The influence of scald and anoxia to the expression of Glycine receptor in rats’ myocardium and the co-localization study of Glycine receptor and endoplasmic reticulum

doi:10.21203/rs.3.rs-3150507/v1

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The influence of scald and anoxia to the expression of Glycine receptor in rats’ myocardium and the co-localization study of Glycine receptor and endoplasmic reticulum

https://doi.org/10.21203/rs.3.rs-3150507/v1

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PURPOSE The objectives of the current study were to demonstrate the influence of scald and anoxia to the expression of glycine receptor in rats’ myocardium and the co-localization of glycine receptor and endoplasmic reticulum.

METHOD (1) Effect of hypoxia on Gly Rα1 expression : Rat cardiomyocytes line H9C2 were cultured and divided into 3 groups randomly: the Glycine group, the Hypoxia group and the Glycine+Hypoxia group and incubated for 0 h, 3 h, 6 h, 12 h, 24 h or 48 h. A total of 24 SD rats were divided into 4 groups: the Control group, the Glycine group, the Scald group and the Glycine+Scald group.The Western blot method detected the expression of Gly Rα1 in each group. (2) Effect of hypoxia on colocalization of GlyR and ER: We labeled ER with Rodamine (red), GlyRα1 (green) with FITC, DNA (blue) with DAPI, and finally, photographed cells with laser confocal microscopy . H9C2 were cultured and divided into 3 groups randomly (the Glycine group, the Hypoxia group and the Gly+Hyp group) and ncubated for 0 h, 3 h, 6 h, 12 h, 24 h or 48 h. Mean density of Gly Rα1 in Each group of time phase was detected.

RESULT glycine obviously decreased the mortality rate of H9C2 cells under anoxic condition. Moreover, the expression level of GlyRα1was found to be increased in a time dependent manner under anoxic condition, and glycine could conspicuously reverse the increased expression of GlyRα1. Furthermore, we confirmed that GlyRα1 localized on ER in cardiomyocyte and the combination of glycine with GlyRα1 on ER caused cytoprotective effect by potential membrane changes. In vivostudies, the protective effects of glycine was also investigated on burned rat models.

CONCLUSION the present study confirmed glycine could protect myocardial cells from being injured through reducing the expression of GlyRα1 under anoxic condition both in vivo and in vitro assays, and combination of glycine with GlyRα1 happened on ER of myocardial cells.

anoxia

glycine receptor

endoplasmic reticulum

myocardial cell

Ischemia and anoxia induced by severe burn may lead to multiple organ failure. Our previous studies have shown that severe burn could cause myocardium ischemia and anoxia injury, which may precede a notable descent of blood volume by increasing capillary permeability. Furthermore, some studies showed that leakage of myocardial-specific structural proteins and histopathologic changes may occur after severe burn, thus cardiac pumping deficit and decreased circulating blood volume could be induced, which are important initiating factors for other organ hypoxic injuries, such as liver, kidney and intestine ⁽¹⁵⁾. In severely burned patients, multiple organ dysfunction syndrome (MODS) has been reported to occur at a 28.1% incidence and is very difficult to reverse ⁽⁵⁾. In addition, MODS is also associated with a high mortality rate, ranging from 78–98%. Despite numerous kinds of prophylactic treatments of MODS has improved survival rate in recent years, MODS remains a main lethal complication in severely burned patients ⁽¹¹⁾.Therefore, effective protection on myocardium are urgently needed, which may prevent organs from ischemia and anoxia, reduce medical complications and improve the prognosis of patients.

In our previous studies, it was revealed that glycine has no effect on the survival rate, examined by trypan blue exclusion, of norm-cultured-cardiomyocytes, wherase it could significantly reduce the mortality rate of hypoxic-cultured-cardiomyocytes in vitro assay. Mechanistically, glycine was discovered to decrease the amount of lactate dehydrogenase (LDH) and creatine kinase in hypoxic cardiomyocyte culture liquid. And different concentrations of glycine could diminish LDH release in myocardial cells. Moreover, animal experiments showed that glycine has a protective effect on the myocardium of severely burned rats.

