hyperglycemia induced changes in zip7 and znt7 expression cause zn 2 release from the sarco endo plasmic reticulum and mediate er stress in the heart

Here, we hypothesized that ZIP7 and ZnT7 transport Zn2+ in opposing directions across the SER membrane in cardiomyocytes and that changes in their activity may play an important role in

1

Manuscript #: DB16-1099

Hyperglycemia-induced Changes in ZIP7 and ZnT7 Expression Cause Zn 2+ Release from the Sarco(endo)plasmic Reticulum and Mediate ER-stress in the Heart

Erkan Tuncay1, Verda C Bitirim1, Aysegul Durak1, Gaelle R J Carrat2, Kathryn Taylor3,

Guy A Rutter2* and Belma Turan1*†

1

Department of Biophysics, Ankara University, Faculty of Medicine, Ankara, Turkey;

2Section of Cell Biology and Functional Genomics, Division of Diabetes Endocrinology and Metabolism, Department of Medicine, Imperial College London, London, UK, and 3School of Pharmacy and Pharmaceutical Sciences, College of Biomedical and Life Sciences, Cardiff University, Cardiff, UK

Running title: Hyperglycemia and zinc-transporters in mammalian heart

Key words: Zinc transporters, cytosolic zinc, diabetic cardiomyopathy, heart, ER stress,

sarco(endo)plasmic reticulum, FRET, Protein kinase-2

or Prof Guy A Rutter Section of Cell Biology and Functional Genomics Department of Medicine

Imperial College London

Du Cane Rad London W12 0NN, U.K

Abstract

Changes in cellular free Zn2+ concentration, including those in the sarco(endo)plasmic

reticulum [S(E)R], are primarily coordinated by Zn2+-transporters whose identity and role in

the heart is not well established Here, we hypothesized that ZIP7 and ZnT7 transport Zn2+ in

opposing directions across the S(E)R membrane in cardiomyocytes and that changes in their

activity may play an important role in the development of ER-stress during hyperglycemia

The subcellular S(E)R-localization of ZIP7 and ZnT7 was determined in cardiomyocytes and

in isolated S(E)R-preparations Markedly increased mRNA and protein levels of ZIP7 were

observed in ventricular cardiomyocytes from diabetic rats or high glucose-treated H9c2 cells

whilst ZnT7 expression was low Additionally, we observed increased ZIP7-phosphorylation

in response to high glucose in vivo and in vitro Using recombinant targeted FRET-based

sensors, we showed that hyperglycemia induced a marked redistribution of cellular free Zn2+,

increasing cytosolic free Zn2+ and lowering free Zn2+ in the S(E)R These changes involve

alterations in ZIP7-phosphorylation and were suppressed by siRNA-mediated silencing of

CK2α Opposing changes in the expression of ZIP7 and ZnT7 were also observed in

hyperglycemia We conclude that sub-cellular free Zn2+ re-distribution in the hyperglycemic

heart, resulting from altered ZIP7 and ZnT7 activity, contributes to cardiac dysfunction in

diabetes

Introduction

Diabetes is an important risk factor for cardiovascular dysfunction via defective Ca2+signaling (1; 2) However, we have previously shown that Zn2+ release during cardiac-cycle results in cytosolic free Zn2+ increase (3), further triggering higher pro-oxidant species-production leading to oxidative damage (4) Conversely, oxidant exposure induces marked cytosolic free Zn2+ increases in cardiomyocytes (5) while hyperglycemia causes oxidative stress and increased cytosolic Zn2+ via underling cardiac dysfunction (6) Similar to Ca2+,

-Zn2+ is essential for cellular functions in mammalian heart (7), serving up as an important secondary-messenger (8)

Excess Zn2+ can be detrimental to cells, particularly that of cardiomyocytes (3; 5-7) Cytosolic

Zn2+ has an important role in excitation-contraction coupling in cardiomyocytes by shaping

Ca2+ dynamics (3; 4; 9) Its level in cardiomyocytes is calculated less than 1-nM while ~5-fold higher in sarco(endo)plasmic reticulum [S(E)R] and less than cytosolic-level in mitochondria demonstrated previously using FRET-based recombinant-targeted Zn2+-probes (10) Elevated cytosolic Zn2+ appears to contribute to deleterious changes in many cellular signaling-pathways including hyperglycemia-challenged cardiomyocytes (3-6; 11)

Cellular Zn2+-fluxes are achieved and controlled by Zn2+-transporters (ZnTs) and importers (ZIPs) (12; 13) while their distributions and functions are not yet well-clarified in cardiomyocytes The Zn2+-selective ion-channel ZIP7 has important role for releasing Zn2+ from S(E)R and Zn2+-associated induction of unfolded-protein-response in yeast (14) and is localized to Golgi apparatus in Chinese-Hamster Ovary-cells, allowing Zn2+-release from Golgi lumen into cytosol (15) ZIP7 facilitates release of Zn2+ from ER (16) and behaves as a critical component in sub-cellular re-distribution of Zn2+ in other systems(17) Additionally, it has been hypothesized that protein kinase-2 (CK2) triggers cytosolic Zn2+-signaling-pathways

by phosphorylating ZIP7 (18) while some studies have also highlighted its important

contribution to Zn2+-homeostasis under pathological conditions (19-21)

