Increased free Zn2+ correlates induction of sarco(endo)plasmic reticulum stress via altered expression levels of Zn2+‐transporters in heart failure

Abstract Zn2+‐homoeostasis including free Zn2+ ([Zn2+]i) is regulated through Zn2+‐transporters and their comprehensive understanding may be important due to their contributions to cardiac dysfunction. Herein, we aimed to examine a possible role of Zn2+‐transporters in the development of heart failure (HF) via induction of ER stress. We first showed localizations of ZIP8, ZIP14 and ZnT8 to both sarcolemma and S(E)R in ventricular cardiomyocytes (H9c2 cells) using confocal together with calculated Pearson's coefficients. The expressions of ZIP14 and ZnT8 were significantly increased with decreased ZIP8 level in HF. Moreover, [Zn2+]i was significantly high in doxorubicin‐treated H9c2 cells compared to their controls. We found elevated levels of ER stress markers, GRP78 and CHOP/Gadd153, confirming the existence of ER stress. Furthermore, we measured markedly increased total PKC and PKCα expression and PKCα‐phosphorylation in HF. A PKC inhibition induced significant decrease in expressions of these ER stress markers compared to controls. Interestingly, direct increase in [Zn2+]i using zinc‐ionophore induced significant increase in these markers. On the other hand, when we induced ER stress directly with tunicamycin, we could not observe any effect on expression levels of these Zn2+ transporters. Additionally, increased [Zn2+]i could induce marked activation of PKCα. Moreover, we observed marked decrease in [Zn2+]i under PKC inhibition in H9c2 cells. Overall, our present data suggest possible role of Zn2+ transporters on an intersection pathway with increased [Zn2+]i and PKCα activation and induction of HF, most probably via development of ER stress. Therefore, our present data provide novel information how a well‐controlled [Zn2+]i via Zn2+ transporters and PKCα can be important therapeutic approach in prevention/treatment of HF.


