Downregulation of the zinc transporter SLC39A13 (ZIP13) is responsible for the activation of CaMKII at reperfusion and leads to myocardial ischemia/reperfusion injury in mouse hearts

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Highlights

  • ZIP13 is downregulated at reperfusion in mouse hearts.

  • Downregulation of ZIP13 leads to CaMKII activation.

  • The heart-specific knockout of ZIP13 exacerbated myocardial infarction via CaMKII in mouse hearts subjected to I/R.

  • Knockout of ZIP13 induced mitochondrial damage in a CaMKII-dependent manner.

Abstract

While Zn2+ dyshomeostasis is known to contribute to ischemia/reperfusion (I/R) injury, the roles of zinc transporters that are responsible for Zn2+ homeostasis in the pathogenesis of I/R injury remain to be addressed. This study reports that ZIP13 (SLC39A13), a zinc transporter, plays a role in myocardial I/R injury by modulating the Ca2+ signaling pathway rather than by regulating Zn2+ transport. ZIP13 is downregulated upon reperfusion in mouse hearts or in H9c2 cells at reoxygenation. Ca2+ but not Zn2+ was responsible for ZIP13 downregulation, implying that ZIP13 may play a role in I/R injury through the Ca2+ signaling pathway. In line with our assumption, knockout of ZIP13 resulted in phosphorylation (Thr287) of Ca2+-calmodulin-dependent protein kinase (CaMKII), indicating that downregulation of ZIP13 leads to CaMKII activation. Further studies showed that the heart-specific knockout of ZIP13 enhanced I/R-induced CaMKII phosphorylation in mouse hearts. In contrast, overexpression of ZIP13 suppressed I/R-induced CaMKII phosphorylation. Moreover, the heart-specific knockout of ZIP13 exacerbated myocardial infarction in mouse hearts subjected to I/R, whereas overexpression of ZIP13 reduced infarct size. In addition, knockout of ZIP13 induced increases of mitochondrial Ca2+, ROS, mitochondrial swelling, decrease in the mitochondrial respiration control rate (RCR), and dissipation of mitochondrial membrane potentialΨm) in a CaMKII-dependent manner. These data suggest that downregulation of ZIP13 at reperfusion contributes to myocardial I/R injury through activation of CaMKII and the mitochondrial death pathway.

Introduction

As a trace element, zinc is essential for the maintenance of normal cellular structure and functions [1]. Studies have demonstrated that zinc dyshomeostasis contributes to myocardial ischemia/reperfusion (I/R) injury in in vitro and in vivo experimental models [2,3]. Intracellular zinc concentration is tightly controlled by zinc transporters and zinc binding molecules. Zinc transporters are encoded by two gene families: SLC39A (ZIP) and SLC30A (ZnT) [4]. The ZIP family zinc transporters increase cytosolic zinc by transporting zinc from the extracellular fluid or from intracellular vesicles into the cytosol, whereas the ZnT family zinc transporters reduce cytosolic zinc by promoting zinc efflux from cells or influx into intracellular compartments. Recently we have reported that SLC39A2 (ZIP2), an important zinc transporter, is cardioprotective against I/R injury in mouse hearts, warranting further investigations on the potential involvements of other SLC39A family zinc transporters in myocardial I/R injury [5].

ZIP13 is expressed in hard and connective tissues and, is mainly localized in Golgi apparatus and other intracellular compartments [6]. Mutation of ZIP13 causes Spondylocheirodysplastic Ehlers–Danlos syndrome (SCD-EDS) [7,8], suggesting that ZIP13 is crucial for the development of hard and connective tissues. However, it has also been reported that ZIP13 is expressed high in the heart [9] and, a latest study demonstrated that knockdown of ZIP13 or ZIP14 significantly inhibited human valve interstitial cell (hVIC) in vitro calcification and osteogenic differentiation [10], implying that ZIP13 may play a role in cardiac physiology or diseases.

The multifunctional protein kinase CaMKII not only regulates the excitation-contraction coupling but also plays an important role in cardiac diseases such as myocardial I/R injury, hypertrophy, and heart failure. CaMKII activity is triggered by an increase in intracellular Ca2+ levels and this activity is sustained by autophosphorylation of the enzyme [11]. In a recent study, Zhang et al. demonstrated that RIP3 induces myocardial necroptosis by phosphorylating CaMKII at Thr287, suggesting that RIP3 is an important upstream kinase of CaMKII [12]. This finding further indicates that CaMKII is phosphorylated not only by autophosphorylation but also by RIP3. In our preliminary study with RNA sequencing analysis, we found that knockout of ZIP13 may alter the Ca2+ signaling events. Nevertheless, little is known about the regulatory effect of ZIP13 on CaMKII activity.

The purpose of this study was to test if ZIP13 plays a role in myocardial I/R injury by modulating CaMKII activity. First, we examined if ZIP13 expression is altered upon reperfusion in in vivo mouse hearts. Then we tested if changes in ZIP13 expression alter CaMKII activity. Third, we determined if ZIP13 downregulation accounts for myocardial I/R injury via CaMKII in mice by genetically altering ZIP13 expression. Lastly, we investigated if ZIP13 downregulation contributes to I/R injury by activating the mitochondrial death pathway via CaMKII.

Section snippets

Chemicals

ZnCl2, CaCl2 and N,N,N′,N′-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) were purchased from Sigma (St.Louis, MO, USA). BAPTA-AM was purchased from MCE (NJ, USA). KN-93 was purchased from Selleck (Houston, TX, USA). Antibodies including anti-TUBULIN, -GAPDH, -CaMKII and the secondary antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-phospho-CaMKII, was purchased from Abcam (Cambridge, UK). Anti-Zip13 was purchased from Biorbyt (Cambridge, UK). Fluorescence dyes

ZIP13 is downregulated at reperfusion or reoxygenation

To test if ZIP13 expression is altered by I/R, we measured its protein expression at reperfusion in in vivo mouse hearts. Ischemia was confirmed by an elevation of the ST segment on EKG (Fig. S1). As shown in Fig. 1A, ZIP13 protein expression was markedly decreased 30 min after the onset of reperfusion. In support, ZIP13 expression was also downregulated in H9c2 cells subjected to 4 h of hypoxia followed by 2 h of reoxygenation (Fig. 1B).

Ca2+ but not Zn2+ accounts for downregulation of ZIP13

Since cardiac Zn2+ is decreased at reperfusion (Figs. 2D

Discussion

This study demonstrates that downregulation of ZIP13 at reperfusion contributes to myocardial I/R injury through activation of CaMKII and the mitochondrial death pathway.

Cellular Zn2+ homeostasis is maintained mainly by the two families of Zn2+ transporters: ZnT (SLC30) and ZIP (SLC39) [4,13]. ZnT transporters reduce intracellular free Zn2+ by promoting Zn2+ efflux from cells or into intracellular vesicles, whereas ZIP transporters increase intracellular free Zn2+ by promoting extracellular Zn2+

Funding

This work was supported by the National Natural Science Foundation of China (NSFC) (grant numbers 81470397, 81970255, and 81802927) and the Key Program of Tianjin Natural Science Foundation (grant number 16JCZDJC35000).

Disclosures

None.

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    These authors contributed equally to the work.

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