Although studies have shown the presence of weakly expressed ZIP7 and ZnT7 in

mammalian heart (22; 23), their subcellular localizations and functional roles are not yet

known well In that regard, previous studies have suggested that either increase or inhibition

of one of them might contribute to cellular dysfunction (11), including defective

insulin-mediated signaling-pathways under hyperglycemia (24) or altered insulin-secretion (25; 26)

ER-stress is one of underlying mechanisms of cardiac dysfunction including

diabetic-cardiomyopathy (27-29) Studies suggest that there is relationship between S(E)R function

and cytosolic Zn2+ level in diabetic rat cardiomyocytes (29; 30) In particular, the latter

studies identified a close association between oxidative-stress, cytosolic Zn2+ increase,

ER-stress and cardiac dysfunction in diabetes However, experimental evidence suggests a

requirement for Zn2+ for proper ER-function, with Zn2+ deficiency leading to ER-stress (14;

31) via decreased Zn2+ in the ER via hypoxia or hypoglycemia (14) However, there are no

clear data as to which Zn2+-transporters play roles in controlling cytosolic Zn2+ increases in

cardiomyocytes during hyperglycemia

It is therefore tempting to hypothesize that disruption of Zn2+-transporters and Zn2+-axis may

contribute to deleterious changes in diabetic cardiomyocytes Here, we aimed first to clarified

their subcellular localizations and then to explore their functional roles in Zn2+-homeostasis

Additionally, we tested their roles in cytosolic Zn2+ re-distribution and development of

ER-stress in hyperglycemic conditions, at most due to activation of CK2(α)

Research Design and Methods

Diabetes Induction

Our study was approved by Ankara University ethic committee (115-449) Type 1 diabetes was mimiced by single injection of streptozotocin (50 mg/kg, i.p.; Sigma-Aldrich) in 3-month-old 15-male Wistar rats while others (CON-group; 10) were injected with vehicle, as described previously (2) Following STZ-injection (7-day), rats with 3-fold higher blood-glucose level comparison to pre-injection level were used as diabetics (DM-group; 13 rats kept for12-week)

Cardiomyocyte Isolation

Cardiomyocytes were isolated from left ventricle using enzymatic-method, as described previously (5) Hearts were cannulated on a Langendorff-apparatus leaving pre-perfusion through the coronary artery with a Ca2+-free solution Following pre-perfusion, it was followed with 1-mg/mL collagenase (Type2, Worthington, USA) containing solution for 30-

35 min Only Ca2+ tolerant rod-shaped cells were used in order to ensure cell viability and excitability, as well to avoid SR Ca2+-overload and terminal-contracture

Cell Culture

The embryonic rat heart-derived H9c2 cell-line was purchased from American Type Culture Collection (Manassas, VA) and was cultured in Dulbecco's modified Eagle's medium, as described previously (10)

Cytosolic and S(E)R Free Zn 2+ Levels

Cytosolic and S(E)R free Zn2+ levels ([Zn2+]Cyt and [Zn2+]ER) in H9c2 cells were measured

using eCALWY sensors (Cyt-eCALWY4 and ER-eCALWY6) delivered with plasmids expressing Cyt-eCALWY4 and ER-eCALWY6 Measurements were performed as described previously (10) Images were captured at 433-nm monochromatic excitation-wavelength and image-analysis was performed with ImageJ-software using a homemade-macro To calculate free Zn2+, maximum (Rmax using a heavy-metal-chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)

ethylenediamine (TPEN, Sigma-Aldrich, USA; 50-µM) and minimum (Rmin using Zn2+

saturation with 100-µM ZnCl2 and Zn2+-ionophore pyrithione (Zn2+/Pyr, 5-µM) fluorescence

ratios were used as described, previously (13)

Imaging Sub-Cellular Localization of ZIP7 and ZnT7

ZIP7 and ZnT7 localizations were determined using anti-ZIP7 and anti-ZnT7 antibodies

(Thermofisher PA5-21072 and Santa-Cruz SC-160946 as 1:50, respectively) in confocal

microscopy (Zeiss LSM510) S(E)R localization was determined by transfection of H9c2 cells

with dsRED-ER (red) plasmid for 24-h After general procedures, cells were incubated with

specific-antibodies to monitor their localizations Following overnight-incubation, cells were

incubated with appropriate secondary-antibodies in presence of BSA (5%; Alexa Fluor-488

Donkey anti-Rabbit and Donkey anti-Goat for ZIP7 and ZnT7 respectively; 1:1000)

The Golgi was labeled with Anti-GM130 antibody cis-Golgi Marker (Abcam; ab52649;