Glycine is a well-documented cytoprotective agent and has cytoprotective and modulatory effects in different cells types. Modulatory effects were mainly described in immune cells, endothelial cells and macroglial cells, where glycine could modulate proliferation, differentiation, migration and cytokine production. Cytoprotective effects were mainly described in renal cells, hepatocytes and endothelial cells, where glycine could protect cells from ischemic cell death. In these cells, glycine has been suggested to stabilize porous defects that develop in the plasma membranes of ischemic cells, and can lead to leakage of macro molecules and subsequent cell death. Although there is some evidence linking these effects to the activation of glycine receptors (GlyRs), different myocardial cells subtypes seem to operate in entirely different modes. GlyRs are pentameric proteins belonging to the cys-loop family of ligand-gated ion channels. GlyRs are able to form homomeric receptors, composed of α subunit, or heteromeric receptors, composed of α and β-subunits and in a putative 3β/2α or 2β/3α stoichiometry ⁽³⁾. Activation of GlyRs leads to post synaptic hyperpolarization induced by chloride influx or a shunt effect characterized by a drop in membrane resistance consecutive to enhanced chloride conductance. Other studies found that human embryonic kidney cells were not protected from ischemic cell death by glycine, unless they were transfected with plasmid constructs expressing the GlyRα1 subunit. GlyRα1 mutants, Y202F or Y202L, which are shown to decrease glycine and strychnine binding, also specifically abolished the effects of glycine, further suggesting the involvement of glycine receptor subunits in cytoprotection ^{(2, 7, 9)}.

The present study aims to confirm GlyRs exists on myocardial cells membrane and glycine could protect myocardial cells in severely burned SD rats. Moreover, colocalization of GlyRα1 and endoplasmic reticulum (ER) was performed to further investigate the mechanism of glycine-induced ER pathway.

Experimental Animals

Healthy 2-month old Sprague-Dawley rats (200 g–250 g, n = 24) were used in this study. The animal experiments were conducted according to protocols reviewed and approved by the Animal Experimental Ethics Committee of the Lanzhou University, Lanzhou, China (Permit Number SYXK(甘)2010-0012 ). Animals were allowed 5 days to acclimate to their surroundings. Food and water for rats were available and libitum throughout the experimental protocol. This study conforms to the NIH Guide for the Care and Use of Laboratory Animals (NIH publication no. 85–23, revised 1996).

H9C2 Cells

The rat myocardial cell line H9C2 was obtained from Chinese Academy of Science. Upon reaching 80% confluence, H9C2 cells were subcultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 5% fetal calf serum at 37°C, 5% CO₂, in humidified air with medium changed every 72 h. And passage 1 cells were frozen in liquid nitrogen, and then stored at -80°C.

Effect of anoxia on the expression of GlyRα1

Passage cells were cultured in 6 cm-plate and incubated for 24 h to allow attachment. Then, cells were subcultured in low-glucose DMEM. Upon reaching 80% confluence, cells were observed and photographed. A total of 72 plates were cultured and divided into 3 groups randomly: the Glycine group, the Hypoxia group and the Glycine + Hypoxia group (n = 24 for each group). The incubator with a hypoxic mode was filled with mixed gas (5% CO_2, 94% N₂) and the concentration of O₂ was monitored. When the concrentration of O₂ decreased to 0%, the plates were placed in the incubator with a hypoxic mode for 0 h, 3 h, 6 h, 12 h, 24 h or 48 h. Cells were harvested and washed by PBS before total protein being extracted. Three independent experiments were performed for each time point.

Effect of anoxia on the colocalization of GlyRα1 and ER

Following incubation, cells grown on glass coverslips were placed in 6 cm-plates after sterilized under ultraviolet light. Firstly, passage cells were incubated for 24 h to allow attachment. Then, the cells were subcultured in low-glucose DMEM. Upon reaching 50% confluence, a total of 48 plates were divided into 3 groups randomly (the Glycine group, the Hypoxia group and the Gly + Hyp group) and incubated for 0 h, 3 h, 6 h, 12 h, 24 h or 48 h (n = 8 for each group) as described previously. Cells were harvested for next experiments. Three independent experiments were performed for each time point.

