Selenium alleviates ER calcium depletion-induced endoplasmic reticulum stress dependent apoptosis via PERK/ATF4/CHOP pathway in chicken myocardium after mercuric chloride exposure

doi:10.21203/rs.3.rs-1991848/v1

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Selenium alleviates ER calcium depletion-induced endoplasmic reticulum stress dependent apoptosis via PERK/ATF4/CHOP pathway in chicken myocardium after mercuric chloride exposure

https://doi.org/10.21203/rs.3.rs-1991848/v1

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published 22 Feb, 2023

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Mercury (Hg) is a highly toxic heavy metal with definite cardiotoxic properties. Selenium (Se) is a heart-healthy trace element and has been demonstrated to attenuate heavy metal induced organ toxicity. This study was designed to explore the antagonistic effect of Se on the cardiotoxicity of mercuric chloride (HgCl₂) in chicken. Histopathological observations demonstrated that Se attenuated HgCl₂-induced myocardial injury, which was further confirmed by the results of serum CK and LDH levels assay and myocardial tissues oxidative stress indexes assessment. The results showed that Se prevented HgCl₂-induced cytoplasmic Ca²⁺ overload and endoplasmic reticulum (ER) Ca²⁺ depletion mediated by Ca²⁺-regulatory dysfunction of the ER. Importantly, ER Ca²⁺ depletion led to unfolded protein response (UPR) and endoplasmic reticulum stress (ERS), resulting in apoptosis of cardiomyocytes via the PERK/ATF4/CHOP pathway. Subsequently, the heat shock proteins expression activated by HgCl₂ through these stress responses, which was reversed by Se. Moreover, Se supplementation partially eliminated the effects of HgCl₂ on the expression of several ER-settled selenoproteins, including selenoprotein K (SELENOK), SELENOM, SELENON and SELENOS. In conclusion, these results suggested that Se alleviated ER Ca²⁺ depletion and oxidative stress induced ERS dependent apoptosis via PERK/ATF4/CHOP pathway in chicken myocardium after HgCl₂ exposure.

Mercuric chloride

Selenium

Calcium ion homeostasis

Endoplasmic reticulum stress

PERK/ATF4/CHOP pathway

Apoptosis

Mercury (Hg) is a persistent bio-accumulative heavy metal, and the public health problems caused by Hg pollution have been of great concern (Bjorklund et al. 2017). Although the signing of the Minamata Convention is expected to assuage Hg pollution amplified by humans activities in the long term, the high risk of Hg exposure is still a concern (Larson 2014). Hg is transformed and transported in the ecological environment in the form of monomers, organic and inorganic (Li &Tse 2015), making it widely distributed and multitoxic. Inorganic Hg, especially mercuric chloride (HgCl₂), was indiscriminately added to certain pesticides, preservatives, laxatives and skin lightening products (Cappelletti et al. 2019, Chan 2011), making it one of the most exposed Hg compounds for humans and animals. Epidemiological investigations and animal studies have revealed that HgCl₂ exposure can be responsible for damage to the structure and function of organs, such as the heart (Baiyun et al. 2018), kidney (Li et al. 2019), liver (Gao et al. 2021) and spleen (Fan et al. 2020). The cardiovascular system accounts for the systemic transport of metals, whereas the cardiotoxicity of HgCl₂ and related mechanisms remain unclear.

Calcium ion (Ca²⁺) is a highly versatile cellular signaling messenger with circulation carefully orchestrated by specific signals, sensors, pumps and ion channels (Eisner 2018, Thiel et al. 2021). The endoplasmic reticulum (ER) is the Ca²⁺ reservoir in the cell and plays an important role in the regulation of Ca²⁺ homeostasis. The sarcoendoplasmic reticulum calcium transport ATPase 2 (SERCA2) is a Ca²⁺ pump on the ER membrane that collects Ca²⁺ from the cytosol against the concentration gradient. The inositol 1,4,5-trisphosphate receptors 1 (IP3R1) and the ryanodine receptor 2 (RYR2) mediate the opposite process, releasing to Ca²⁺ into the cytoplasm when required (Carreras-Sureda et al. 2018). Additionally, stromal interaction molecule 2 (STIM2)/Orai1-mediated store operated calcium entry (SOCE) can drive extracellular Ca²⁺ inward flow in response to ER Ca²⁺ deficiency (Correll et al. 2015). A growing number of studies have highlighted the role of Ca²⁺-regulatory dysfunction of the ER in promoting myocardial diseases (Dickhout et al. 2011, Dridi et al. 2020, Michalak &Opas 2009). A recent study in fish found that HgCl₂ exposure limited the contribution of the ER to the rise in cytoplasmic Ca²⁺ during myocardial contraction, suggesting that Ca²⁺ storage and release of ER is restricted. Therefore, HgCl₂ might contribute to myocardial Ca²⁺ disorder by disrupting the precise regulation of Ca²⁺ by the ER.