1:750) Then, cells were incubated either with anti-ZIP7 or anti-ZnT7 and Golgi-marker

GM130-antibody for ZIP7 and ZnT7 (secondary-antibodies: Alexa Fluor 488 Donkey

anti-Rabbit for ZIP7; 1:1000 and Alexa Fluor 568 Goat anti-Mouse for GM130; 1:1000, Alexa

Fluor 488 Donkey anti-Goat for ZnT7; 1:1000 and Alexa Fluor 647 Rabbit anti-Mouse for

GM130; 1:1000) Finally, cells were mounted with medium containing DAPI (blue) Images

were deconvolved and analyzed for their colocalizations using Huygens-software and

processed with ImageJ (https://svi.nl/HuygensProfessional)

Sarco(E)R Isolation

Left ventricular S(E)R-fractionation of hearts was performed using ER-isolation kit (Sigma,

E0100) Briefly, hearts were homogenized in isotonic extraction-buffer and then centrifuged

Crude microsomal-fraction was isolated from post-mitochondrial fraction using

ultracentrifugation For further purification and separation for RER (rough-ER) and SER

(smooth-ER), self-generating density-gradient procedure was performed according to manufacturer’s instructions Western (immuno-) blot analysis was carried out using primary antibodies against ZIP7 and ZnT7 To confirm S(E)R-isolation, SERCA2 (Santa Cruz, SC-

8094), Golgi 58K Protein (Abcam, ab-27043) and cyclin E (Santa Cruz, SC481) were used as

S(E)R, Golgi or nuclear markers

ZIP7-silencing in H9c2-Cells with Stable Lentiviral-Infection

H9c2 cells were stably transfected with 4-unique 29-mer shRNA constructs in lentiviral vector (Origene, SR02938179A, B, C and D) and non-effective 29-mer scrambled shRNA cassette in pRFP-CB-shLenti Vector as control (Origene, TR30033) Following 24-h before transfection, cells were seeded into separate dishes Every shRNA construct with packaging and enveloping plasmids (psPAX; Addgene-12260 and pMD2G; Addgen-12259) were mixed into a solution contained CaCl2 (375-mM) and then incubated for 30-min DNA mixtures were added into HBS-buffer (mM; 12 Dextrose, 50 HEPES, 10 KCl, 280 NaCl, 1.5 Na2HPO4.H2O) while every mixture added into separate 15-cm dishes and incubated for 12-h Viral supernatants were harvested following 24-h and 48-h and then cells were infected with each lentivirus produced from every shRNA-construct including scrambled-sequences at 3-PFU/cell (incubation with 10-µg/mL blasticidin for antibiotic selection) They were seeded into 6-well plates and harvested for knock-down efficiency measuring ZIP7 mRNA level (Suppl Table 1) A mixture of 4-ZIP7 gene-specific shRNA-constructs was used

RFP-QRT-PCR Analysis

Total-RNA was prepared using RNA Isolation-kit (Macherey–Nagel, 740955.10) and purified total-RNA was reverse transcribed with ProtoScript First-Strand cDNA Synthesis-kit (New England Biolobs, E6300S) First strand-cDNAs were quantified with GoTaq® qPCR Master Mix (Promega, A6001) The amplified fragment size of PCR-products for each primer and primers’ specificity were controlled with NCBI and ENSEMBL databases Primer sequences

for cyclophilin, ZIP7 and ZnT7 are listed in Suppl.Table2 The fold changes in the genes were

analyzed based on comparative (2-∆∆Ct) method

Western (immuno-) blot Analysis

The lysates were extracted with NP-40 lysis buffer (250 mM NaCl, 1% NP-40, and 50 mM

Tris-HCl; pH 8.0 and 1X PIC) from homogenized samples Protein concentration of

supernatants (centrifuged 12,000×g, 5-min, at 4°C) was measured with BCA assay-kit

(Pierce) Equal amount of protein were separated on 12% SDS-PAGE Tris-glycine or 4-12%

Bis-Tris gels (Life Technologies) The membranes were probed with antibodies against

GRP78 Cruz sc-13968; 1:200), Calregulin Cruz sc-11398; 1:200), ZIP7

(Santa-Cruz sc-83858; 1:200), ZnT7 (Santa (Santa-Cruz sc-160946; 1:200), SERCA-2 (Santa-(Santa-Cruzsc-8094,

1:200), 58K Golgi Protein (Abcam ab-27043; 1µg/ml), cyclin E (Santa-Cruz SC481),

GAPDH (Santa-Cruz sc-365062; 1:1000) and β-actin (Santa-Cruz sc-47778; 1:500) in

BSA/PBS/Tween-20 solution Specific bands were visualized with HRP-conjugated

compatible secondary antibodies (anti-mouse: 1:2000, anti-goat: 1:7500, anti-rabbit: 1:7500)

and detected by ImmunoCruz Western-Blotting Luminol-Reagent (Santa Cruz, sc-2048) The

band densities were analyzed using ImageJ-software

Co-immunoprecipitation

Cells were treated as indicated, lysed in co-immunoprecipitation (co-IP) buffer (mM: 50 Tris,