Introduction
Advanced HF is an irreversible process while numerous different signalling pathways and mechanisms are involved during its development. The association between defective cardiac activity and altered cellular Ca 2+ -homoeostasis is well characterized in HF [1]. Emerging evidence suggests a central role of intracellular-free Zn 2+ ([Zn 2+ ] i ) in excitation-contraction coupling in cardiomyocytes by shaping Ca 2+ dynamics [2,3]. Experimental and clinical studies have also shown that several proteins, having pivotal role in controlling cardiac contractility, are also potential targets of [Zn 2+ ] i as well as [Ca 2+ ] i [4,5]. Zinc as Zn 2+ is required for structure and function of cells and its availability, via interprotein Zn 2+ -binding sites, influences functions of numerous proteins in mammalian cells [6]. Therefore, any impairment in [Zn 2+ ] i homoeostasis may result in a variety of cellular dysfunction including cardiomyocytes, which may in turn induce serious cardiovascular pathologies [7]. Cellular [Zn 2+ ] i is tightly regulated against its adverse effects through either Zn 2+ transporters, Zn 2+ -binding molecules or Zn 2+ sensors, and, therefore plays a critical role in cellular signalling pathways [8,9]. In addition, it has been suggested that [Zn 2+ ] i associated with cellular signalling mechanisms can be classified by time windows, such as early Zn 2+ signalling within several minutes as well as late Zn 2+ signalling within several hours following its intracellular increase [7]. However, it remains unclear whether these suggested signalling effects are independent, combinatory or cell-dependent. Most of [Zn 2+ ] i -associated cellular events arised due to either the effect of mobile reactive/free Zn 2+ but not on the non-exchangeable Zn 2+ . Free Zn 2+ participates in redox homoeostasis on sarco(endo)plasmic reticulum {S(E)R} function and mitochondrial metabolism, as well [10][11][12][13][14]. Only few studies have documented the role of Zn 2+ homoeostasis in cardiovascular dysfunction. These studies reported the role of Zn 2+ dyshomeostasis in the pathogenesis of myocardial ischaemia/reperfusion injury [15], the role of a Zn 2+ transporter, ZIP12, in the regulation of the pulmonary vascular response to chronic hypoxia [16] and the role of crosstalk between ZnT-1 and the L-type Ca 2+ channels in cardiac electrical remodelling of adult rat atrium and in humans with atrial fibrillation [17,18].
ER stress is one of the underlying mechanisms of major diseases associated with cardiac dysfunction including diabetic cardiomyopathy [10,19]. Indeed, we have previously shown that S(E)R function can be normalized when [Zn 2+ ] i is kept at normal level via enhancement of the antioxidant defence in diabetic rats [3,11]. Recently, we have shown that Zn 2+ decrease in S(E)R leads to the up-regulation of ER stress confirming the requirement of Zn 2+ for proper S(E)R function [20]. However, there are no clear data on the role of Zn 2+ transporters controlling [Zn 2+ ] i in cardiomyocytes during the progression of HF, and, further studies are needed to clarify this important possible relation.
Excess [Zn 2+ ] i could affect the function of the ZIP and ZnT families, including the enhancements of protein kinase C (PKC) signalling and activation in Akt and ERK pathways [21]. Particularly, free [Zn 2+ ] i can increase the activity of PKC by contributing to its binding to plasma membrane and hence play a crucial role in many signal transduction pathways [21][22][23]. Furthermore, one isoform of PKC, PKCa, has been shown to have unique properties among other PKC isotypes in terms of induction of cardiac hypertrophy, and regulation of contractility and HF susceptibility [24][25][26]. Moreover, we recently have shown that hyperglycaemia induced marked changes in ZIP7 and ZnT7 expression levels underlined excess Zn 2+ release from S(E)R and could mediate ER stress in the heart [20].
Therefore, in this study, we first aimed to test the possible roles of some Zn 2+ transporters in the development of HF via induction of ER stress. For this purpose, we, for the first time, monitored subcellular localizations of Zn 2+ transporters such as ZIP8, ZIP14 and ZnT8 in cardiomyocytes. We also examined the role of [Zn 2+ ] i in HF-modelled cardiomyocytes with doxorubicintreated cells (embryonic left ventricular rat heart cell line, H9c2 cells) and protein expressions levels of these Zn 2+ transporters in human HF samples (end-stage failing hearts, due to dilated or ischaemic cardiomyopathy) and in HF-modelled cells. Lastly, we monitored the onset of ER stress using its markers such as GRP78, CHOP/Gadd153 and calnexin as well as phosphorylation level of PKCa in heart preparations or in directly [Zn 2+ ] i increased cardiomyocytes.

Patients and tissues
Patients, who were scheduled to undergo orthotopic heart transplantation for end-stage HF at the Department of Cardiovascular Surgery of Ankara University Faculty of Medicine, were eligible to participate in the study. The Local Ethics Committee of Ankara University approved the study protocol (1003201404-142-14). The investigation conforms to the principles outlined in the Declaration of Helsinki. All participating patients signed informed written consent before surgery. Left ventricular tissues were collected from discarded hearts of advanced HF patients. The cause of HF was dilated cardiomyopathy (DCMP) in two patients, and one patient had ischaemic cardiomyopathy (ICMP). Control left ventricular tissues were obtained from deceased donors that were unsuitable for cardiac transplantation. Donor families were also consented to use of the cardiac tissues for research. Clinical characteristics of patients and controls are given in Table 1.

Cell line and treatment of cells with doxorubicin
We used cardiac myoblasts from left ventricle of the embryonic rat heart (ATCC CRL1446; purchased from American Type Culture Collection, Manassas, VA). Cells were grown at a density of about 10 5 cells/ cm 2 in DMEM modified using 5.5 mM glucose instead of 25 mM and supplemented with 10% foetal calf serum, 50 U/ml penicillin-G and 50 lg/ml streptomycin in a humidified atmosphere of 95% air and 5% CO 2 at 37 ο C.
To model HF in cardiomyocytes, the procedure as described previously was performed [27]. The cultured H9c2 cardiomyocytes in media containing 0.5% bovine serum albumin were treated with a doxorubicin analog agent adriamycin (1 lM for 24 hrs) at 37°C. Cell viability and apoptosis were evaluated by MTT cell viability assay and some apoptosis-related signal proteins.