ER is one of the most multifunctional organelles, responsible for key physiological processes such as protein processing, Ca²⁺ storage and lipid metabolism (Schwarz &Blower 2016). Accumulating evidence suggested that Ca²⁺ disruption can lead to ER dysfunction and increase the probability of protein misfolding, which may cause endoplasmic reticulum stress (ERS) characterized by unfolded protein response (UPR) (Dridi et al. 2020, Hetz et al. 2020). During the UPR, the glucose regulated protein 78 (GRP78) is occupied by unfolded proteins, allowing the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), the inositol requiring enzyme 1 α (IRE1α) and the activating transcription factor 6 (ATF6) to be exposed and adjust a series of transcriptional and translational events to alleviate the folding pressure in the ER. However, if ER homeostasis cannot be re-established, the C/EBP homologous protein (CHOP) pathway-mediated apoptosis may be activated (Zhang et al. 2019). In addition, oxidative stress caused by reactive oxygen (ROS) accumulation, which is inevitably induced by heavy metals (Dai et al. 2018, Liu et al. 2019a, Paithankar et al. 2021), is an important factor contributing to ERS (Cao &Kaufman 2014, Zeeshan et al. 2016). Studies have reported that ERS-mediated apoptosis plays an important role in the neurotoxicity of methylmercury (Liu et al. 2019b). Moreover, a recent study confirmed that HgCl₂ can cause apoptosis in chicken embryonic kidney cells via ERS (Ma et al. 2020a). Therefore, we hypothesized that ERS might mediate apoptosis of HgCl₂ in cardiomyocytes after Ca²⁺ homeostasis imbalance and oxidative stress occur.

Selenium (Se) is an essential nutrient for humans and animals, and its deficiency is associated with a variety of cardiac diseases (Oropeza-Moe et al. 2015). Recent studies have found that Se mitigated the organ toxicity of many heavy metals through its bioactivities of anti-oxidant, anti-inflammatory, anti-apoptotic and regulating metabolic homeostasis (Gao et al. 2021, Huang et al. 2021). Se exercises its function by embedding in selenoproteins, 25 of which have been identified as differentially distributed in tissues and cells, and the precise functions of specific selenoproteins are being gradually revealed (Zhang et al. 2020c). Among them, seven selenoproteins are assigned to the ER, including selenoprotein K (SELENOK), SELENOM, SELENON, SELENOS, SELENOF, SELENOT and deiodinase 2 (DIO2), which have been shown to be directly or indirectly involved in critical ER functions such as regulation of Ca²⁺ homeostasis, maintenance of the redox environment, and auxiliary protein folding (Pitts &Hoffmann 2018). It was reported that Ca²⁺ homeostasis imbalance and ERS caused by cadmium (Cd) involved perturbation of ER-settled selenoproteins expression (Zhang et al. 2020a, Zhang et al. 2020b). In addition, Se can form stable Hg-Se complex with Hg at the cost of availability (Yoneda &Suzuki 1997). Therefore, functional assessment centered on the ER is expected to provide key information to reveal the mechanism of cardiotoxicity of HgCl₂ and antagonism of Se. In this study, we demonstrated that Se alleviated HgCl₂-induced myocardial damage in chicken by mechanisms involving Ca²⁺ disorder, oxidative stress, ERS dependent apoptosis via PERK/ATF4/CHOP pathway, activation of heat shock proteins and expression profile changes of ER-settled selenoproteins.

2.1 Animals

All animal experiments in this study were approved by Care and Use of Laboratory Animals in Shandong Agricultural University (NO: SDAUA-2019-057). Ninety male Hyline brown chickens (one-day-old, initial body weight 39.5 ± 0.28 g) were purchased from Dongyuezhongqin Co. Ltd. (Taian, China).

