Upregulation of Zip14 correlates with induction of endoplasmic reticulum stress (ERS) in hypertrophied hearts of Dahl saltsensitive rats

Zinc is a trace element involved in maintaining cellular structure and function. Although zinc is associated with left ventricular hypertrophy (LVH), there have been few reports on this association. This study aimed to evaluate the correlation between Zip14 and expression of endoplasmic reticulum stress (ERS) associated molecules in hypertrophied hearts of rats. Dahl salt-sensitive rats were fed a high salt diet to establish a left ventricular hypertrophy (LVH) rat model. RT-PCR was used to determine Zip14, activating transcription factor (ATF4), ATF6, x-box-binding protein 1 (xBP1), C/EBP homologous protein (CHOP), immunoglobulin-binding protein (BiP) mRNA expression. Western blotting was used to evaluate Zip14, BiP, CHOP, GAPDH expression. Zinc levels were measured by Inductively Coupled Plasma Optical Emission Spectroscopy. The results indicated that compared with the Control group, Zip14 mRNA and protein expression in LVH rat hearts were markedly increased (P < 0.01). Zinc content in rat heart tissue was significantly increased in the LVH group compared with the Control group (P < 0.05). ATF4, ATF6, xBP1 mRNA expressions were increased in LVH rat hearts compared with Control hearts (P < 0.001). Compared with the Control group, CHOP and BiP mRNA and protein expression were markedly increased in LVH rat hearts (P < 0.05, P < 0.01). Linear regression models showed that Zip14 mRNA expressions were positively correlated with zinc concentration, ATF4 and ATF6 mRNA expressions in Control hearts (P = 0.0005, P = 0.0052, P = 0.0026, respectively) and LVH rat hearts (P < 0.0001, P = 0.0119, P = 0.0033, respectively). In conclusion, upregulation of Zip14 in LVH rat hearts correlated with zinc accumulation and induction of ERS.


Introduction
Zinc is a trace element involved in maintaining cellular structure and function (Ryul et al., 2015). High zinc levels have irreversible effects on proteins and lead to the dysfunction of many proteins. Low levels of zinc are also detrimental to cells because it is a cofactor for more than 300 enzymes and 2000 transcription factors, as well as mediating cell signaling (Roshanravan et al., 2015;Huang et al., 2017). Therefore, the balance of intracellular zinc concentration, termed zinc homeostasis, is critical. Under physiological conditions, the regulation of zinc homeostasis mainly depends on zinc transporters, zinc-binding molecules, and zinc sensors (Foster and Samman, 2010). Zinc transporters are divided into two major families, Zip and ZnT. The 14 zinc transporters of the Zip family are responsible for transferring extracellular zinc into cells, while 10 zinc transporters of the ZnT family have the opposite role (Lichten and Cousins, 2009). Zip14 is located in the cell membrane and promotes extracellular zinc into the cytosol and increases the zinc concentration in the cytoplasm (Taylor et al., 2007).
The endoplasmic reticulum (ER) is an important organelle widely present in cells and an important site for the folding, assembly, and modification of protein molecules (Ron and Walter, 2007;Kim et al., 2008). When intracellular zinc is deficient, ER stress (ERS) occurs, causing dysfunction of the ER. Therefore, zinc is necessary to maintain normal ER function. In addition, ERS and cell dysfunction can be induced by oxidative stress and acute ischemia-reperfusion (IR) injury (Zhang, 2010;Zhang et al., 2014a;Zhang et al., 2014b).
Zinc is associated with a variety of cardiovascular diseases, such as atherosclerosis and thrombosis of atherosclerotic plaque rupture, diabetic cardiomyopathy, arrhythmia, myocardial infarction, and congestive heart failure. Although zinc is associated with left ventricular hypertrophy (LVH), there have been few reports on this association (Little et al., 2010). LVH is characterized by pathological remodeling of the heart and is a good predictor of cardiovascular diseases such as myocardial infarction, congestive heart failure, sudden cardiac death, stroke, and overall CVD mortality (Desai et al., 2012). Our previous studies reported that serum zinc ion concentrations were significantly lower in patients with LVH compared with normal patients (Huang et al., 2017). Furthermore, the zinc trafficking and the activity of the Zip14 transporter are important for adapting to the ERSassociated chronic metabolic disorders, and the Zip14mediated transport of zinc is necessary for adapting ERS (Kim et al., 2017). Olgar et al. (2018a) also reported that Zn 2+ correlates the induction of ERS through altering expressions of Zn 2+ -transporter, Zip14, in heart failure. Therefore, there might be a correlation between the Zip14 and the ERS. The present study aimed to investigate the mechanism of zinc transporter Zip14 and endoplasmic reticulum stress in the development of LVH in rats.