Statistical analysis
Continuous data were expressed as mean (AES.E.M.), and categorical data as percentages. Groups were tested and compared using one-way ANOVA and Tukey post hoc test. A value of P ≤ 0.05 was considered statistically significant. For all data, no statistics were used for predetermination of the sample size, randomization or blinding.

Outcome of patients
We used left ventricular samples from three patients and two controls. Three patients underwent orthotopic heart transplantation with end-stage HF. One of these patients had ICMP while the other two had DCMP (HF group). We obtained control group samples from deceased donors that were unsuitable for cardiac transplantation (DD group). Both controls have good left ventricular function. One control deceased donor had Hepatitis C, and the other heart of the deceased donor was discarded because of persistant hypovolemic shock due to multiple traumatic injuries. The haemodynamic characteristics of patients and controls are summarized in Table 1. As mentioned previously, an important biochemical marker in heart failure is B-type natriuretic peptide (BNP), which is produced in heart ventricles in response to increased mechanical load and wall stretch [28]. Therefore, before undergoing orthotopic heart transplantation with endstage HF, the plasma BNP levels were measured in patients. The BNP level was in the range of 3000-4000 pg/ml in group of HF while this value was 2000-2400 pg/ml in DD group. The difference between these two groups is significantly different from each other (P < 0.001).

Demonstration of subcellular localizations of ZIP8, ZIP14 and ZnT8 in cardiomyocytes
To test whether ZIP8, ZIP14 and ZnT8 are localized to either on S(E) R, sarcolemma or both, the cells were co-incubated with plasma membrane marker antibody (PMCA) and ZIP8, ZIP14 or ZnT8 primary antibodies after fixation and permeabilization. Images were captured using confocal microscopy and then merged ( Our PCs' values strongly support the localization of these three Zn 2+ transporters on sarcolemma as about over 60% in mammalian ventricular cardiomyocytes. Additionally, our data demonstrated that these transporters were also localized on the S(E)R of the cardiomyocytes as about 40-50% due to calculated PCs' values.

Altered expression levels of the Zn 2+ transporters in heart failure
We examined first the expression levels of Zn 2+ transporters ZIP8, ZIP14 and ZnT8 in human heart tissue. Western blot analysis showed that the expression levels of ZIP14 and ZnT8 were significantly higher than those of controls, whereas ZIP8 level was significantly lower comparison to the control ( Fig. 2A, right) while the representative Western blot images are given also in the same figure, as well ( Fig. 2A, left). To validate the human heart data related with changes in expression levels of ZIP14, ZIP8 and ZnT8, we also examined the expression levels of these transporters in doxorubicin-treated rat ventricular cells. The observed changes in these transporters were similar to the human data (HF). The expression levels of ZIP14 and ZnT8 were also significantly higher than those of controls while the ZIP8 level was markedly low (Fig. 2B, right). Representative Western blot images are given in the left part of Figure 2B.
Validation of heart failure in human heart tissue and doxorubicin-treated cells To correlate diastolic and systolic dysfunction in human HF and deposition of non-sarcomeric alpha (a)-actinin (ACTN) in HF and HF-modelled cells, we first measured the ACTN level in underwent orthotopic heart transplantation with end-stage HF with respect to unsuitable for cardiac transplantation patients (CON group). As can be seen from Figure 3A, expression level of ACTN in human tissue (HF group) was significantly high with respect to CON group (left). To confirm further the HF in heart tissues and cell line, we measured the expression level of this biomarker protein, ACTN in doxorubicin-treated H9c2cells. As can be seen in Figure 3A, ACTN level was markedly high in the treated group compared to the untreated group (right). We also examined the expression level of PUMA (as a proapoptotic member of the Bcl-2 family) to further confirmation of heart failure in both human tissues and doxorubicin-treated H9c2 cells. As can be seen in Figure 3B, PUMA protein levels (with respect to GAPDH) were increased markedly in HF group and HFmodelled H9c2 cells comparison with those of their controls (left and right, respectively).

