Related Experiment Video
Updated: Jul 6, 2026
Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart
Published on: May 19, 2017
Glycolytic inhibition and calcium overload as consequences of exogenously generated free radicals in rabbit hearts
M C Corretti1, Y Koretsune, H Kusuoka
1Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Abstract:
Free radicals have been implicated in the pathogenesis of reperfusion injury, but it is unclear how they exert their deleterious effects on cellular metabolism. Several lines of indirect evidence suggest that free radicals elevate intracellular Ca2+ concentration ([Ca2+]i) and inhibit glycolysis as part of their mechanism of injury. We tested these ideas directly in hearts subjected to hydroxyl radicals produced by the Fenton and Haber-Weiss reactions. Nuclear magnetic resonance spectra were obtained from Langendorff-perfused rabbit hearts before, during, and after 4 min of perfusion with H2O2 (0.75 mM) and Fe(3+)-chelate (0.1 mM). Isovolumic left ventricular pressure exhibited progressive functional deterioration and contracture after exposure to H2O2 + Fe3+. Phosphorus nuclear magnetic resonance (NMR) spectra revealed partial ATP depletion and sugar phosphate accumulation indicative of glycolytic inhibition. To measure [Ca2+]i, fluorine NMR spectra were acquired in a separate group of hearts loaded with the Ca2+ indicator 5F-BAPTA [5,5'-difluoro derivative of 1,2-bis-(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid]. Mean time-averaged [Ca2+]i increased from 347 +/- 14 nM in control to 1,026 +/- 295 nM 4 min after free radical generation (means +/- SEM, n = 7), and remained elevated thereafter. We conclude that free radicals induce clear-cut, specific derangements of cellular metabolism in the form of glycolytic inhibition and calcium overload. The observed increase in [Ca2+]i suggests that the deleterious effects of free radicals are at least partially mediated by secondary changes in cellular calcium homeostasis.
Insights
Free radicals cause cell damage by increasing intracellular calcium and inhibiting glycolysis, leading to reperfusion injury. This study directly demonstrates these metabolic disruptions in heart cells.
Area of Science:
- Biochemistry
- Cellular Metabolism
- Cardiovascular Physiology
Background:
- Free radicals are suspected in reperfusion injury pathogenesis.
- Indirect evidence suggests free radicals increase intracellular calcium ([Ca2+]i) and inhibit glycolysis.
Purpose of the Study:
- To directly investigate the effects of hydroxyl radicals on cellular metabolism in heart cells.
- To determine if free radicals elevate intracellular calcium and inhibit glycolysis.
Main Methods:
- Utilized Langendorff-perfused rabbit hearts exposed to hydroxyl radicals generated by Fenton and Haber-Weiss reactions.
- Employed phosphorus Nuclear Magnetic Resonance (NMR) spectroscopy to assess metabolic changes.
- Used fluorine NMR spectroscopy with a calcium indicator (5F-BAPTA) to measure intracellular calcium levels.
Main Results:
- Exposure to H2O2 + Fe3+ caused functional deterioration and contracture.
- Phosphorus NMR showed ATP depletion and sugar phosphate accumulation, indicating glycolytic inhibition.
- Fluorine NMR revealed a significant increase in mean time-averaged intracellular calcium ([Ca2+]i) from 347 nM to 1,026 nM.
Conclusions:
- Free radicals induce specific metabolic derangements: glycolytic inhibition and calcium overload.
- The observed increase in intracellular calcium suggests a key role in free radical-induced cellular damage.
- These findings highlight the impact of free radicals on cellular calcium homeostasis during reperfusion injury.