Establishment of the LVH rat model
To establish the LVH rat model, we chose Dahl salt-sensitive rats and fed them a high salt diet. The control group was fed a normal salt diet. The content of sodium chloride in the high salt feed was 8%, while the normal diet group contained 0.3%. We purchased 5-week-old rats of SPF grade from Beijing Weitong Lihua Experimental Animal Technology Co. Ltd., (license key SCXK (Beijing) 2012-0001). Blood pressure and heart rate were measured by a noninvasive blood pressure detector (BP-2010AUL Softron, Tokyo, Japan) every two weeks. All animals were exposed to a 12-h light-dark cycle and were given free access to tap water and standard chow daily.
The rats in this study were randomly divided into the Control group (N = 14, 7 males and 7 females) and LVH group (N = 22, 11 males and 11 females). At weeks 6, 12, and 18, rats were anesthetized by the intraperitoneal injection of 30 mg/kg 10% chloral hydrate. Then, the rats were fixed in a supine position, excluding the neck and chest hair, and coated with appropriate coupling agents. Mild sedation was maintained through a nasal tube with a continuous low flow of oxygen. The heart rate was maintained at about 300-350 bpm and was detected by an animal-specific ultrasound system (VEVO 2100 Imaging System, Toronto, Canada). LVH was successfully induced in high salt-fed Dahl rats at about 18 weeks. Left ventricular myocardium specimens were removed from successful LVH model rats and the Control group at various time points, immediately frozen in liquid nitrogen, and stored at −80°C until use.

RT-PCR assay
Total RNA was extracted from myocardial tissues using Trizol Reagent (Invitrogen) following the manufacturer's recommendations. RNA concentrations were determined by spectrophotometry at 260 nm and 280 nm, and RNA (3 μg) was reverse transcribed to obtain complementary DNA (cDNA) in a 20 μL reaction mixture with Promega Reverse Transcription System (Sigma-Aldrich.) according to manufacturer recommendations. Primers used for RT-PCR amplification were designed with Software Primer 5.0 and synthesized by Invitrogen Company (Beijing, China). Realtime PCR was performed with the primer sequences listed in Table 1 using a CFX96 Real-Time PCR System (Bio-Rad, Singapore) and a SYBR® Green PCR Kit (Invitrogen) according to a standard application protocol and the manufacturer's instructions. The cycling parameters were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s, and the annealing/extension temperature and time is 60°C for 60 s. All samples were assayed in triplicate. The control gene GAPDH RNA was used to normalize the results. The comparative threshold cycle method was used for data analyses.
The binding of the primary antibody was detected with secondary antibodies (anti-rabbit, 1:2500) and visualized by the ECL method. The intensities of the bands were analyzed using Image J software.

Measurement of zinc concentrations
Fifty mg myocardial tissue and 1 mL of 65% concentrated nitric acid was added to a small beaker for digestion and placed on a graphite-controlled thermoelectric plate at 120°C for 30 min. Finally, the nitric acid volume was made to 1 mL, and 4 mL of distilled water was added. The zinc levels were measured by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES optima 8300, Perkin Elmer, MA, USA).

Statistical analysis
Data are expressed as the mean ± SEM and were obtained from 6 to 10 separate experiments. Statistical analysis of experimental data was performed using SPSS 22.0 software (IBM Corp., Armonk, NY, USA). Statistical significance was determined using the Student's t-test or repeated-measures analysis of variance test. Linear regression analysis was used for relationships between variables. A value of P < 0.05 was considered statistically significant.