100 NaCl, 1 EDTA, 1% Triton-X-100, 10% glycerol, pH=7.4) containing protease inhibitors

(1-mM PMSF, 1-µg/mL each of leupeptin, aprotinin and pepstatin) and phosphatase inhibitors

(mM: 10 sodium-fluoride, 1 sodium-orthovanadate) for 30-min at 4°C The 600-µg protein

lysates from aliquots (1-mL lysis buffer) were pre-cleared via incubation with 30-µL of

protein A/G Sepharose (Sigma USA) for 1-h at 4°C The pre-cleared samples were incubated

with specific primary antibody (anti-CK2α, 10-µg/mL; Santa-Cruz sc-6480) in lysis-buffer

for 2-h at 4°C and then 30-µL of protein A/G beads were added Then, samples were incubated for overnight at 4°C and then beads were washed 5 times with lysis-buffer, boiled and separated by 10% SDS-PAGE

CK2α- silencing in H9c2 Cells

CK2α (1 and 2) was silenced in H9c2 cells using Lipofectamine2000 ™ according to the manufacturer’s siRNA transfection protocol Briefly, cells were seeded into 6-well plates and cultured for 48- or 72-h with a mixture of 25 nM CK2α1 and CKα2 or non-targeted siRNAs (Dharmacon; ON-TARGETplus SMARTpool Csnk2a1 and Csnk2a2-siRNA, ON-TARGETplus Non-targeting Pool; L-096197-02-0005, L-092756-02-0005 and D-001810-10-

05 respectively) with serum-free medium including 5-µL/mL Lipofectamine After incubation, the cells were extracted into buffers to examine protein and mRNA expression levels The primer sequences designed for CK2α1 and CKα2 are given in Suppl.Table2

Protein and mRNA levels of ZIP7 (at 50-kDa) and ZnT7 (at 42-kDa) in isolated rat ventricular-cardiomyocytes are given in Fig.1A and B Both mRNA and protein levels of ZIP7 were significantly increased in diabetic (DM) rat cardiomyocytes with significantly decreased ZnT7 levels To test these changes directly arising via hyperglycemia, we incubated isolated-cardiomyocytes with high-glucose (33-mM) for 3-h and then examined ZIP7 and ZnT7 comparison to those of mannitol (23-mM)-incubated cells There were similar changes

in mRNAs to those of DM-cells except their protein levels (about 10%) (Suppl.Fig.1), most

probably due to shorter high-glucose expose comparison to diabetes

For further validation, we used high-glucose (25-mM) incubated (24-h)-H9c2 cells and

measured protein and mRNA levels As can be seen from Fig.1C and D, both levels of ZIP7

increased significantly in high-glucose incubated cells while ZnT7 mRNA level (Fig.1D) is

markedly (∼75%) decreased with relatively small decrease (∼25%) in its protein level

Next we performed experiments to determine whether the expression of other Zn2+

-transporters, such as ZIP1, ZIP6, ZnT1, ZnT5, may be affected in

hyperglycemic-cardiomyocytes Using QPCR, we demonstrated that mRNA levels of these transporters were

not significantly altered in high-glucose incubated versus control cells (25-mM for 24-h;

Suppl.Fig.2A and B)

High-Glucose Induces ZIP7-Phosphorylation

Since using a specific antibody developed by one of us (KT) in breast-cancer cells (18),

ZIP7-phosphorylation, we examined possible dependency of ZIP7-phosphorylation to

hyperglycemia in both diabetic-rat cardiomyocytes and high-glucose incubated H9c2 cells

(25-mM for 24-h or 48-h) As shown in Fig.1E, phospho-ZIP7 level measured at about

50-kDa in DM-cells was about 7-fold higher comparison to the controls Furthermore, the

phospho-ZIP7 level in high-glucose incubated H9c2 cells was higher comparison to

non-incubated cells, as a time-dependent manner (Fig.1F)

The pZIP7/ZIP7 ratio was similar in high glucose-incubated H9c2 cells while higher for

diabetic heart (0.96±0.04 vs 4.79±0.39, respectively)

Zn 2+ -transporters ZIP7 and ZnT7 Localize to the S(E)R

Since the existence of ZIP7 and ZnT7 has previously been reported in mammalian heart (22;

23) and proposed to localize to ER (17; 21; 25) and/or perinuclear-vesicles associated with

