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

Figures

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  Figure 1. Visualization of ZIP8, ZIP14 and ZnT8 localizations in H9c2 cells. Localizations either to the S(E)R or to the sarcolemma/plasma membrane of ZIP8 (A), ZIP14 (B) and ZnT8 (C) were visualized using confocal micrographs in H9c2 cells. To examine the subcellular localizations of these transporters to S(E)R, H9c2 cells were transfected with ER‐resident discosoma red fluorescent protein (dsRed‐ER) to label ER or incubated with PMCA antibody to label sarcolemma/plasma membrane and with DAPI (blue) as well. Cells were incubated only with secondary antibodies and mounted with mounting medium contains DAPI (blue), and then images were merged.

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  Figure 2. Expression levels of ZIP8, ZIP14 and ZnT8 in heart tissue homogenates and doxorubicin‐treated H9c2 cells. Representative Western blotting bands for protein expression levels are given left part of figures in (A) and (B). The densitometry analysis showing the proteins of ZIP8, ZnT8 and ZIP14 is expressed at 50 kD, 41 kD and 50 kD, respectively, in doxorubicin‐treated H9c2 cells (DOXO group) with respect to untreated cells (CON group) (A) and underwent orthotopic heart transplantation with end‐stage heart failure (HF group) with respect to unsuitable for cardiac transplantation patients (CON group) (B) GAPDH at 37 kD as reference protein. Bars represent mean (±S.E.M.) for each group. Number of human samples, n = 3 for HF group and n = 2 for CON group. All measurements with double assays in each sample from each group for each type of measurement. Significance level accepted at *P < 0.05 versus CON.

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  Figure 3. Validation of HF in human heart tissue and doxorubicin‐treated cells and demonstration of intracellular‐free Zn2+ increase under HF. (A) Expression level of α‐actinin, ACTN and PUMA in both human heart tissue with heart failure (HF) (A) and doxorubicin (DOXO)‐treated H9c2 cells (B) with respect to their controls (CON). Representative Western blotting bands for protein expression levels are given upper parts of figures in (A) and (B). (C) Representing of measurement protocol of intracellular‐free Zn2+, [Zn2+]i in doxorubicin‐treated (failing heart modelled, DOXO group) cells, loaded with Zn2+ selective fluorescent dye FluoZin‐3. Zn2+ ionophore pyrithione, Zn2+/Pyr (1‐μM) exposure (3 min.) and high‐affinity heavy‐metal Zn2+‐chelator, N,N,N',N'‐tetrakis(2‐pyridylmethyl)ethane‐1,2‐diamine, TPEN (50 μM). The mean (±S.E.M.) intracellular Zn2+level, as FluoZin‐3 intensity, respectively. (D) Bar graphs representing the [Zn2+]i levels calculated as nM (see Methods and materials) in the treated cells comparison with the untreated cells (CON group). Bars represent mean (±S.E.M.) for each group. Number of human samples, n = 3 for HF group and n = 2 for CON group. All measurements with double…

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  Figure 4. Western blot analysis showing the expression levels of ER stress/chaperone proteins in human and doxorubicin‐treated H9c2 cells. The densitometry analysis showing the protein levels of GRP78, CHOP/Gadd153 and calnexin at 94 kD, 30 kD and 90 kD, respectively, as well as PKCα activation status detected as protein expression level at 80 kD (with respect to GAPDH at 37 kD) in human heart failure (HF) (A) and in doxorubicin (DOXO)‐treated H9c2 cells (B) with respect to their controls (CON group). Representative Western blotting bands for protein expression levels are given upper parts of figures in (A) and (B). Bars represent mean (±SEM) for each group. Number of human samples, n = 3 for HF and n = 2 for CON. Significance level accepted at *P < 0.05 versus CON and # P < 0.05 versus TUDCA group.

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  Figure 5. Verification of the role of altered expression levels of Zn2+ transporters on increased [Zn2+]i‐associated ER stress in HF. The ER stress marker proteins such as GRP78, CHOP/Gadd153 and calnexin at 94 kD, 30 kD and 90 kD (with respect to GAPDH at 37 kD) in directly [Zn2+]i increased H9c2 cells with Zn2+ ionophore pyrithione, Zn2+/Pyr (0.1 μM for 24 hrs) incubation (without or with an ER stress inhibitor tauroursodeoxycholic acid, TUDCA, 50 μM for 18 hrs) H9c2 cells with respect to their controls (CON) (A). The densitometry analysis showing the protein levels of GRP78, CHOP/Gadd153 and calnexin at 94 kD, 30 kD and 90 kD, respectively (with respect to GAPDH at 37 kD). (B) The expression levels of ZIP8, ZIP14 and ZnT8 in a direct ER stress activator tunicamycin incubated H9c2 cells (TUN; 10 μM for 18 hrs). All measurements with double assays in each sample from each group for each type of measurement. Significance level accepted at *P < 0.05 versus CON.

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  Figure 6. Endogenous kinases CK2 and PKC in the human heart tissues. The densitometry analysis of the protein expression levels (Western blotting) given as original bands and mean (±S.E.M.) values for CK2 (α1/α2) in (A), total PKC in (B), phospho‐PKCα (right) and PKCα (left) in (C) with respect to GAPDH at 37 kD given for human (HF) and group comparison with the controls. The data were obtained after duplicate assays of each sample from each group for each type of measurement. The number of samples used are the same given in Figure 4 for groups. (D) The [Zn2+]i levels measured in doxorubicin‐treated cells with and without a total PKC inhibitor (IPKC GF109203X; 0.1 μM for 24‐hrs incubation). Bars represent mean (±S.E.M.). Significance level accepted at *P < 0.05 versus CON group, # P < 0.05 versus CON group and † P < 0.05 versus DOXO group.

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  Figure 7. A putative summarized presentation of our data on a possible role of Zn2+ transporters on an intersection‐pathway associated with increased cytosolic free Zn2+ and PKCα‐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 PKCα and increased [Zn2+]i, intracellular‐free Zn2+ concentration in cells under heart failure. The thickness of red arrows related with localization of Zn2+ transporters represent increase/decrease expression levels. S(E)R represents sarco(endo)plasmic reticulum.