Hemodynamic and echocardiographic parameters of rats at different ages
The parameters of the rats are presented in Table 2. There was no significant difference in each index between LVH and Control group rats at week 6. The mortality rate of the LVH group rats was 22.7% from weeks 6 to 12. The mean systolic blood pressure (SBP) and late systolic thickness of the posterior wall of the left ventricle (LVPWs) in the LVH group were significantly higher than in the Control group (respectively, P < 0.05). However, the body weight (BW) was lower in the LVH group than in the Control group (P < 0.05) at week 12.
The mean systolic blood pressure (SBP) and mean diastolic blood pressure (DBP) were significantly increased in the LVH group compared with the Control group (respectively, P < 0.05) at week 18. However, the SBP arose in 12-week and 18-week-old rats in the Control group, which might be caused by the increased body weight or obesity. The interventricular septum thickness at enddiastole (IVSd), left ventricular end-diastolic diameter (LVDd), the end-diastolic thickness of the left ventricular wall (LVPWd), and late systolic thickness of the posterior wall of the left ventricle (LVPWs) in the LVH group were significantly higher than in the Control group (all P < 0.05). However, body weight (BW) was lower in the LVH group compared with the Control group (P < 0.05) at week 18.
Successful establishment of a LVH rat model As shown in Fig. 1, the left ventricular mass (LVM) and left ventricular hypertrophy index (LVM/BW) were significantly higher in the LVH group compared with the Control group at week 18 (744.83 ± 104.74 vs. 635.83 ± 119.06 mg, 2.29 ± 0.34 vs. 1.56 ± 0.32, respectively, P < 0.05). In addition to being a LVH rat model, it is also a classic rat model of hypertension.
Expression of Zip14 and zinc contents are increased in LVH rat hearts As shown in Fig. 2A, Zip14 mRNA expression was markedly higher in LVH compared with Control rats (P < 0.01). A representative western blotting band for Zip14 is shown in Fig. 2B. Western blot analysis demonstrated that the expression of Zip14 proteins was increased in the LVH rat heart compared with Control hearts (P < 0.01) (Fig. 2B). In addition, as shown in Fig. 2C, the zinc concentration was significantly increased in LVH rat hearts compared with Control hearts (1.28 ± 0.29 vs. 1.00 ± 0.23 mg/L, P < 0.05).

Induction of ERS in LVH rat hearts
When ERS occurs, plenty of molecules are involved in this process. Therefore, to demonstrate the occurrence of ERS,  we detected signal transduction molecules by western blotting and RT-PCR. As shown in Figs. 3A-3C, the mRNA expressions of activating transcription factor 4 (ATF4), activating transcription factor 6 (ATF6), and x box-binding protein 1 (xBP1) were increased in the LVH rat heart compared with Control hearts (P < 0.001). As shown in Fig. 3D, CHOP mRNA expression was markedly higher in LVH hearts compared with Control hearts (P < 0.001). A representative western blotting band for CHOP is shown in Fig. 3E. Western blot analysis demonstrated that the expression of CHOP proteins was increased in the LVH rat heart compared with Control hearts (P < 0.05) (Fig. 3E). As shown in Fig. 3F, BiP mRNA expression was markedly higher in LVH hearts compared with Control hearts (P < 0.05). A representative western blotting band for BiP is shown in Fig. 3G. Western blot analysis demonstrated that the expression of BiP proteins was increased in the LVH rat heart compared with Control hearts (P < 0.05) (Fig. 3G).
Zip14 mRNA expressions are positively correlated with zinc contents, ATF4, and ATF6 mRNA expression To confirm the relationship between Zip14 and zinc, ATF4, and ATF6, we performed linear regression analyses. As shown in Figs. 4A and 4B, the linear regression models showed a significant positive relationship between Zip14 mRNA expressions and zinc concentration in Control hearts (R 2 = 0.8027, P = 0.0005) and LVH rat hearts (R 2 = 0.8769, P < 0.0001).