Golgi (22; 24) in cells but not cardiomyocytes and furthermore has been shown previously localization of ZIP6 and ZIP7 to ER regulating cytosolic Zn2+-homeostasis in dispersed pancreatic islet-cells (19), here, we postulated first the similar endogenous subcellular localization for ZIP7 and ZnT7 to S(E)R in H9c2 cells To examine this postulate cells,

co-following transfaction with ER-resident Discosoma-red fluorescent protein (See Methods), are

visualized for ZIP7 or ZnT7 localizations to S(E)R Individual and merged confocal images are given in Fig.2A and B The merge visual-inspections can be attributed to presence of majority of ZIP7 and ZnT7 to S(E)R For colocalization, Pearson-coefficient values were calculated for ZIP7 and ZnT7 associated with S(E)R by using Huygens programme as 0.67±0.07 (n=3) and 0.72±0.13 (n=3) , respectively

To test whether ZIP7 and ZnT7 localizes to Golgi, we used combination antibodies for either GM130 and ZIP7 or GM130 and ZnT7 Images are shown in Fig.2C and D The merge visual inspections can be attributed to the presence of minority of either ZIP7 or ZnT7 to Golgi For colocalization, Pearson-coefficient values for ZIP7 and ZnT7 associated with Golgi are found

in Suppl.Fig.2A to D For colocalization of ZIP7 and ZnT7 in the S(E)R, Pearson-coefficient values were calculated as 0.42±0.09 (n=3) and 0.54±0.08 (n=3) For colocalization of ZIP7 and ZnT7 in Golgi, Pearson-coefficient values were 0.11±0.02 (n=3) and 0.70±0.08 (n=3) For a comparison of the effects of hyperglycemia on subcellular localizations of ZIP7 and ZnT7, we compared the calculated Pearson-coefficient vales in normal and hyperglycemic

cells Exception of ZIP7 locatization to Golgi, localizations of other transporters was

unaffected by hyperglycemia (Suppl.Fig.3E)

Validation of ZIP7 and ZnT7 Localizations Using Western (immuno-) Blot in Isolated

S(E)R

The S(E)R localization of ZIP7 and ZnT7 was further investigated by Western (immuno-) blot

in isolated S(E)R fractions (ranging from 1 to 4) obtained from rat heart The intensities of

protein bands associated with either ZIP7 or ZnT7 showed both a gradually increasing

strength from rough to smooth fractions of isolated S(E)R preparation (Fig.2E) These data

strongly demonstrated the presence of these two-transporters in the smooth part of S(E)R For

further validation of their presence in S(E)R fractions, we assessed SERCA2a (110kDa) as

positive control and Golgi marker (Golgi-58K protein) and a nuclear marker cyclin E (53kDa)

as negative controls in the same S(E)R preparations (Fig.2E) The last two markers were

clearly observed in total cell lysate, as well

FRET-based Measurement of Cytosolic and S(E)R Free Zn 2+ Levels

Using FRET Zn2+-probes (13; 39), we measured free Zn2+ levels in cytosol and S(E)R in H9c2

cells after culture at normal (5.5-mM) or high (25-mM) glucose for 24-h Representative free

Zn2+ measurements (See Methods) are given in Fig.3 (A for cytosol and B for S(E)R) Using

quantification from fluorescence-intensity (10), Cyt-eCALWY4-infected and high-glucose

incubated H9c2 cells have significantly high cytosolic free Zn2+ level comparison to controls

(1.74±0.15 vs 0.97±0.14 nM) while S(E)R free Zn2+ level compared to the controls was

ranging 4.88±1.00 nM vs 2.52±0.36 nM

Of note, the total cellular free Zn2+ level in high glucose-treated cells was lower in comparison

to that of control cells There are several possible reasons for these differences First, ZnTs

located on sarcolemma might be activated by hyperglycemia Alternatively high cytosolic

Zn2+ might result Zn2+-increases in other organelles such as mitochondria through the kinetic activation of uptake-pathways (10)

Re-distribution of Cellular Free Zn 2+ in Hyperglycemic Cardiomyocytes Depends on ZIP7-Phosphorylation

Since the demonstration of ZIP7 and ZnT7 localizations to the S(E)R provides further evidence of S(E)R role as an intracellular Zn2+-pool in cardiomyocytes (3), with high mRNA and protein levels of ZIP7 and high ZIP7-phosphorylation under hyperglycemia (rather than ZnT7), we assessed its direct role in redistribution of cellular free Zn2+ in hyperglycemic cardiomyocytes We first silenced ZIP7 in H9c2 cells The mRNA levels of ZIP7 and ZnT7 in ZIP7-silenced cells comparison to sc-shRNA cells are given in Fig.4 (A and B; left).The ZIP7 mRNA level was reduced (>60%) in comparison to controls while ZnT7 mRNA level was not changed As expected, ZIP7 immunoreactivity was reduced by >90% in silenced-cells with no change in ZnT7 protein level (Fig.4A and B; right)