Western blotting

After abandoning the culture solution, hypoxia-treated-myocardial cells were washed thrice by PBS and homogenized by RIPA lysate and PMFS (300 µl per plate). The homogenates were then centrifuged for 30 min at 12, 000 × g at 4°C, the clarified supernatant was removed and protein concentration was determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL). Finally, SDS buffer was added and the mixture was heated at 95°C for 5 min. For western blotting, 20 µg protein was loaded per lane and resolved by SDS-PAGE. Resolved protein was then transferred onto a PVDF membrane (Millipore, Bedford, MA). Non-specific binding was blocked by PBS supplemented with 10% non-fat dry milk for 2 h at room temperature. Then, the membrane was incubated with primary antibody, including anti-β-actin antibody at a dilution of 1:1000 and anti-GlyRα1 antibody at a dilution of 1: 500 at 4°C overnight. Next, membrane was washed 5 times in TBS-T and incubated with horseradish peroxidase secondary antibody at a dilution of 1:10, 000 in TBS-T for 2 h at 37°C. Protein bands were visualized using enhanced chemiluminescence. The time of exposure was depended on the indication. Results were photographed and scant by Image-Pro 6.0 software.

Indirect immunofluorescent staining and laser confocal microscopy

H9C2 cells grown on glass coverslips were placed in 3.5 cm-plates and fixed with 4% paraformaldehyde. Then, the cells were made permeable with 0.1% TritonX-100 treatment. After that, the cells were blocked in immune fluorescence blocking buffer (1 ml/plate) for 1 h at room temperature, followed by anti-GlyRα1 primary antibody treatment at a dilution of 1:500 at 4°C overnight. For detection, the cells were treated with FITC-conjugated goat anti-rabbit secondary antibodies at a dilution of 1:500, DAPI nucleolus stains at a dilution of 1:10000 and Rhodamine-conjugated Cyto Painter ER Staining. Finally, the cells were photographed by laser confocal microscopy. Excitation light was FITC excitation light, Rhodamine excitation light and ultraviolet excitation light (Olympus FV1000, Tokyo, Japan).

Groups and Burn Procedure

A total of 24 rats (2-month old) were divided into 4 groups randomly: the Control group, the Scald group, the Glycine group and the Scald + Glycine group (n = 6 for each group). Animals of the Control group and the Scald group were injected with normal saline intraperitoneally. Animals of the Glycine group and the Scald + Glycine group were injected with 1 g/L 2.2% glycine intraperitoneally. Animals of the Scald group and the Scald + Glycine group were injected with 10% chloral hydrate (30 mg/kg) intraperitoneally; next,, hair on the back of the rats was shaved under anesthesia, and the nude skin was scalded through a 98°C water bath for 18 s, sufficient to cause a 3rd degree burn encompassing 30% total body surface area, which was confirmed post-mortem. The other groups were performed in the same procedure, except that the temperature of the water bath was 37 ℃. Six hours later, myocardial tissues were harvested under anesthesia, washed by PBS, frozen in liquid nitrogen and stored at -80°C.

Myocardium assay

Myocardium tissues were homogenized with liquid nitrogen, then the tissues were centrifuged for 30 min at 12, 000 × g at 4°C with RIPA lysate and PMFS added. Protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL). Finally, SDS buffer was added and the mixture were heated at 95°C for 5 min. For western blotting, 20 µg protein was loaded per lane and resolved by SDS-PAGE and processed for detection as described previously.