Discussion
Our research provides new and interesting insights into the complex relationship between zinc and LVH. A model of LVH by feeding Wistar germline Dahl salt-sensitive rats a high salt diet has been successfully established. At week 18, the left ventricular mass and left ventricular hypertrophy index of the experimental group were significantly higher than in the control group. We also detected a marked increase in zinc concentration and a remarkable upregulation of zinc transporter Zip14 expression in myocardial tissues of LVH rats. Furthermore, the mRNA expressions of ATF4, ATF6, CHOP, BiP, and xBP1 in LVH rats under ERS were significantly upregulated and partially related to CHOP protein levels in the myocardial tissues of LVH rats. A marked increase in the expression of BiP confirmed the occurrence of ERS in the myocardial tissues of LVH rats.
Zinc plays an important role in cardiomyocyte protection by involving lots of signaling pathways, such as the cGMP/PKG pathway (Jang et al., 2007), NO/cGMP/PKG and glycogen synthase kinase-3β (GSK-3β) signaling pathway (Xi et al., 2010). Moreover, the zinc transporter Zip14 is closely related to inflammation and production of proinflammatory cytokines (Aydemir et al., 2012;Eizirik et al., 2012;Troche et al., 2016). Zip14 is also expressed in cardiac tissues and located in the plasma membrane, which promotes extracellular zinc into the cytosol and increases the concentration of zinc in the cytosol (Taylor et al., 2007;Olgar et al., 2018a). We observed higher expression levels of Zip14 and ICP-measured myocardial tissue zinc ions in the LVH rat model compared with the Control group. Similarly, Olgar et al. (2018b) used a rat hypertrophic heart model induced by transverse aortic coarctation (TAC) to show that the expression of Zip14 and the concentration of zinc ions in cardiomyocytes were increased compared with the SHAM group. We found that Zip14 plays an important role in the increased zinc concentration in the myocardium of LVH rats through linear regression analysis. ERS occurs when cells are affected by external factors such as inflammation, oxidative stress, and intracellular zinc homeostasis. ERS refers to misfolding and folding disorders of newly synthesized proteins leading to unfolded and misfolded proteins accumulating in the ER, affecting the normal function of the ER (Schroder and Kaufman, 2005;Kim et al., 2017). Activation of the UPR-related signaling pathway increased the expression of ER chaperones such as BiP and induced apoptosis independently (Walter and Ron, 2011); therefore, we determined the expression of Bip molecule in our study. Recently, Kim et al. (2017) confirmed that Zip14 is closely related to the adaptation of ER to livermediated stress and reduces hepatic steatosis and apoptosis by activating the ATF4 and ATF6α and UPR reactions (Kim et al., 2017). Therefore, when ERS occurs in cells, increased intracellular Zn 2+ concentrations play an important role in cell protection. Our findings showed that expressions of ATF4, ATF6, xBP1, and BiP were increased in LVH rat hearts compared with Control hearts. Meanwhile, ERS were significantly upregulated and partially related to CHOP protein levels in the myocardial tissues of LVH rats. All these results suggest that Zip14 is associated with the ERS in the LVH rat models. Moreover, according to the previous studies, many key biomarkers (Rutkowski et al., 2006;Wang et al., 2016) (such as PKR-like ER 1 kinase (PERK), eukaryotic initiation factor 2α (eIF2α)) and signaling pathways (such as p-eIF2α/ATF4/CHOP pathway) (Rutkowski et al., 2006), also involve in ERS and UPR. Meanwhile, the Zn 2+ participated metabolism of cardiomyocytes is also associated with the ERS, which involving Zip14, Zip7, and other molecules (MacDonald, 2000;Yoshida, 2007;Murakami and Hirano, 2008;Wang et al., 2016;Tuncay et al., 2017;Xing et al., 2017). Therefore, the association between Zip14 (or other Zn 2 + -transporters) and ERS in LVH animal models should be verified in future investigations.
Furthermore, linear regression analysis indicated that the expression of Zip14 mRNA expression in the myocardial tissues of the Control and experimental groups was positively correlated with zinc concentration. The mRNA expressions of Zip14, ATF4, and ATF6 in the myocardial tissues of the Control and experimental groups were also positively correlated. However, the correlation between Zip14 expression and CHOP or BiP has not been verified in our study, which is needed to be detected in further studies.
Therefore, we hypothesized that ERS in the myocardium of LVH rats induced by external factors such as inflammation and oxidative stress upregulates the expression of ATF4 and ATF6 by activating UPR-related pathways or the activation of other pathways to increase the expression of Zip14 to increase the zinc concentration in cardiomyocytes. A shortterm increase in zinc concentration allows cells to adapt to ERS and protects cardiomyocytes. Sustained ERS and the long-term increase of the zinc concentration in cardiomyocytes may cause myocardial cell apoptosis and dysfunction, which might be a pathophysiological mechanism in the development of LVH.
There were some limitations in this study. Firstly, in the process of studying how Zip14 regulates zinc homeostasis, there was no intervention study of zinc and Zip14; thus, it was not clear which was the originating factor. To clarify the role of zinc in the pathogenesis of LVH, we will establish Zip14 gene knockout LVH rats fed with different concentrations of zinc and will use the ER inhibitor TUDCA to investigate the relationship between the Zip14 regulation of zinc homeostasis and ERS. Also, we would examine whether the ERS is inhibited by zinc supplementation in the LVH rat model in the following studies. Secondarily, this study mainly focused on the correlation between Zip14 and ERS induction of LVH animal model; however, the unfolded protein response (UPR) pathway and associated sensors, such as IRE phosphorylation, PERK (or eIF2α) phosphorylation or ATF6 cleavage, have not been determined here. In a future study, we would determine more contents such as PERK/eIF2α, cleaved ATF6, spliced XBP1 signaling pathways and associated molecules to enrich the findings and conclusions. Thirdly, the link between ERS and Zip upregulation has not been clearly investigated in this study. A previous study reported that under pharmacologically induced ERS or chemically alleviated protein misfolding, the Zip14 and zinc content are upregulated (Kim et al., 2017). Therefore, knocking down the Zip14 gene, such as siRNA targeting, would be better for clarifying the link between Zip14 and ERS. Meanwhile, the potential common transcription factor binding sites (TFBS) clusters in the promoter regions of gene Zip14, ATF6, ATF4 might be significant for demonstrating the correlation between Zip14 and ATF6 or ATF4. Fourthly, in the process of modeling, the body weight of rats was lower in LVH group compared to the Control group, which is another limitation of our study. We speculated that the body weight loss might be caused by the treatment of high salt fed (might affect the appetite of the rats). Finally, the Zn 2+ contents might affect the effects of Zip14; however, Zn 2+ contents in the blood of rats in both groups have not been determined.