To test further the role of ZIP7 in subcellular Zn2+ distribution, we measured cytosolic and S(E)R free Zn2+ levels in non-targeted and ZIP7-targeted shRNA-infected cells using

previously mentioned protocols (See Methods) As shown in Fig.4C (left), cytosolic free Zn2+

was increased over 3-fold in hyperglycemic cells in comparison to the controls (0.92±0.14 nM

vs 3.28±0.65 nM; n cell=36-45) Furthermore, the cytosolic free Zn2+ in ZIP7-silenced cells under hyperglycemia was not different from that of ZIP7-silenced cells under physiological conditions (Fig.4C, right) However, the S(E)R free Zn2+ in non-targeted shRNA infected hyperglycemic cells had tended less (36%; Fig.4D, left) Furthermore, the free Zn2+ level in S(E)R in ZIP7-silenced cells under hyperglycemia was not different from that of ZIP7-silenced cells under physiological condition (Fig.4D, right) These results are consistent with

an important role for S(E)R-localized ZIP7 in re-distribution of subcellular Zn2+ in cardiomyocytes under hyperglycemia

Alterations in ZIP7 and ZnT7 Expressions Cause ER-stress via Loss of S(E)R Free Zn 2+

Under Hyperglycemia

Previously, marked ER-stress and increased intracellular free Zn2+ in isolated left ventricular

cardiomyocytes from diabetic rats have been shown (4; 29; 30) and also demonstrated an ER

Zn2+-deficiency associated disruption in its function and induction of ER-stress in eukaryotes

(14) We, therefore, hypothesized possible similar changes in cardiomyocytes that might lead

to ER-stress under hyperglycemia To test this prediction, we first measured ER-stress

chaperones levels GRP78 and calregulin (CALR) in high-glucose incubated cells (25-mM for

24-h) comparison to those of controls The GRP78 and CALR levels were increased

significantly in hyperglycemic cells, similar to those diabetic rat heart-tissue (Fig.5A) (32)

We also examined GRP78 induction in response to a direct ER-stress activator tunicamycin

(TUN; 10-µM; 18-h) and showed again increased GRP78 expression levels (Fig.5B)

To demonstrate a hypothesis related with hyperglycemia-associated changes in protein

expression levels of these transporters can underlie the induction of ER-stress in

cardiomyocytes, we used directly ER-stress induced cardiomyocytes with TUN-incubation

and then measured ZIP7 and ZnT7 expression levels which are not significantly different

from those of controls (Fig.5C and D)

Hyperglycemia-Associated ZIP7 Phosphorylation

Since we observed a markedly higher ZIP7-phosphorylation level in diabetic rat

cardiaomyocytes (Fig.1E) and high-glucose incubated cells (Fig.1F) versus their respective

controls, we aimed to clarify whether these changes arose as a direct consequence of

stress To address this prediction, we treated cells with 10-µM TUN (24-h) to induce

ER-stress directly and then measured phospho-ZIP7 levels There were no significant differences

between treated and untreated cells, implying no direct ER-stress effect on

ZIP7-phosphorylation (Fig.5E) For further support associated with ZIP7-induced ER-stress, we monitored expression levels of GRP78 and CALR in ZIP7-silenced cells under hyperglycemia whereas none of them were different from those of respective controls (Fig.5F and G) For further support to association between activation of ZIP7-related increased cytosolic Zn2+under hyperglycemia and ER-stress induction, we increased cytosolic free Zn2+ via Zn2+/Pyr (100-nM for 24-h) Then we measured markedly increased GRP78 and CALR levels while these levels could be reversed to control levels with ER-stress inhibitortauroursodeoxycholic acid (TUDCA; 50-µM for 24-h) (Suppl.Fig.5A and B) The above results thus provide further support for the view that hyperglycemia-associated changes in pZIP7 and ZIP7 levels underlie

an induction of ER-stress

CK2αα Activation in Diabetic-Heart Associated with Both Hyperglycemia and

Hyperglycemia-Induced Cytosolic Free Zn 2+ Increase

CK2α is an unusual protein kinase being constitutively active and undergoes autophosphorylation (33) Both CK2 activity and expression areincreased with low Zn2+ in a concentration-dependent manner(34) Since CK2α-activation has been shown previously to

prompt cytosolic Zn2+ signaling (18) as well as high glucose (0–25 mM) treatment (for 4-h) of cells induced significant CK2α activity in a concentration-dependent manner (35), we first examined CK2α expression in isolated cardiomyocytes from diabetic rat heart and then in

Zn2+/Pyr (1-µM; 20-min)-incubated rat cardiomyocytes comparison to those of controls Both CK2α expressions were significantly higher comparison to their controls (Fig.5F and G) Furthermore, we repeated the similar measurements in H9c2 cells maintained at high-glucose (25-mM for 24-h) or Zn2+/Pyr (1-µM for 20-min) High-glucose incubation for 24-h induced a significant increase in CK2α-expression (2-fold) while there was 8-fold increase in CK2α-expression in 1-µM Zn2+/Pyr incubated cells for 20-min (Fig.6E) These data demonstrate that

