the D2 and D3 groups, it was also observed that the microstructure of myocardial tissue blocks was unclear and incomplete, including damaged nucleolus and swell mitochondria. However, in the D2 group, the microstructure of myocardial tissue blocks was better than that in the D1 and D3 group with the integrity of structure, regular assay of endoplasmic reticulum and amount of mitochondria (Figure 1 (c)).

The analysis of caspase-12 by immunohistochemistry assay

Caspase-12 was located on the endoplasmic reticulum in the myocardial cells, distributed by macrobeads. The positive proteinic expression of caspase-12 was identified by blue staining in the cytoplasm. The expression of caspase-12 obviously decreased in the normal control group compared with that in the simple scald group, with the least amount. The amount of caspase-12 was the most in the simple scald group. However, the expression of caspase-12 obviously increased in the D1 and D3 groups compared with that in the D2 group. The amount of caspase-12 in the D1 group was nearly equal to that in the normal control. This indicated that the effect of 30μg/kg dexmedetomidine on the inhibition of the myocardial cellular apoptosis may be the better than that in the D2 and D3 groups (Figure 2).

Figure 2.

The effect of dexmedetomidine on caspase-12 by immunohistochemistry assay.

The expression of GRP78 and CHOP by western blot assay

The proteinic expression of GRP78 increased in the B and D1 groups, which were higher than that in the normal control (p < 0.05). The proteinic expression of GRP78 in the D2 and D3 groups was less than that in the B group (p < 0.01). The proteinic expression of GRP78 in the D2 group was less than that in the D3 group (p < 0.05).The proteinic expression of CHOP in the B, D1, D2, and D3 groups was more than that in the normal control (p < 0.05). The proteinic expression of CHOP in the D2 and D3 groups was less than that in the B group (p < 0.01). The results demonstrated that the inhibitory effect of 30 μg/kg dexmedetomidine on the expression of GRP78 and CHOP may be the better than that in the 50 μg/kg and 10 μg/kg dexmedetomidine groups (Figure 3).

Figure 3.

The effect of dexmedetomidine on the proteinic expression of GRP78 and CHOP by western blot assay.

Discussion

Dexmedetomidine (DEX) is an α₂-adrenergic agonist that shares physiologic similarities with clonidine. It currently is approved by the FDA for continuous infusions up to 24 h in adult ICU patients who are initially intubated and received mechanical ventilation as well as for being monitored anesthesia care.^13,14 DEX cardioprotective effects on global ischemia in an isolated rat heart model. DEX administration prior to global ischemia and reperfusion decreased coronary flow and decreased myocardial infarct size. DEX-induced coronary vasoconstriction by α2-adrenergic receptor stimulation decreased coronary flow, induced myocardial ischemia and triggered ischemic preconditioning of the heart. Previous work in human volunteers showed coronary vasoconstriction and reduction of coronary blood flow, but a parallel reduction of myocardial oxygen demand. No ischemic episodes were reported in this trial. ¹⁵ From a molecular point of view, almost every cardioprotective strategy available activates the canonical signaling pathways associated with cell survival. Erk, Akt, and eNOS are normally activated during I/R events, but at insufficient levels to produce cardioprotection. A more powerful stimulus, either mechanical or pharmacological, is required to confer protection. Several agents have shown to confer cardioprotection by binding to their specific G-protein coupled receptor. Ligand binding at the G-protein coupled receptor results in PI3K/Akt and Ras-MEK1-2-Erk1/2 signaling cascades activation. DEX is a known potent agonist of the α2-adrenergic receptor. The impact of these effects must be considered especially for the patient with CHD. There are very limited data available describing the impact of dexmedetomidine on patients with myocardial damage induced by grave scald or burn.^16,17 In contrast, for specific patient populations such as patients with myocardial damage induced by grave scald or burn, relative slowing of the heart rate may be beneficial.

Our studies demonstrated that dexmedetomidine could step down HR within the normal range, compared with the simple scald group, which indicated that dexmedetomidine could prevent the heart from the damage of shock heart induced by scalding. On the other hand, dexmedetomidine could inhibit the expression of GRP78 (glucose-regulated protein 78) and caspase-12 induced by scalding, compared with the simple scald group. Dexmedetomidine could inhibit the myocardial cellular apoptosis, also evidenced by the result of TUNEL assay.

It was reported that caspase-12 play an important role in myocardial ischemia- reperfusion injury by ERS (endoplasmic reticulum stress), could interfere in the activity of ERS and caspase-12 excessively, therefore, it may be the new drug target to prevent and cure the myocardial ischemia- reperfusion injury.^18–21 Caspase-12 is in the endoplasmic reticulum as one of the plasmalemmal composite protein, which is located in the hyalomitome of the endoplasmic reticulum as the form of inactive prosoma. It could be activated by endoplasmic reticulum stress. ERS is one of the cellular self-care mechanism, which could mediate the up-regulation of GRPs, CRT, and protein foldase, enhance the capacity of cellular stress tolerance, recover the function of endoplasmic reticulum. However, excessive duration of ERS could lead to ERAD (endoplasmic reticulum associated dead) and the damage of myocardial tissue and cells. The activation of caspase-12 could make the ERS induce apoptosis all by itself, which is the onset factor of apoptosis. Caspase-12 plays a critical role in the process of ERAD.^22,23 The inhibitory effect of dexmedetomidine on the expression of caspase-12 and GRP78 may be one of the mechanism of myocardial injury healing. GRP78 is also located in the endoplasmic reticulum, separated from the compound without binding to the unfold protein, when ERS happens, which could make the activation and release of caspase, leading to the myocardial apoptosis, ¹⁸ Excessive ERS could result in the clearage of the GRP78, caspase-7, and caspase-12 composites, inducing the myocardial apoptosis. ¹⁸

The present study shows that dexmedetomidine produces cardioprotection against myocardial apoptosis injury. Also, it demonstrates activation of cardiac kinases associated with cellular survival in a receptor-mediated manner. These facts support the idea that DEX is not only a useful sedative, but also plays a pivotal role in anesthetic cardioprotection. The potential benefits of DEX protection in high risk cardiovascular patients undergoing surgery are enormous.