Differential contribution of mitochondria, NADPH oxidases, and glycolysis to region-specific oxidant stress in the anoxic-reoxygenated embryonic heart

Abstract

The ability of the developing myocardium to tolerate oxidative stress during early gestation is an important issue with regard to possible detrimental consequences for the fetus. In the embryonic heart, antioxidant defences are low, whereas glycolytic flux is high. The pro- and antioxidant mechanisms and their dependency on glucose metabolism remain to be explored. Isolated hearts of 4-day-old chick embryos were exposed to normoxia (30 min), anoxia (30 min), and hyperoxic reoxygenation (60 min). The time course of ROS production in the whole heart and in the atria, ventricle, and outflow tract was established using lucigenin-enhanced chemiluminescence. Cardiac rhythm, conduction, and arrhythmias were determined. The activity of superoxide dismutase, catalase, gutathione reductase, and glutathione peroxidase as well as the content of reduced and oxidized glutathione were measured. The relative contribution of the ROS-generating systems was assessed by inhibition of mitochondrial complexes I and III (rotenone and myxothiazol), NADPH oxidases (diphenylene iodonium and apocynine), and nitric oxide synthases (N-monomethyl-L-arginine and N-iminoethyl-L-ornithine). The effects of glycolysis inhibition (iodoacetate), glucose deprivation, glycogen depletion, and lactate accumulation were also investigated. In untreated hearts, ROS production peaked at 10.8 ± 3.3, 9 ± 0.8, and 4.8 ± 0.4 min (means ± SD; n = 4) of reoxygenation in the atria, ventricle, and outflow tract, respectively, and was associated with arrhythmias. Functional recovery was complete after 30-40 min. At reoxygenation, 1) the respiratory chain and NADPH oxidases were the main sources of ROS in the atria and outflow tract, respectively; 2) glucose deprivation decreased, whereas glycogen depletion increased, oxidative stress; 3) lactate worsened oxidant stress via NADPH oxidase activation; 4) glycolysis blockade enhanced ROS production; 5) no nitrosative stress was detectable; and 6) the glutathione redox cycle appeared to be a major antioxidant system. Thus, the glycolytic pathway plays a predominant role in reoxygenation-induced oxidative stress during early cardiogenesis. The relative contribution of mitochondria and extramitochondrial systems to ROS generation varies from one region to another and throughout reoxygenation.

A hypoxic episode during cardiogenesis downregulates the adenosinergic system and alters the myocardial anoxic tolerance

“…The fact that H6 hearts displayed the lowest tolerance to 30 min anoxia and the most delayed recovery at reoxygenation relative to N hearts could be partly explained by glycogen depletion (30%). Indeed, we previously showed that glycogen depletion augments the reoxygenation-induced ROS production and/or weaken the defense against oxyradicals (37). Such observations and our present findings suggest that ADO-induced preservation of glycogen can afford cardioprotection against anoxia/reoxygenation following hypoxia.…”

“…Protein and glycogen determination. Protein and glycogen were determined according to our previous works (37,42). Briefly, protein content was determined using BCA protein assay kit (Thermo Scientific Pierce) and BSA as the standard.…”

“…We previously showed that reoxygenation of the embryonic heart after 30 min anoxia produces a burst of reactive oxygen species (ROS) in A, V, and OT (37). Furthermore, in chick embryonic cardiomyocytes (10-day-old embryo) the peak of ROS at reoxygenation is reduced by ADO through A 1 AR and A 2A R (60).…”

NADPH Oxidase 4 Regulates Cardiomyocyte Differentiation via Redox Activation of c-Jun Protein and the cis-Regulation of GATA-4 Gene Transcription

“…The developing heart is known to be differentially sensitive to oxidants in a spatial and temporally defined way in vivo (61). The challenge ahead is to delineate the precise sources of ROS and opposing antioxidant systems within the developing embryo and to determine their roles in coordinating the diversity of cellular transcriptional programs during mammalian development.…”

Chronic hypoxia increases peroxynitrite, MMP9 expression, and collagen accumulation in fetal guinea pig hearts

“…Hypoxia has been shown to generate oxidizing species in cardiac tissue through several mechanisms, including upregulation of nicotinamide adenine dinucleotide phosphate oxidase (35) and/or inhibition of mitochondrial function (35). Although each mechanism may generate superoxide anions (O 2 − ), the mechanisms that contribute to increased lipid peroxidation in HPX fetal hearts are not known.…”