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Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion

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Abstract

The role of zinc ion in cytotoxicity following ischemic stroke, prolonged status epilepticus, and traumatic brain injury remains controversial, but likely is the result of mitochondrial dysfunction. We describe an excitation ratiometric fluorescence biosensor based on human carbonic anhydrase II variants expressed in the mitochondrial matrix, permitting free zinc levels to be quantitatively imaged therein. We observed an average mitochondrial matrix free zinc concentration of 0.2 pM in the PC12 rat pheochromacytoma cell culture line. Cytoplasmic and mitochondrial free zinc levels were imaged in a cellular oxygen glucose deprivation (OGD) model of ischemia/reperfusion. We observed a significant increase in mitochondrial zinc 1 h following 3 h OGD, at a time point when cytosolic zinc levels were depressed. Following the increase, mitochondrial zinc levels returned to physiological levels, while cytosolic zinc increased gradually over a 24 h time period in viable cells. The increase in intramitochondrial zinc observed during reoxygenation after OGD may contribute to bioenergetic dysfunction and cell death that occurs with both in vitro and in vivo models of reperfusion.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD, 21201, USA

    Bryan J. McCranor, Rebecca A. Bozym, Michele I. Vitolo & Richard B. Thompson

  2. University of Maryland Marlene and Stewart Greenebaum NIH Cancer Center, University of Maryland, 22 S. Greene St., Baltimore, MD, 21201, USA

    Michele I. Vitolo

  3. Department of Chemistry, University of Michigan, 930 N. University Ave., Ann Arbor, MI, 48109, USA

    Carol A. Fierke

  4. Department of Anesthesiology and the Center for Shock, Trauma, and Anesthesiology Research, University of Maryland School of Medicine, 685 W. Baltimore St., Baltimore, MD, 21201, USA

    Linda Bambrick, Brian M. Polster & Gary Fiskum

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  1. Bryan J. McCranor
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  2. Rebecca A. Bozym
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  3. Michele I. Vitolo
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  8. Richard B. Thompson
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Correspondence to Richard B. Thompson.

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McCranor, B.J., Bozym, R.A., Vitolo, M.I. et al. Quantitative imaging of mitochondrial and cytosolic free zinc levels in an in vitro model of ischemia/reperfusion. J Bioenerg Biomembr 44, 253–263 (2012). https://doi.org/10.1007/s10863-012-9427-2

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  • DOI: https://doi.org/10.1007/s10863-012-9427-2

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