[Zn 2+ ] i increases in doxorubicin-treated cardiomyocytes
To determine [Zn 2+ ] i in HF-modelled cells, we measured the [Zn 2+ ] i changes in H9c2 cells loaded with a Zn 2+ selective and highly sensitive fluorescent dye FluoZin-3. Representative fluorescence intensity changes of FluoZin-3 and experimental protocol are given in Figure 3C. Our data showed that Zn 2+ -ionophore pyrithione, Zn 2+ /Pyr (1 lM) exposure (3 min.) induced significant increase in FluoZin-3 intensity. Further addition of high-affinity heavy-metal Zn 2+ chelator, N,N,N',N'-tetrakis(2-pyridylmethyl) ethane-1,2-diamine, TPEN (50 lM), FluoZin-3 intensity decreased below its initial value. Of note, in our experiments, the fluorescence loading and Zn 2+ /Pyr-induced changes in [Zn 2+ ] i changes as well as [Zn 2+ ] i chelation of [Zn 2+ ] i in the cells were very sharp and confirmed to be homogenous using confocal images of the loaded cells. The differences between calculated intracellular-free Zn 2+ levels as nM from normal and doxorubicin-incubated H9c2 cells are given in Figure 3D. As can be seen from the bar graphs, the [Zn 2+ ] i of the treated cells is about fourfold higher compared to the controls.

ER stress sensors as molecular chaperones in human HF and doxorubicin-treated H9c2 cells
As GRPs (ER-targeted cytoprotective chaperones, the unfolded-protein response signalling-proteins), as molecular chaperones, are regulated by signal transduction pathways originating in the ER, we first examined the protein expression levels of GRP78, CHOP/Gadd153 and calnexin in the human tissues. Expression levels of GRP78 and CHOP/Gadd153 in human HF group were significantly increased with respect to the controls while no change in calnexin expression level ( Figure 4A).
To test whether the inhibition of an endogenous kinase, protein kinase C (PKC), which is generally activated under pathological condition due to different cellular alterations including cellular Zn 2+ level [22,23] lead to the suppression of ER stress, we performed another group of measurements. As can be seen in Figure 4C, PKC inhibition with an inhibitor (IPKC; incubation of H9c2 cells with 0.1 lM GF109203X hydrochloride for 24 hrs) prevented the induction of ER stress, significantly.

Verification of increased [Zn 2+ ] i -associated ER stress in doxorubicin-treated H9c2 cells
To verify whether increased [Zn 2+ ] i underlies the induction of ER stress in HF-modelled H9c2 cells, we first incubated H9c2 cells with Zn 2+ -ionophore pyrithione, Zn 2+ /Pyr (0.1 lM for 24 hrs) and then measured the levels of ER stress chaperones mentioned the previous section. As can be seen from Figure 5A, the expression levels of GRP78, CHOP/Gadd153 and calnexin increased significantly when [Zn 2+ ] i are increased.
For further confirmation of this experimental approach, in another set of experiments, we measured the expression levels of these markers in the presence of ER stress inhibitor tauroursodeoxycholic acid (TUDCA; 50 lM for 18 hrs) in Zn 2+ /Pyr pre-treated cardiomyocytes. As can be seen from Figure 5A (last columns), the expression of these chaperone proteins was not different from those of controls. These data can suggest a possible association between ER stress induction and increased [Zn 2+ ] i in human HF.
To understand the involvement of PKCa either under increased [Zn 2+ ] i or with TUDCA which directly inhibits ER stress, we measured PKCa expression level in these groups. As can be seen in Figure 5A (last column), PKCa expression level was increased significantly under increased [Zn 2+ ] i with respect to the controls while its level was not changed in ER stress inhibited cells.

The [Zn 2+ ] i increase but not a direct ER stress induction underlies the changes in the altered expression levels of Zn 2+ transporters in HFmodelled doxorubicin-treated H9c2 cells
To verify whether ER stress response activation accounts for cardiomyocyte dysfunction obtained in doxorubicin-incubated H9c2 cells, we examined the expression levels of these chaperones in a direct ER stress activator tunicamycin incubated H9c2 cells (TUN; 10 lM for 18 hrs). As seen in Figure 5B, the expression levels of none of these Zn 2+ transporters were not changed, significantly. From these data, one can provide a hypothesis that ER stress due to any sources cannot directly induce changes in the expression levels of ZIP8, ZnT8 and ZIP14 except increased [Zn 2+ ] i . Therefore, one can further hypothetize that ER stress can be the end process following the changes in these transporters, most probably due to increased [Zn 2+ ] i in mammalian HF condition.