2.2 Experimental Protocol

The HgCl₂ and sodium selenite (Na₂SeO₃) of high purity were purchased from Sigma-Aldrich (Carlsbad, CA). The chickens were randomly divided into three groups of 30, namely Con group (normal drinking water and standard commodity diet), HgCl₂ group (drinking water with 250 mg/L HgCl₂ and standard commodity diet), and HgCl₂ + Se group (drinking water with 250 mg/L HgCl₂ and commodity diet with 10 mg/kg Na₂SeO₃). The doses of HgCl₂ and Na₂SeO₃ were referenced to several recent exposure experiments (Fan et al. 2020, Kim &Kil 2020, Zhou et al. 2020). All chickens were allowed to acclimatize to the laboratory environment (25 ± 2°C, 12 h light/dark cycle, ad libitum diet) for one week prior to the start of the experiment. The chickens were euthanized with sodium pentobarbital and weighed after seven weeks. Blood samples were collected into procoagulant tubes by cardiac blood collection and rested to collect serum samples for serum biochemical analysis. Next, the myocardial tissues were quickly removed, washed with saline, and then divided into 3 parts for kits detection, histopathology sections preparation, and storage for backup (-80℃).

2.3 Histopathological Analysis

Myocardial tissues were trimmed into small pieces and soaked in 4% formaldehyde solution for 14 d. The myocardial tissues that had been fixed were rinsed overnight in a beaker of running water and then the water was removed from the tissues with a low to high concentration of alcohol (75%-100% in that order). After xylene immersion (3 min) and paraffin embedding, myocardial tissues were cut into 5–8 µm slices for hematoxylin and eosin (H&E) staining. The stained sections were dehydrated with anhydrous ethanol and transparent with xylene. Finally, the sections were fixed with neutral resin to make sections for observation under a light microscope (Nikon Eclipse E600 W, Suzhou, China).

2.4 Blood Biochemical Analysis

In brief, the isolated serum samples were tested for creatine kinase (CK) and lactic dehydrogenase (LDH) by a fully automated blood biochemistry analyzer BC-2600Vet (Mindray, Shenzhen, China).

2.5 Oxidative Stress Parameters Measurement

Myocardial tissues of 20 mg and cold saline were homogenized with a mechanical homogenizer, and the supernatants were collected after centrifugation at 1000 × g for 10 min. The levels of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX), malondialdehyde (MDA) and total protein were measured and calculated with the appropriate kits according to the manufacturer's instructions. All these kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

2.6 Western Blot Analysis

Total proteins were isolated from myocardial tissues using RIPA lysate (Applygen Technologies, Beijing, China) containing protease inhibitor and protein concentrations were determined by BCA method. According to the measured concentrations, protein samples (200 µg) were mixed with the loading buffer (5 ×) and boiled at 100°C for 5 min. The prepared protein samples (20 µg) were separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in tris buffered saline tween (TBST) buffer of 5% skimmed milk for 90 min at room temperature and then incubated overnight at 4°C with diluted primary antibodies against SERCA2, phospholamban (PLN), IP3R1, RYR2, STIM2, Orai1, sodium/calcium exchanger 1 (NCX1), plasma membrane Ca²⁺-ATPase (PMCA), Ca²⁺/calmodulin-dependent kinase II delta (CAMK2D), GRP78, X-box binding protein 1 (XBP1), ATF6, PERK, eukaryotic initiation factor 2 alpha (elf2α), ATF4, CHOP, Bcl-2 antagonist/killer 1 (BAK1), Caspase-3, heat shock protein 27 (HSP27), HSP40, HSP60, HSP70, SELENOK, SELENOM, SELENON, SELENOS, GAPDH and Tubulin. The membranes were then incubated with horseradish peroxidase (HRP)-labeled secondary antibody against rabbit IgG or mouse IgG for 1 h at room temperature. All primary and secondary antibodies were purchased from ABclonal Technology Co., Ltd. (Wuhan, China) and the dilution ratio of GAPDH, and two secondary antibodies are 1:10000 and both the rest antibodies are 1:1000. Finally, the fluorescent signal was detected using the image VCD gel imaging system (Clinx Science Instruments, Shanghai, China).

2.8 Statistical Analysis

The data of this experiment were expressed as mean ± SEM. Experimental groups were compared using a two-way analysis of variance followed by Scheffe’s test when data were normally distributed and by the Kruskal-Wallis test when data were not normally distributed analyzed by SPSS 22.0 (SPSS Inc., Chicago, USA). P < 0.05 was regarded as statistically significant.