Effect of anoxia on the expression of GlyRα1 and the protection of glycine on hypoxic myocardial cells

As mentioned above, anoxia could lead to a higher mortality rate and a higher expression level of GlyRα1 in myocardial cells in a time dependent manner (illustrated in Figs. 1 and 2). Besides, the rat myocardial cell line H9C2 has been extensively studied as a model for investigating the relationship among glycine, GlyRα1 and anoxia. In the present study, we found that glycine decreased the mortality rate of H9C2 cells (Fig. 1). It has been shown that glycine significantly reduced the death of hypoxic cells but had no effect on normal myocardial cells (Fig. 1). Additionally, we observed the expression of GlyRα1 of each group in different time points by Western blotting and densitometry, and found that the expression of GlyRα1 increased gradually in a time dependent manner under hypoxic conditions, especially at 6 h, 12 h, 24 h and 48 h (Fig. 2B). Moreover, the levels of GlyRα1 of the Hypoxic + Glycine group statistically increased from 0 h to 6 h, while the trend was slowed from 12h to 48h, strikingly no difference between 0h and 48h (Fig. 4B). Furthermore, the expression of GlyRα1 in the Glycine group displayed no obvious difference among different time points (Fig. 3B). Together, these results suggested that anoxia could promote the expression of GlyRα1 in myocardial cells and glycine could inhibit the death of myocardial cells. However, the detailed mechanisms by which glycine exerts its protective effect remain obscure.

Effect of glycine on the co-localization of GlyRα1 and ER

In our previous studies, we characterized glycine and ER by quantum dot to colocalized via laser microscope and found that numerous glycine was localized on ER in cardiomyocytes, indicating glycine-receptors may localize on cardiomyocyte ER ^{(19, 20)}. In this study, we marked the ER by Rodamine (red), GlyRα1 by FITC (green) and DNA by DAPI (blue). The results showed that GlyRα1 was presented on the ER (Fig. 5). Furthermore, to further explore the function of anoxia and glycine on the co-localization of ER and GlyRα1, we cultured H9C2 cells with hypoxia, glycine, hypoxia and glycine for 12 h and 24 h. Then, colocalization detection was performed. It was observed that colocalization of Glyα1 and ER was presented in 3 groups; and that the expression of ER was the same among the 3 groups. Besides, the expression of GlyRα1 was upregulated in the Hypoxia group, while the Glycine group had a much lower expression level. What’s more, the increased GlyRα1 expression was reversed notably with glycine treatment under hypoxic condition (Fig. 6). When we extended the hypoxic time to 24 h, we found that the colocalization of ER and GlyRα1 still existed, and the Hypoxia group had a much higher expression level of GlyRα1 than other groups, and the increased level of GlyRα1 caused by hypoxia was notably reversed with glycine treatment (Fig. 7). Taken together, GlyRα1 exists on the ER of myocardial cells, and the combination of glycine with GlyRα1 could reverse the myocardial cell injury caused by hypoxia.

Expression of GlyRα1 in rats’ myocardial cells treated with scald

Severe burn could lead to anoxia and ischemia of myocardial cells, which may further induce inconvertible damage of myocardium. As demonstrated above, anoxia could induce the expression of GlyRα1. However, it is still unknown weather scald and glycine could influence the expression of GlyRα1 in rats’ myocardium. To verify this speculation, a total of 24 SD rats were divided into 4 groups: the Control group, the Glycine group, the Scald group and the Glycine + Scald group. The results showed that the expression of GlyRα1 in the Scald group was conspicuously increased compared with the Control group, ranging from 0.4 to 0.95. As expected, the increased expression of GlyRα1 caused by scald was inhibited strikingly by glycine treatment, ranging from 0.95 to 0.56. There was a significant difference between the Scald group and the Scald + Glycine group (Fig. 8). These results suggested that scald could lead to increased expression of GlyRα1, while glycine could notably reverse this effect. In addition, the expression of GlyRα1 in the Scald and Glycine group was lower than the Glycine group, which indicated that glycine cytoprotection may be due to glycine uptake.

