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ZnT-1 protects HL-1 cells from simulated ischemia–reperfusion through activation of Ras–ERK signaling

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Abstract

Activation of ERK signaling may promote cardioprotection from ischemia–reperfusion (I/R) injury. ZnT-1, a protein that confers resistance from zinc toxicity, was found to interact with Raf-1 kinase through its C-terminal domain, leading to downstream activation of ERK. In the present study, we evaluated the effects of ZnT-1 in cultured murine cardiomyocytes (HL-1 cells) that were exposed to simulated-I/R. Cellular injury was evaluated by lactate dehydrogenase (LDH) release and by staining for pro-apoptotic caspase activation. Overexpression of ZnT-1 markedly reduced LDH release and caspase activation following I/R. Knockdown of endogenous ZnT-1 augmented the I/R-induced release of LDH and increased caspase activation following I/R. Phospho-ERK levels were significantly increased following I/R in cells overexpressing ZnT-1, while knockdown of ZnT-1 reduced phospho-ERK levels. Pretreatment of cells with the MEK inhibitor PD98059 abolished the protective effect of ZnT-1 following I/R. Accordingly, a truncated form of ZnT-1 lacking the C-terminal domain failed to induce ERK activation and did not protect the cells from I/R injury. In contrast, expression of the C-terminal domain by itself was sufficient to induce ERK activation and I/R protection. Interestingly, the C-terminal of the ZnT-1 did not have protective effect against the toxicity of zinc. In the isolated rat heart, global ischemic injury rapidly increased the endogenous levels of ZnT-1. However, following reperfusion ZnT-1 levels were found to be decreased. Our findings indicate that ZnT-1 may have important role in the ischemic myocardium through its ability to interact with Raf-1 kinase.

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Acknowledgments

This work was supported by Israel Science Foundation (ISF) grant 992/07 (AM and YE) and the Deutsche Forschungsgemeinschaf (DFG) grant MO 1932/1-1 (AM).

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Authors and Affiliations

  1. Department of Physiology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel

    Ofer Beharier, Shani Dror, Shiri Levy, Joy Kahn, Merav Mor, Daniel Gitler & Arie Moran

  2. Center for Cardiovascular Research, John Milliken Department of Medicine, Washington University School of Medicine, St. Louis, MO, 63110, USA

    Anthony J. Muslin

  3. Department of Cardiology, Barzilai Medical Center, Ashkelon, Israel

    Amos Katz

  4. Cardiac Arrhythmia Research Laboratory, Soroka University Medical Center, Beer-Sheva, Israel

    Ofer Beharier, Shani Dror, Merav Mor, Sharon Etzion & Amos Katz

  5. Cardiac Arrhythmia Research Laboratory, Faculty of Health sciences, Ben-Gurion University of the Negev & Soroka University Medical Center, P.O. Box 151, Beer-Sheva, 84101, Israel

    Yoram Etzion

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  1. Ofer Beharier
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  2. Shani Dror
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Correspondence to Yoram Etzion.

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Authors Ofer beharier and Shani Dror contributed equally to this work. Authors Arie Moran and Yoram Etzion contributed equally to this work.

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Beharier, O., Dror, S., Levy, S. et al. ZnT-1 protects HL-1 cells from simulated ischemia–reperfusion through activation of Ras–ERK signaling. J Mol Med 90, 127–138 (2012). https://doi.org/10.1007/s00109-011-0845-0

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