The endogenous kinases in failing human heart
To extend our testing on possible association between HF, increased [Zn 2+ ] i and role of protein kinase-2 (CK2) on triggering Zn 2+ -signalling pathways by phosphorylating Zn 2+ transporters [29] and contributing to Zn 2+ homoeostasis, particularly under pathological conditions [20,30], we measured the expression level of CK2a. As can be seen from Figure 6A, the CK2a level in human HF was not significantly different from that of controls.
As a PKC isoform, PKCa, has been shown to have unique properties among the PKC isotypes such as induction of cardiac hypertrophy [25], regulation of contractility and heart failure susceptibility [26], we aimed to examine this parameter in human HF samples.
The expression level of PKCa was markedly high in human HF group compared to the CON group (Fig. 6C). Furthermore, we also determined the phosphorylation level of PKCa (phospho-PKCa) in the same samples. As can be seen in Figure 6D, the expression level of phospho-PKCa was significantly increased in HF group comparison with the CON group.

Validation of the role of PKCa-phosphorylation under increased [Zn 2+ ] i in human HF
To test further the role of PKCa-phosphorylation in [Zn 2+ ] i increase in mammalian HF, we measured [Zn 2+ ] i level in both control and doxorubicin-treated cells with and without a total PKC inhibitor (IPKC; 0.1 lM for 24 hrs incubation). As shown in Figure 6E, the increased [Zn 2+ ] i level in doxorubicin-treated H9c2 cells was significantly decreased while there was also decrease in the untreated cells.