Multiple studies have reported that glycine was able to protect tissues from injury caused by ischaemia–reperfusion⁽⁶⁾, and it has also been demonstrated that it can prevent or reverse the induction of colitis caused by TNBS, dextran sulphate or abdominal irradiation in rodents ⁽¹³⁾. In this study, we confirmed that GlyRα1 localized on ER in cardiomyocyte and the combination of glycine with GlyRα1 on ER caused cytoprotective effect by potential membrane changes. However, the mechanisms by which ER pathway exert its protective effects needs further illumination because the available evidence differs greatly. Therefore, to provide a more tractable system for such studies, rat cell models with scald myocardium injury were established in this study, which can be used to investigate glycine mediated cytoprotection.

During the last twenty years, numerous studies have reported the protective effective of glycine in a broad range of tissues, including kidney, liver and lung. In addition, glycine can also fight against a number of injury agents, such as hypoxia, ischemia-reperfusion and septicopyemia ^{(6, 12, 14, 18)}. Additionally, glycine could prevent ischemic myocardial cells from being damaged by reducing the lipid peroxide productions of myocardial tissues, malondialdehyde and the superoxide dismutase. Glycine may acquire resistance to hepatic cells anoxia by inhibiting Na + influx in hepatic cells via Na+/H + exchangers. A clinical trail has shown beneficial effects of glycine in protecting right ventricle compliance from early ischemia injury and thus improved cardiac function and patients’ prognosis. What’s more, glycine supplementation cloud reverse lipopolysaccharide caused damage in rat myocardial cells by activating glycine-receptor on cardiomyocyte membrane, which could further block Ca⁺ influx in myocardial cell via detecting lactate dehydrogenase and Ca^{+ (4, 16)}.

As a potent stimulating factor, sever burn could induce cardiomyocyte ER pathway, including activation of activating transcription factor 6α (ATF6α) that acts on Golgi apparatus and participation in XBP-1 mRNA splicing. The second signaling pathway is the activation of inosital-requiring enzyme-1α (IRE1α) that participates in XBP-1 mRNA splicing and degrades protein-synthesis related mRNA to depress protein transcription and translation. The third signaling pathway is the activation of PKR2like ER Kinase (PERK) that activate eukaryotic translation initiation factor 2α (eIF2α) to depress protein transcription and translation ^{(1, 8, 17)}. ER can activate unfold protein response (UPR) -a group of intracellular signal transduction pathways. Endoplasmic UPR as a part of the global ER stress response could activate cell death program by 3 pathways: CHOP/JNK and caspase-12 when endoplasmic overload beyond ERS compensatory mechanism to ensure the essential organs survival ⁽¹⁰⁾. Studies reported that hypoxic myocardial cells have ERS in early sever burn and this injury involves a spectrum from reversible to irreversible damage. Therefore, early treatment is critical for optimal outcomes.

Funding

This work was supported by National Natural Science Foundation of China: The study of glycine’s myocardial protective effect and mechanism in severely burned rats via the endoplasmic reticulum pathway (Grant numbers 81460294).

Author information

Yue Wu and Ruonan Lu have contributed equally to this work. Corresponding authors: Hui Cai; Junli Zhou.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Yue Wu and Ruonan Lu. The first draft of the manuscript was written by Yue Wu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

The data supporting this study can be obtained from the corresponding author [Hui Cai].

Ethics approval

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethical Review Committee of Gansu Provincial Hospital, Lanzhou, China.

Consent to participate

Informed consent was obtained from all the participants included in the study.

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The influence of scald and anoxia to the expression of Glycine receptor in rats’ myocardium and the co-localization study of Glycine receptor and endoplasmic reticulum

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Abstract

Figures

Introduction

Materials and Methods

Experimental Animals

H9C2 Cells

Effect of anoxia on the expression of GlyRα1

Effect of anoxia on the colocalization of GlyRα1 and ER

Western blotting

Indirect immunofluorescent staining and laser confocal microscopy

Groups and Burn Procedure

Myocardium assay

Results

Effect of glycine on the co-localization of GlyRα1 and ER

Expression of GlyRα1 in rats’ myocardial cells treated with scald

Discussion

Declarations

References

Additional Declarations

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