CK2α expression increases in response to hyperglycemia or elevated cytosolic free Zn2+ and

might contribute in a feed-forward cycle to further Zn2+ release into cytoplasm Most

importantly, with given data in Fig.1F, it should be emphasized the importance of a

time-dependency of hyperglycemia associated elevated cytosolic free Zn2+ effect on

pZIP7-expression

CK2αα Contributes to Activation of ZIP7 via ZIP7-phosphorylation in Hyperglycemia

Since an association between CK2α-activation/expression, ZIP7-phosphorylation and Zn2+

influx into the cytoplasm from the ER was previously reported in TamR cells (18), we

performed co-immunoprecipitation measurements in H9c2 cells (Fig.6C) These data reveal

an association between CK2α-activation (at most, due to hyperglycemia) and increased

cytosolic free Zn2+-dependent phosphorylation and activation of ZIP7 in response to

hyperglycemia

To test whether ZIP7 may be phosphorylated by other kinases, which are also activated by

increased intracellular Zn2+ in cardiomyocytes (4; 30; 36), we performed further

co-immunoprecipitation experiments using different antibodies against them We couldn’t

observe any co-immunoprecipitation between ZIP7 and any of other kinases

To validate the essentiality of CK2α activation for ZIP7-phosphorylation, we silenced CK2α

in normal- or high glucose-treated (25-mM for 24-h) H9c2 cells and then measured pZIP7 and

ZIP7 expression levels CK2α protein level is markedly reduced in CK2α-silenced cells

Fig.6D) while the mRNA levels of CK2α1 and CK2α2 are decreased (Suppl.Fig.6)

Additionally, there is no ZIP7-phosphorylation in CK2α-silenced cells under control or

hyperglycemia while 7-fold increased pZIP7 in nontarget-siRNA transfected hyperglycemic

cells is observed (Fig.6E and F) The ratio of pZIP7/ZIP7 is 5-fold higher in hyperglycemia

compared to normo-glycemia in nontargeting-siRNA cells However, silencing of CK2α has

not changed this ratio under control and hyperglycemic conditions (Suppl.Fig.7), suggesting the importance of increased ZIP7-phosphorylation in response to hyperglycemia

Discussion

Our overall aim was to examine roles of Zn2+-carriers ZIP7 and ZnT7 in cardiomyocytes under physiological condition and as potential mediators of ER-stress in hyperglycemia and hyperglycemia-induced cardiac dysfunction Our results suggest that these Zn2+-carriers are likely to carry Zn2+ in opposite directions across S(E)R membrane Furthermore, we showed that increased ZIP7 level together with increased ZIP7-phosphorylation were likely to drive changes in S(E)R lumen and cytosol with deleterious consequences for cell function in both cases Although not assessed directly, lowered ZnT7 expression under hyperglycemia, particularly under chronic condition, is likely to further exacerbate these changes and contribute to deleterious consequences of Zn2+-redistribution between compartments Importantly, these transporters were shown to localize into S(E)R membrane and may thus operate as a functional proteins catalyzing Zn2+-release and uptake, respectively from S(E)R

To best of our knowledge, the current study is the first to be conducted to assess both localization and functional roles of ZIP7 and ZnT7 as working opposing-direction in mammalian heart, and most importantly their contributions to diabetic cardiac dysfunction via ER-stress Additionally, we demonstrated markedly increased CK2α-expression under hyperglycemia (at most via by hyperglycemia-activation of CK2α; (35) and provided evidences that this can lead to phosphorylation of the channel on residues previously implicated in control of Zn2+-release (18) Particularly, our experiments using CK2α-silenced cells provide further support for this view Thus, a combination of transcriptional and post-transcriptional mechanisms may contribute to ZIP7-activation in hyperglycemia Indeed, increases in both expression and phosphorylation of ZIP7 under hyperglycemia are time dependent and ZIP7-phosphorylation increase is significantly higher comparison to ZIP7

increase in chronic-hyperglycemia, indicating a role of ZIP7-activation in cardiomyocytes

under diabetes

Our data provide further evidence of a role for S(E)R as an intracellular Zn2+-pool,

contributing to Zn2+-regulation in cardiomyocytes, in a process controlled by ZIP7 and ZnT7

In this regard, it has been shown how S(E)R Zn2+ deficiency induces

unfolded-protein-response (14) and Zn2+-homeostasis is involved, via a putative Zn2+-transporter, localized to

S(E)R (37) Moreover, it has been demonstrated that high-glucose induces a stable increase in

cytosolic Zn2+ in pancreatic β-cells and leads to profound alterations in expression of genes

important for Zn2+-homeostasis such as ZIP7 increase (25) Therefore, our data are

noteworthy when considering ZIP7 and ZnT7 roles located into S(E)R and responsible for

ER-stress, in part, due to depressed S(E)R free Zn2+

Cytosolic Zn2+ increase in cardiomyocytes can induce marked high-phosphorylation in many

kinases and oxidation of proteins responsible for heart function (3; 4; 30; 38) This statement

is consistent with previous studies on role of ZIP7 in control of Zn2+ release from S(E)R in

cancer cells (17; 18) Supporting these earlier findings, we showed here that CK2α is

activated in hyperglycemic cardiomyocytes and activated also directly with increased

cytosolic Zn2+ More importantly, these observations also support our present hypothesis