Conclusions
Zinc accumulation and the upregulation of Zip14 and ERS were observed in myocardial tissues of rats with LVH. The upregulation of Zip14 in LVH rat hearts correlated with zinc accumulation and induction of ERS. However, the exact mechanism of these interactions needs to be investigated further.
Acknowledgement: We thank the support and help of the team of Professor Xu Zhelong from the School of Basic Medicine, Tianjin Medical University.
Availability of Data and Materials: All data generated or analyzed during this study are included in this published article (and its supplementary information files).
Author Contribution: The authors confirm contribution to the paper as follows: study conception and design: QY and YS; data collection: JH, TT, BB, YX, LH, ZX; analysis and interpretation of results: JH, LH, ZX, YS; draft manuscript preparation: JH, QY and YS. All authors reviewed the results and approved the final version of the manuscript.
Ethics Approval: All animal treatments were strictly in accordance with international ethical guidelines and the National Institute of Health Guide concerning the Care and Use of Laboratory Animals. The experiments were carried out with the approval of the Committee of Experimental Animal Administration of the University (ethical approval code: ZYY-IRB-SOP-016(F)-002-02, date of approval: 30th, April, 2015).
Funding Statement: This work was supported by the key projects of Tianjin Natural Science Foundation (Grant No. 17JCZDJC34800).

Conflicts of Interest:
The authors declare that they have no conflicts of interest to report regarding the present study.