3.1 Se protects against HgCl₂-induced myocardial damage

To determine the protective effect of Se on HgCl₂-induced myocardial injury, we first assessed the histopathological changes by H&E staining. As shown in Fig. 1A, the Con group exhibited normal myocardial structure, including closely aligned and uniformly sized myocardial fibers. Significant pathological damage was observed in the myocardium after HgCl₂ exposure, such as myocardial fibers dissolving, gap enlargement and texture blurring, and these pathological changes were considerably improved in the HgCl₂ + Se group.

Serum levels of CK and LDH can be used to characterize myocardial injury, which are released into the blood by dead cardiomyocytes. The results showed a significant increase in serum CK and LDH activities after HgCl₂ exposure compared to the Con group (Fig. 1B and C), suggesting impaired myocardium. Consistent with morphological observations, Se inhibited the rise of both myocardial injury indicator enzymes induced by HgCl₂. These results showed a significant protective effect of Se against HgCl₂-induced myocardial injury.

3.2 Se alleviates HgCl₂-induced oxidative stress in the myocardium

Accumulating evidence suggested that oxidative stress is closely related to the biotoxicity of HgCl₂ (Baiyun et al. 2018, Ma et al. 2020b) and the biofunction of Se (Zhang et al. 2020c). Our results showed that HgCl₂ significantly increased the content of lipid peroxidation product MDA in the myocardium compared to the Con group, and this increase was inhibited by Se (Fig. 2A). Furthermore, the activities of two important antioxidant enzymes, SOD and GSH-PX, were significantly decreased after HgCl₂ exposure, which were partially restored by Se (Fig. 2B and C). These results suggested that Se alleviated the oxidative stress in the myocardium caused by HgCl₂.

3.3 Se improves HgCl₂-induced dysregulation of myocardial Ca²⁺ homeostasis

Ca²⁺ regulates important physiological and pathological processes in cells in a concentration-dependent manner, but how it is involved in the myocardial cytotoxicity of HgCl₂ remains outstanding. A recent study demonstrated that HgCl₂ impaired the regulation of cytoplasmic Ca²⁺ concentration by ER in cardiomyocytes (Monteiro et al. 2017), which prompted us to evaluate this event initially. The results showed that the protein expression of SERCA2 was decreased while that of its inhibitory substrate PLN was increased in the HgCl₂ group compared to the Con group (Fig. 3A and B), implying that HgCl₂ resulted in a decrease of Ca²⁺ pumped into ER stores. In addition, the protein expression levels of IP3R1 and RYR2, which control the release of Ca²⁺ from the ER into the cytosol at the ER membrane in the myocardium, were significantly upregulated by HgCl₂. The decrease in ER Ca²⁺ refill and increase in efflux may lead to ER Ca²⁺ depletion, and for confirmation of this speculation, we examined the expression of SOCE-associated protein, which is initiated in ER Ca²⁺ depletion. As expected, the protein expression levels of STIM2 and Orai1 were significantly increased in the HgCl₂ group compared with the Con group. However, these changes in ER Ca²⁺ handling-related protein expression were reversed by Se, suggesting that Se prevented HgCl₂-induced ER Ca²⁺ depletion.

We further examined NCX1 and PMCA, which are responsible for pumping cytoplasmic Ca²⁺ out of the cell at the cardiomyocyte membrane, and their protein expression levels were significantly upregulated by HgCl₂ compared with the Con group (Fig. 3C and D), which might be a compensatory response caused by ER Ca²⁺ depletion and cytoplasmic Ca²⁺ overload. CAMK2D contacts multiple signals in the heart in a cytoplasmic Ca²⁺ level-dependent manner and is chronically overactive in a variety of myocardial diseases (Hegyi et al. 2019, Mustroph et al. 2017). The result showed that its protein expression level was upregulated in the HgCl₂ group compared to the Con group. Notably, Se inhibited the protein expression of NCX1, PMCA and CAMK2D that were upregulated by HgCl₂. Collectively, Se ameliorated HgCl₂-induced Ca²⁺ depletion in the ER and overload in the cytoplasmic.