Indeed, CK2α plays a key role in the regulation of pro-survival as well as the pro-apoptotic

ER-stress signalling by directly modulating the activities of ER-stress signalling actors by

phosphorylation, regulating expression of the key factors of signalling-pathways or binding to

regulator-proteins (39)

Little is known about cytosolic Zn2+, Zn2+-transporters and cardiovascular complications

Early studies demonstrated that ZIP7 is involved in Zn2+-homeostasis of Golgi apparatus (15)

Although it has been shown weakly-expressed ZIP7 and ZnT7 in mammalian heart (22; 23),

here, we observed marked ZIP7 and ZnT7 protein levels in diabetic rat heart whereas others

demonstrating a correlation between increased-ROS, ER-stress and CK2α-activation (40; 41) via cellular control of Zn2+-distribution with CK2α-mediated ZIP7-phosphorylation (18) Therefore, our data represent the first investigation in the role of CK2α-mediated ZIP7-phosphorylation and Zn2+-homeostasis in cardiomyocytes under hyperglycemia and further proposed on protective role for ZnT7 against oxidative-stress or high susceptibility to diet-induced glucose intolerance/insulin resistance (24; 42)

Hyperglycemia induces cardiac dysfunction due to increased oxidative-stress and ER-stress (43; 44) Our data reinforce prediction of a S(E)R role as an important Zn2+-pool in cardiomyocytes with ZIP7 and ZnT7 localization into S(E)R and making an important contribution to Zn2+-homeostasis Nonetheless, we recognize also possible contribution of other Zn2+-transporters contributing to ER-stress in cardiomyocytes under hyperglycemia Our data, therefore, provide important information related to Zn2+-homeostasis via ZIP7 and ZnT7 functioning as opposing Zn2+-transporters and localized to S(E)R

in cardiomyocytes Perturbed expression of either or both transporters may lead to altered

Zn2+-distribution across S(E)R and leading to persistent ER-stress

Zn2+-homeostasis in cardiomyocytes under physiological and hyperglycemic conditions is summarized (Fig.7) Regulation of sub-cellular free Zn2+-distribution is proposed to occur via ZIP7/ZnT7 system localized to S(E)R When cardiomyocytes are exposed to high-glucose, CK2α becomes activated inducing Zn2+-influx into cytosol from S(E)R via ZIP7-phosphorylation (18) while a down-regulated ZnT7 may further contribute to this process High-glucose stimulates also cytosolic free Zn2+ increase via mobilization of Zn2+ from metalloproteins (5; 30; 45; 46) which in turn induces phosphorylation of several proteins (30; 46; 47) Of note, the latter might further stimulate ZIP7-activity and may lead to accelerated

Zn2+-influx into cytosol from S(E)R Consequently, cytosolic free Zn2+ increases driven by multiple mechanisms lead to ER-stress via and the overexpression of ER-stress chaperones in

cardiomyocytes during hyperglycemia Our data provide here novel insights into regulation of

cellular-Zn2+ and its role in hyperglycemia/diabetes-associated cardiac dysfunction

Additionally, our findings may provide new targets such as cellular Zn2+-regulation via

mediation of Zn2+-transporters and suggest that modulation of CK2α may provide a novel

means to correct diabetes-induced cardiac dysfunction

Acknowledgements

The authors would like to thank Mr Yusuf Olgar for his contribution to some of Western

blotting analysis We thank Dr Angeles Mondragon (Imperial College London) for assistance

with plasmid amplification and virus production and Mr Steve Rothery (Imperial College

London) for assistance with imaging experiments

Funding

B.T thanks to The Scientific and Technological Research Council of Turkey (TUBITAK) for

grant SBAG-113S466 and COST action TD1304 G.A.R thanks the Medical Research

Council (UK) for Programme grant MR/J0003042/1, the Biotechnology and Biological

Sciences Research Council (UK) for a Project grant (BB/J015873/1), the Royal Society for a

Wolfson Research Merit Award and the Wellcome Trust for a Senior Investigator Award

(WT098424AIA) E.T thanks to COST action TD1304 short scientific mission grant (STSM)

and European Foundation for the Study of Diabetes (EFSD) Albert Renold Travel Fellowship

Duality of Interest

No potential conflicts relevant to this article were reported

Authors Contributions

B.T designed the study, wrote the manuscript, researched data, and provided funding for the

study G.A.R supported the experimental data in his lab and reviewed and edited the

manuscript E.T performed the electrophysiological and imaging experiments and contributed

all biochemical and molecular biology experiments and analyzed the all experimental data and confocal images V.B and A.D performed all biochemical and molecular experiments G.R.J.C contributed to knock down experiments K.T provided the antibody for ZIP7 and contributed to the discussion B.T is the guarantor of this work and, as such, had full access

to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis

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