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Superoxide production during ischemia-reperfusion in the perfused rat heart: a comparison of two methods of measurement.
J Mol Cell Cardiol 2012; 53(6):906-15JM

Abstract

Reactive oxygen species (ROS) have been implicated in many aspects of tissue/cellular metabolic signaling and pathology, including cardioprotection against ischemia-reperfusion damage. Recent reports of enhanced ROS production under global or simulated ischemia in intact heart or isolated cardiomyocytes, respectively, and its decrease again upon reperfusion are paradoxical. Mechanisms for increasing ROS production with decreasing reactant (oxygen) concentration remain elusive, making it important to critically evaluate the experimental methods used to measure ROS production. In the present paper superoxide production in isolated perfused rat hearts was monitored by lucigenin chemiluminescence or dihydroethidine (DHE) oxidation product fluorescence in parallel with redox state of flavin and cytochrome oxidase. Lucigenin luminescence decreased in ischemia and increased again upon reperfusion, transiently reaching values eightfold the control value coincidently with an overshoot of mitochondrial oxygen concentration. Hypoxic perfusion decreased lucigenin chemiluminescence in spite of coronary flow increase, whereas change in lucigenin concentration in the perfusate had negligible effect. In contrast to lucigenin luminescence, the fluorescence of the DHE oxidation product increased continuously during a 30-min global ischemia and decreased precipitously upon reperfusion, this change is coincident with absorption changes of the oxygen-binding protein myoglobin. The time course of DHE oxidation product fluorescence during ischemia and reperfusion was similar to that of the mitochondrial membrane potential probe safranin as shown in perfused heart previously [Ylitalo KV, Ala-Rämi A, Liimatta EV, Peuhkurinen KJ, Hassinen IE. J Mol Cell Cardiol 2000;32:1223-38]. In solution under high oxygen partial pressure DHE was mainly oxidized to a product, whose fluorescence, absorbance and mass spectra were similar to ethidium, and this product behaved like a mitochondrial membrane potential probe in isolated mitochondria. As a membrane permeable cation it accumulates into the mitochondria when the membrane potential is high (high intramitochondrial concentration quenches fluorescence) and then is released (increased fluorescence) during hypoxia/ischemia. Upon reperfusion it is re-accumulated in the mitochondria as the membrane potential recovers. The non-specific oxidation of DHE makes this dye less suitable for superoxide detection in experiments on isolated perfused hearts that necessitate high oxygen partial pressure in the perfusate. The time course of lucigenin luminescence during ischemia/reperfusion is consistent with decreased ROS production during ischemia/hypoxia, while the oxygen concentration is decreased, followed by an overshoot when the heart tissue is reperfused and the oxygen pressures return to normal or above normal.

Authors+Show Affiliations

Institute of Diagnostics, Departments of Pathology, University of Oulu, and Oulu University Hospital, FIN-90014 Oulu Finland.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23036824

Citation

Näpänkangas, Juha P., et al. "Superoxide Production During Ischemia-reperfusion in the Perfused Rat Heart: a Comparison of Two Methods of Measurement." Journal of Molecular and Cellular Cardiology, vol. 53, no. 6, 2012, pp. 906-15.
Näpänkangas JP, Liimatta EV, Joensuu P, et al. Superoxide production during ischemia-reperfusion in the perfused rat heart: a comparison of two methods of measurement. J Mol Cell Cardiol. 2012;53(6):906-15.
Näpänkangas, J. P., Liimatta, E. V., Joensuu, P., Bergmann, U., Ylitalo, K., & Hassinen, I. E. (2012). Superoxide production during ischemia-reperfusion in the perfused rat heart: a comparison of two methods of measurement. Journal of Molecular and Cellular Cardiology, 53(6), pp. 906-15. doi:10.1016/j.yjmcc.2012.09.011.
Näpänkangas JP, et al. Superoxide Production During Ischemia-reperfusion in the Perfused Rat Heart: a Comparison of Two Methods of Measurement. J Mol Cell Cardiol. 2012;53(6):906-15. PubMed PMID: 23036824.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Superoxide production during ischemia-reperfusion in the perfused rat heart: a comparison of two methods of measurement. AU - Näpänkangas,Juha P, AU - Liimatta,Erkki V, AU - Joensuu,Päivi, AU - Bergmann,Ulrich, AU - Ylitalo,Kari, AU - Hassinen,Ilmo E, Y1 - 2012/10/02/ PY - 2012/08/16/received PY - 2012/09/24/revised PY - 2012/09/24/accepted PY - 2012/10/6/entrez PY - 2012/10/6/pubmed PY - 2013/4/23/medline SP - 906 EP - 15 JF - Journal of molecular and cellular cardiology JO - J. Mol. Cell. Cardiol. VL - 53 IS - 6 N2 - Reactive oxygen species (ROS) have been implicated in many aspects of tissue/cellular metabolic signaling and pathology, including cardioprotection against ischemia-reperfusion damage. Recent reports of enhanced ROS production under global or simulated ischemia in intact heart or isolated cardiomyocytes, respectively, and its decrease again upon reperfusion are paradoxical. Mechanisms for increasing ROS production with decreasing reactant (oxygen) concentration remain elusive, making it important to critically evaluate the experimental methods used to measure ROS production. In the present paper superoxide production in isolated perfused rat hearts was monitored by lucigenin chemiluminescence or dihydroethidine (DHE) oxidation product fluorescence in parallel with redox state of flavin and cytochrome oxidase. Lucigenin luminescence decreased in ischemia and increased again upon reperfusion, transiently reaching values eightfold the control value coincidently with an overshoot of mitochondrial oxygen concentration. Hypoxic perfusion decreased lucigenin chemiluminescence in spite of coronary flow increase, whereas change in lucigenin concentration in the perfusate had negligible effect. In contrast to lucigenin luminescence, the fluorescence of the DHE oxidation product increased continuously during a 30-min global ischemia and decreased precipitously upon reperfusion, this change is coincident with absorption changes of the oxygen-binding protein myoglobin. The time course of DHE oxidation product fluorescence during ischemia and reperfusion was similar to that of the mitochondrial membrane potential probe safranin as shown in perfused heart previously [Ylitalo KV, Ala-Rämi A, Liimatta EV, Peuhkurinen KJ, Hassinen IE. J Mol Cell Cardiol 2000;32:1223-38]. In solution under high oxygen partial pressure DHE was mainly oxidized to a product, whose fluorescence, absorbance and mass spectra were similar to ethidium, and this product behaved like a mitochondrial membrane potential probe in isolated mitochondria. As a membrane permeable cation it accumulates into the mitochondria when the membrane potential is high (high intramitochondrial concentration quenches fluorescence) and then is released (increased fluorescence) during hypoxia/ischemia. Upon reperfusion it is re-accumulated in the mitochondria as the membrane potential recovers. The non-specific oxidation of DHE makes this dye less suitable for superoxide detection in experiments on isolated perfused hearts that necessitate high oxygen partial pressure in the perfusate. The time course of lucigenin luminescence during ischemia/reperfusion is consistent with decreased ROS production during ischemia/hypoxia, while the oxygen concentration is decreased, followed by an overshoot when the heart tissue is reperfused and the oxygen pressures return to normal or above normal. SN - 1095-8584 UR - https://www.unboundmedicine.com/medline/citation/23036824/Superoxide_production_during_ischemia_reperfusion_in_the_perfused_rat_heart:_a_comparison_of_two_methods_of_measurement_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2828(12)00360-4 DB - PRIME DP - Unbound Medicine ER -