3.4 Se attenuates HgCl₂-induced PERK/ATF4/CHOP mediated myocardial ERS-dependent apoptosis

High concentration of Ca²⁺ stored in the ER not only act as a buffer for cytosolic Ca²⁺, but also participate in protein folding there. Alterations in the redox environment may also lead to protein misfolding and trigger UPR (Zhang et al. 2019). GPR78, a marker of UPR, showed a significant increase in its protein expression level after HgCl₂ exposure and a decrease after Se and HgCl₂ co-exposure (Fig. 4A and B), indicating that Se mitigated the HgCl₂-induced UPR. Any of the three branches of ERS can be activated by the UPR and may produce differential effects. Our results showed that HgCl₂ activated PERK-mediated ERS, not IRE1α and ATF6, as only PERK protein expression level was increased in response to HgCl₂ exposure. Meanwhile, the results also indicated that PERK-mediated ERS was attenuated by Se.

Persistent ERS may push cells toward apoptosis by activating CHOP (Tabas &Ron 2011). As shown in Fig. 4C and D, the PERK downstream proteins elf2α, ATF4 and CHOP were significantly upregulated in the HgCl₂ group compared to the Con group, suggesting that HgCl₂ activated ERS-dependent apoptosis. High expression of proteins of BAK1 and Caspase-3 after HgCl₂ exposure also confirmed the activation of the apoptotic program. However, Se significantly inhibited HgCl₂-increased protein expression levels of elf2α, ATF4, CHOP, BAK1 and Caspase-3, indicating that Se suppressed HgCl₂-induced PERK/ATF4/CHOP-mediated ERS-dependent cardiomyocyte apoptosis.

3.5 Heat shock proteins and ER-settled selenoproteins are involved in the antagonism of Se to HgCl ₂ cardiotoxicity

As molecular chaperones, heat shock proteins are activated when cells under various stresses. As shown in Fig. 5A and B, increased protein expression of HSP27, HSP40, HSP60 and HSP70 corroborated the myocardial toxicity of HgCl₂. In parallel, Se significantly inhibited the heat shock proteins activation induced by HgCl₂ exposure, suggesting the attenuation of stress.

Selenoproteins execute the biofunctions of Se, and ER-settled selenoproteins have been shown to regulate the ER redox environment, Ca²⁺ handling and protein folding (Pitts &Hoffmann 2018). Our results showed that HgCl₂ significantly inhibited the expression of SELENOK and promoted the expression of SELENOM, SELENON and SELENOS, while this effect of HgCl₂ was partially abolished by Se (Fig. 5C and D).

With the growing problem of Hg pollution, the risk of humans and animals exposure to HgCl₂ is on the rise (Ajsuvakova et al. 2020, Bjorklund et al. 2017, Larson 2014). This study was dedicated to further clarify the cardiotoxicity of HgCl₂ and to explore its specific mechanisms and prevention. Se has the potential to antagonize HgCl₂ cardiotoxicity, and the current study confirmed the effectiveness of Se in antagonizing HgCl₂-induced cardiotoxicity in chickens by histopathological observation and serum biochemical analysis. Our further results reveal a possible mechanism for HgCl₂ cardiotoxicity that imbalance in Ca²⁺ homeostasis and oxidative stress activate UPR and PERK-mediated ERS, and persistent ERS leads to cardiomyocyte apoptosis via the CHOP pathway. It is exciting to note an effective antagonistic effect of Se on the negative effects produced by HgCl₂ was observed in all these aspects.

The cytoplasmic Ca²⁺ concentration is much lower than that in the extracellular and ER, creating a potential energy for many physiological activities (Prakriya 2020, Thiel et al. 2021). For instance, a rapid influx of extracellular and ER Ca²⁺ into the cytoplasm to drive myocardial contraction, followed by an equal amount of Ca²⁺ being pumped back during myocardial diastole, completes a cardiac excitation-contraction coupling (Eisner et al. 2017). An increasing body of evidence suggested that heavy metals tend to disrupt the precise control of Ca²⁺ homeostasis. Tang et al. found a rapid increase in Ca²⁺ content in the cytoplasm of neurons after Cd exposure (Tang et al. 2021). The exposure to arsenic (As) and antimony (Sb) resulted in ER Ca²⁺ depletion and cytoplasmic Ca²⁺ overload in mouse cardiomyocytes, caused by upregulation of IP3R, RYR2 and CAMK2 and downregulation of SERCA2α (Jiang et al. 2021). A recent study showed that HgCl₂-induced contraction of rat uterine myometrium was caused by an increase in Ca²⁺ channel protein-dependent cytoplasmic Ca²⁺ levels (Koli et al. 2019). The present study evaluated the changes in major Ca²⁺ handling proteins after HgCl₂ treatment for the first time, and the results converged to ER Ca²⁺ depletion and cytoplasmic Ca²⁺ overload, which is consistent with the findings of previous related studies. Deprivation of part of the contribution of the ER in the regulation of Ca²⁺ flux might link HgCl₂ to the downstream timing of Ca²⁺ homeostasis imbalance such as arrhythmias, heart failure, and hypertrophic cardiomyopathy (Dickhout et al. 2011, Dridi et al. 2020). Therefore, we suggested that the examination of Ca²⁺-regulated function of the ER should be included in the priority examination cohort for heavy metal toxicity assessment, especially for the heart.