Discussion
In the present study, we provide evidence to support the crucial role of increased [Zn 2+ ] i on mammalian heart dysfunction via alterations of protein expression levels of Zn 2+ transporters and induction of ER stress, most probably due to phosphorylation of PKCa. Additionally, our present data further suggest that expression level of some Zn 2+ transporters together with PKCa activation may play important role in human HF via induction of ER stress. Therefore, our observation related with the activation of PKCa under an increased [Zn 2+ ] i but no change in ER stress markers under PKC inhibition as well as no increase in [Zn 2+ ] i of the doxorubicin-treated cells under PKC inhibition suggests the possible role of these Zn 2+ -transporters. These information led us to have a conclusion of increase [Zn 2+ ] i and PKCaactivation (on a way of intersection-pathway associated with them) induced HF, via induction of ER stress. Therefore, they have a paramount importance since [Zn 2+ ] i via Zn 2+ transporters and PKCa may be novel therapeutic approach in prevention/treatment of human HF. Zinc, particularly as Zn 2+ , is essential for human health, and disturbances in its homoeostasis can contribute and/or exacerbate the pathology observed in many chronic conditions including cardiovascular diseases [31][32][33]. Although Zn 2+ is required by all cell types, and its toxicity is relatively rare, [Zn 2+ ] i is tightly controlled under physiological conditions by several ways including specific Zn 2+ transporters, at most, in redox homoeostasis of the cells [34]. Yet, the overall physiological importance of [Zn 2+ ] i and Zn 2+ -transporters at the whole-organism level, particularly their interaction with pathological conditions, remains unclear. We examined possible roles of the Zn 2+ transporters ZIP8, ZIP14 and ZnT8 in cardiomyocytes under failing condition. For this essential aim, we first examined their protein levels in failing heart preparations. Under any types of heart failure, cardiomyocytes significantly decreased protein level of ZIP8 with markedly increased level of ZIP14 and ZnT8. Additionally, high [Zn 2+ ] i existence of ER stress together with marked apoptotic status validated the failing heart in our samples. We also, for the first time, presented the subcellular localizations of these transporters in cardiomyocytes. Here, we demonstrated that ZIP8, ZIP14 and ZnT8 localized to both S(E)R and sarcolemma in H9c2 cells, further indicating multiple localization sides for these Zn 2+ transporter in cardiomyocytes. Taken into consideration a possible crosstalk between increased [Zn 2+ ] i , induction of ER stress and apoptosis in cardiomyocytes, although not assessed directly, it is very logical to hypothesize that any change in any Zn 2+ transporter under pathological conditions is likely to further exacerbate these changes and contributes to the deleterious consequences of Zn 2+ redistribution between compartments. Importantly, these transporters are shown to localize into S(E)R membrane and may thus operate as a functional couple catalysing Zn 2+ release and uptake respectively from the S(E)R while S(E)R is a very good candidate as Zn 2+ pool [2,5,20].
Herein, we have shown a close correlation between Zn 2+ transporters, [Zn 2+ ] i and ER stress in failing heart. Our present data demonstrated that ER stress is not induced directly but, our data showed that increased [Zn 2+ ] i due to alterations in the Zn 2+ transporters induces ER stress chaperons. ER stress is associated with a range of diseases, including ischaemia/ reperfusion injury and other heart diseases, making ER stress a probable instigator of pathological cell death and dysfunction [35]. The role of ER stress in heart disease has not been extensively studied, but it is well accepted that disturbances in the normal functions of the ER lead to an evolutionarily conserved cell stress response, which is aimed initially at compensating for damage but can eventually trigger cell death if ER dysfunction is severe or prolonged. The principal challenge with any strategy for blocking cell death caused by ER stress lies with the multitude of parallel pathways potentially leading to downstream cell death mechanisms. Thus, blocking only one cell death pathway emanating from the ER may be inadequate to preserve cell survival. Further studies of genes and gene products involved in ER stress-initiated cell death are needed to fully validate targets for drug discovery.
In the present study, we also confirmed the development of HF in human tissue and HF-modelled cardiomyocytes by measuring high level of non-sarcomeric alpha (a)-actinin (ACTN). Indeed, correlation between morphological changes, including development of fibrosis, and diastolic and systolic dysfunction together with deposition of ACTN in cardiomyocytes from patients with dilated cardiomyopathy or chronic pressure overload has been shown, previously [36,37]. Although the precise mechanism of ER stress-induced cardiomyocyte apoptosis remains elusive; it is believed that the mitochondrial apoptotic machinery is recruited through up-regulation of PUMA, a proapoptotic member of the Bcl-2 family. Importantly, we and others have shown that any suppression of PUMA activity could prevent both ER stress and ischaemia/reperfusion-induced or diabetes-induced cardiomyocyte loss [38]. In parallel to these data, we also found PUMA levels were increased markedly in all failing heart models compared to the controls suggesting a signalling mechanism related with ER stress-mediated apoptosis.
