title: Spectroscopic analysis of myoglobin and cytochrome c dynamics in isolated cardiomyocytes during hypoxia and reoxygenation.
abstract: Raman microspectroscopy was applied to monitor the intracellular redox state of myoglobin and cytochrome c from isolated adult rat cardiomyocytes during hypoxia and reoxygenation. The nitrite reductase activity of myoglobin leads to the production of nitric oxide in cells under hypoxic conditions, which is linked to the inhibition of mitochondrial respiration. In this work, the subsequent reoxygenation of cells after hypoxia is shown to lead to increased levels of oxygen-bound myoglobin relative to the initial levels observed under normoxic conditions. Increased levels of reduced cytochrome c in ex vivo cells are also observed during hypoxia and reoxygenation by Raman microspectroscopy. The cellular response to reoxygenation differed dramatically depending on the method used in the preceding step to create hypoxic conditions in the cell suspension, where a chemical agent, sodium dithionite, leads to reduction of cytochromes in addition to removal of dissolved oxygen, and bubbling-N2 gas leads to displacement of dissolved oxygen only. These results have an impact on the assessment of experimental simulations of hypoxia in cells. The spectroscopic technique employed in this work will be used in the future as an analytical method to monitor the effects of varying levels of oxygen and nutrients supplied to cardiomyocytes during either the preconditioning of cells or the reperfusion of ischaemic tissue.
authors.affiliation: Department of Physics, University of Leicester, Leicester LE1 7RH, UK.
authors.forename: A
authors.initials: A
authors.lastname: Almohammedi
authors.affiliation: Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK.
authors.forename: S M
authors.initials: SM
authors.lastname: Kapetanaki
authors.affiliation: Centre for Biospectroscopy and School of Chemistry, Monash University, Wellington Road, Clayton, Victoria 3800, Australia.
authors.forename: B R
authors.initials: BR
authors.lastname: Wood
authors.affiliation: Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK.
authors.forename: E L
authors.initials: EL
authors.lastname: Raven
authors.affiliation: Department of Cell Physiology and Pharmacology, University of Leicester, Leicester LE1 9HN, UK ns140@leicester.ac.uk.
authors.forename: N M
authors.initials: NM
authors.lastname: Storey
authors.affiliation: Department of Chemistry, University of Leicester, Leicester LE1 7RH, UK andrew.hudson@leicester.ac.uk.
authors.forename: A J
authors.initials: AJ
authors.lastname: Hudson
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology*; D045304:Cytochromes c / Q000378:metabolism*; D008297:Male; D032383:Myocytes, Cardiac / Q000378:metabolism*; D009211:Myoglobin / Q000378:metabolism*; D009572:Nitrite Reductases / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000008:administration & dosage / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013059:Spectrum Analysis, Raman
chemical_list: D009211:Myoglobin; D045304:Cytochromes c; D009572:Nitrite Reductases; D010100:Oxygen
keywords: Raman spectroscopy; biophotonics; cardiomyocytes; ex vivo biospectroscopy; hypoxia and reoxygenation