A high concentration of Ca²⁺ and a unique redox environment are maintained within the ER, and the absence of optimal conditions increases the probability of being misfolded protein, which may lead to UPR. Numerous studies have documented ERS and eventual activation of apoptosis due to ER Ca²⁺ depletion and oxidative stress (Farrukh et al. 2014, Liu et al. 2019b, Tabas &Ron 2011). The status of ER Ca²⁺ depletion and overall redox imbalance has been demonstrated in our results, and we are interested in whether the UPR and ERS were activated. Interestingly, detection of the proteins within the three branches revealed that only PERK, which shuts down the mRNA translation process via elf2α (Mekahli et al. 2011), mediates ERS activation upon HgCl₂ exposure and pushes cells toward apoptosis via CHOP cascade signaling. A recent study confirmed that the PERK/ATF4/CHOP axis mediated HgCl₂-induced apoptosis in chicken embryonic kidney cells (Ma et al. 2020a). However, several studies have also reported that specific heavy metals can also activate ERS via the IRE1α and ATF6 branches, such as Cd (Zhang et al. 2020a), As and Sb (Jiang et al. 2021). Thus, based on the clarification of ERS sensitivity to heavy metals, we speculated that different branches might have biased effects depending on the source, timing, intensity and location of the stimulus. In addition, activation of heat shock proteins is considered a marker of cells subjected to adverse stress (Fan et al. 2020, Jacob et al. 2017), and we detected a set of heat shock proteins (HSP27/40/60/70) upregulated by HgCl₂, which can be attributed to Ca²⁺ homeostasis imbalance, oxidative stress and ERS. Overall, these results suggested that HgCl₂ initiated the UPR through ER Ca²⁺ depletion and oxidative stress, triggering the PERK-dependent ERS that lead to apoptosis and activating heat shock proteins in myocardial cell.

As the mechanisms of heavy metal toxicity are revealed, a number of mechanism-specific supplements have been shown to mitigate heavy metal toxicity, including Se (Fan et al. 2020), curcumin (Garcia-Nino &Pedraza-Chaverri 2014), luteolin (Xu et al. 2021) and melatonin (Cao et al. 2017). In particular, Se, as a biologically essential trace element, has been increasingly studied to confirm its superior detoxification of heavy metals at the multi-organ and multi-cellular levels, and its biological functions of anti-oxidant, anti-inflammatory, anti-apoptotic and regulation of substance metabolism have been intensively emphasized (Gao et al. 2021, Jin et al. 2017, Zhang et al. 2020b). Recent studies have shown that Se worked in the regulation of Ca²⁺ homeostasis (Zhang et al. 2020b, Zheng et al. 2021) and ER function (Cao et al. 2021, Ma et al.). In this study, we demonstrated that Se attenuated HgCl₂-induced ERS and apoptosis in cardiomyocytes, involving the correction of Ca²⁺ handling proteins and the alleviation of oxidative stress. Notably, ER-settled selenoproteins have been shown to be directly or indirectly involved in the regulation and functional exercise of the ER internal environment (Pitts &Hoffmann 2018). Previous studies suggested that abnormal ER selenoproteins expression contributed to Cd-induced Ca²⁺ disorder and ERS, which can be reversed by Se supplementation (Zhang et al. 2020a, Zhang et al. 2020b). Considering the unique physical affinity of Hg and Se and the crossover of their associated biological processes (Yoneda &Suzuki 1997), which may act on the expression of selenoproteins, we further focused on the expression patterns of the four ER-settled selenoproteins. SELENOK is a transmembrane small molecule protein containing a Sec residue that acts as a cofactor in the palmitoylation of IP3R to regulate ER Ca²⁺ flux (Fredericks &Hoffmann 2015). Another of our studies found that SELENOK deficiency induced skeletal muscle satellite cell apoptosis via ERS (unpublished), which is consistent with changes following HgCl₂ exposure. However, no consistent changes were obtained for SELENOK and IP3R, impling different regulatory mechanisms. Whether and how other selenoproteins are involved in ER function is not fully understood, and the available evidence suggested that they may exert some antioxidant function and participate in protein folding through shared structural Sec residues (Zhang et al. 2020c). The organism tends to preferentially synthesize important selenoproteins (Tang et al. 2020), and we found that the protein expression of SELENOM, SELENON and SELENOS was significantly increased after HgCl₂ exposure, which might be related to specific functional requirements. Moreover, we noted the reversal of HgCl₂-induced SELENOK, SELENOM, SELENON, and SELENOS changes by Se, possibly related to the elimination of common or different upstream crises.