Although there are several studies related with ZIP8, ZIP14 and ZnT8, there are missing data associated with these transporters and cardiovascular complications. As summary of role of ZIP8 in mammalian cells, Galvez-Peralta et al. have shown a critical role of ZIP8mediated Zn 2+ transport during in utero and neonatal growth, organ morphogenesis and hematopoiesis while its critical importance was demonstrated for Zn 2+ cytoprotection in lung epithelia [39]. ZIP14 associated studies pointed out the expression of this transporter in heart tissue besides the other organs in mammalians. Previous studies have shown that ZIP14 is localized at the plasma membrane and in transferrin-containing endosomal compartments [40,41]. Recent studies demonstrated that the level of ZIP14 protein is increased in the liver of rats fed a high-iron diet and in iron-loaded human hepatoma cells, suggesting that ZIP14 contributes to tissue iron loading under high-iron conditions [41]. A tissue expression array shows that ZIP14 mRNA is ubiquitously expressed with high levels in the liver, pancreas and heart [40].
The ZnT8 is exclusively expressed in pancreatic b-cells and encodes a protein that transports Zn 2+ from cytoplasm to insulin secretory vesicles [42]. Additionally, genomewide-associated studies and recent meta-analysis studies demonstrated that a polymorphism Fig. 7 A putative summarized presentation of our data on a possible role of Zn 2+ transporters on an intersection-pathway associated with increased cytosolic free Zn 2+ and PKCa-activation and induction of heart failure via development of ER stress. The thickness of the arrows related with the transporters show the alterations in their expression levels. Here, thick red arrows represent increased level of pPKC as phosphorylated PKCa and increased [Zn 2+ ] i , intracellular-free Zn 2+ concentration in cells under heart failure. The thickness of red arrows related with localization of Zn 2+ transporters represent increase/decrease expression levels. S(E)R represents sarco(endo)plasmic reticulum.  1953 in ZnT8 has closely associated with increased risk of impaired glucose regulation and type 2 diabetes [12,43,44]. In studies on multiple Zn 2+ transporters, such as ZIP8 and ZIP14 or ZIP14 and ZnT10, it has been mentioned that they can be functioning together and thus enhancing their roles in cellular signalling mechanisms [45].
It is well documented that several endogenous substrate proteins of mammalian heart can be phosphorylated with PKC isozymes while they can be activated under pathological conditions including high oxidative stress status via thiol oxidation and release of Zn 2+ from cysteine-rich region of PKC [46,47]. In here, we have shown that PKCa can be activated under increased [Zn 2+ ] i in cardiomyocytes while its activation can be prevented by ER stress inhibition. Additionally, we have also shown that if we inhibited PKCa, then ER stress can also be inhibited. Therefore, one can suggest that ER stress can be prevented if we have a well-controlled [Zn 2+ ] i via inhibition of PKCa even if under HF. Indeed, studies pointed out the important role of activated PKCa in the heart, as an important mediator of induction of ventricular arrhythmias [48]. It has also been shown that PKCa along with [Zn 2+ ] i and Zn 2+ transporters function as effectors of oxidative tissue injury, in part, via induction of ER stress [49,50]. Particularly, PKCa is a specific member of the protein kinase family and is unique in its mode of regulation compared to other kinases within this family. However, it seems CK2 does not underlie any Zn 2+ -related mechanisms in mammalian heart failure, although it has important role in function of cancer cells via ZIP7 [40].
A schematic presentation of our present data, given in Figure 7, shows the subcellular distribution of Zn 2+ transporters, ZIP8, ZIP14 and ZnT8 in ventricular cardiomyocytes. Three of them are localized to both S(E) and sarcolemma and seem to be responsible for the regulation of cellular distribution of free Zn 2+ under physiological condition. When cardiomyocytes are under pathological condition, leading to heart failure, the expression levels of these three transporters are affected through most probably phosphorylation/activation of PKCa, consequently, [Zn 2+ ] i is increased in cardiomyocytes. In this regard, it has been shown that PKC has ability to regulate many cardiovascular functions via targeting many cardiovasotropic growth factors [51], whereas [Zn 2+ ] i increases the interaction between PKC and actin flaments [23,52]. Therefore, PKC isoforms could be assessed as possible Zn 2+ targets and as important regulators of cardiac function under the conditions of ischaemia reperfusion [53]. The increase in [Zn 2+ ] i under pathological condition further underlies induction of ER stress via overexpression of ER stress chaperons in cardiomyocytes which is nicely confirmed with measurement of [Zn 2+ ] i under a PKC inhibition in doxorubicin-treated cardiomyocytes.
Overall, our present data provide an important insight into the HF in humans, in part, due to the importance of [Zn 2+ ] i , Zn 2+ transporters and proper excitation-contraction coupling in cardiomyocytes, via an association with phosphorylation of PKCa. Indeed, Zn 2+ is an essential cofactor for normal cell function and herein, we have shown that altered expression and function of Zn 2+ transporters can contribute to the pathogenesis of cardiac disorders through increased ER stress and apoptosis. The expression and regulation of Zn 2+ transporters in the heart and the toxicity of high [Zn 2+ ] i to these cells will open new insights into the HF in mammalians as proposing new therapeutic strategy as well as the development of Zn 2+ containing new markers/ sensors to better handle HF in humans.

Limitations
Further studies are needed to evaluate the role of [Zn 2+ ] i , and Zn 2+ transporters in advanced HF patients with different aetiologies. Duration of advanced HF and medications also may affect the results.