In this study, we confirmed the significant cardiotoxicity of HgCl₂ that ER Ca²⁺ depletion and oxidative stress activated the UPR, and the PERK/ATF4/CHOP pathway pushed chicken cardiomyocytes from ERS to apoptosis. Deficits in Ca²⁺-regulated function of the ER might link HgCl₂ to numerous myocardial diseases. In parallel, our results also suggested that dietary supplementation with Se represents a promising measure to counteract long-term HgCl₂ toxicity for humans and animals at high risk of HgCl₂ exposure, although supplementation modalities and doses still need further optimization.

Ethical Approval

Not applicable.

Consent to Participate

All individual or guardian participants gave their oral and written informed consent.

Consent to Publish

Not applicable.

Authors Contributions

Rui-Feng Fan and Pei-Chao Gao contributed to the study conception and design. Material preparation, data collection and analysis were performed by Pei-Chao Gao, An-Qi Wang, Xue-Wei Chen, Han Cui and Yue Li. The first draft of the manuscript was written by Pei-Chao Gao and An-Qi Wang, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (grant number 31902330), China Postdoctoral Science Foundation (grant number 2020M672098), Youth Innovation and Technology Program in Colleges and Universities of Shandong Province (grant number 2020KJF009), and Funds of Shandong “Double Tops” Program, and Funds of Shandong “Double Tops” Program.

Competing Interests

The authors declare no competing interests.

Availability of Data and Materials

All data generated or analyzed during this study are included in this published article and its additional files.

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Selenium alleviates ER calcium depletion-induced endoplasmic reticulum stress dependent apoptosis via PERK/ATF4/CHOP pathway in chicken myocardium after mercuric chloride exposure

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Animals

2.2 Experimental Protocol

2.3 Histopathological Analysis

2.4 Blood Biochemical Analysis

2.5 Oxidative Stress Parameters Measurement

2.6 Western Blot Analysis

2.8 Statistical Analysis

3. Results

3.1 Se protects against HgCl₂-induced myocardial damage

3.2 Se alleviates HgCl₂-induced oxidative stress in the myocardium

3.3 Se improves HgCl₂-induced dysregulation of myocardial Ca²⁺ homeostasis

3.4 Se attenuates HgCl₂-induced PERK/ATF4/CHOP mediated myocardial ERS-dependent apoptosis

4. Discussion

5. Conclusion

Declarations

References

Status:

Journal Publication

Version 1

Selenium alleviates ER calcium depletion-induced endoplasmic reticulum stress dependent apoptosis via PERK/ATF4/CHOP pathway in chicken myocardium after mercuric chloride exposure

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Animals

2.2 Experimental Protocol

2.3 Histopathological Analysis

2.4 Blood Biochemical Analysis

2.5 Oxidative Stress Parameters Measurement

2.6 Western Blot Analysis

2.8 Statistical Analysis

3. Results

3.1 Se protects against HgCl2-induced myocardial damage

3.2 Se alleviates HgCl2-induced oxidative stress in the myocardium

3.3 Se improves HgCl2-induced dysregulation of myocardial Ca2+ homeostasis

3.4 Se attenuates HgCl2-induced PERK/ATF4/CHOP mediated myocardial ERS-dependent apoptosis

4. Discussion

5. Conclusion

Declarations

References

Status:

Journal Publication

Version 1

3.1 Se protects against HgCl₂-induced myocardial damage

3.2 Se alleviates HgCl₂-induced oxidative stress in the myocardium

3.3 Se improves HgCl₂-induced dysregulation of myocardial Ca²⁺ homeostasis

3.4 Se attenuates HgCl₂-induced PERK/ATF4/CHOP mediated myocardial ERS-dependent apoptosis