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Role of myoglobin in the antioxidant defense of the heart

ULRICH FLÖGEL^*,1, AXEL GÖDECKE^*, LARS-OLIVER KLOTZ and AND JÜRGEN SCHRADER^*

^* Institut für Herz- und Kreislaufphysiologie
Institut für Biochemie und Molekularbiologie I, Heinrich-Heine-Universität Düsseldorf, Germany

¹Correspondence: Institut für Herz- und Kreislaufphysiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany. E-mail: floegel{at}uni-duesseldorf.de

SPECIFIC AIMS

The aim of the present study was to explore the role of myoglobin (Mb) in cardiac metabolism of reactive oxygen species (ROS). We analyzed the functional and energetic effects of ROS either pharmacologically applied or endogenously generated in beating hearts of Mb-deficient (myo^–/–) and control mice.

PRINCIPAL FINDINGS

1. Mb attenuates the effect of exogenously induced oxidative stress on cardiac contractile force
Langendorff-perfused hearts were exposed to pharmacologically induced oxidative stress by stepwise increasing the concentrations of intracoronary infused H₂O₂ or 2,3-dimethoxy-1,4-naphtoquinone (DMNQ), a compound continuously releasing superoxide by redox cycling. Left ventricular developed pressure (LVDP) progressively decreased at [DMNQ] >0.3 µM, resulting in a 30% reduction of cardiac contractility at the maximal [DMNQ] applied. In the DMNQ dose range of 1–10 µM, a significantly greater decrease in LVDP was observed in myo^–/– than in wild-type (WT) hearts. Comparable differences between the WT and myo^–/– groups were detected for dP/dt. Similar to the results with DMNQ, we found the Mb-deficient group was more sensitive to exogenously applied H₂O₂. Up to [H₂O₂] = 10 µM, there was a significant LVDP decrease in myo^–/– hearts only (90.4±4.2 vs. 98.1±0.7% of control, n=6, P<0.05). Again, comparable differences were observed for dP/dt between the WT and myo^–/– groups in the H₂O₂ dose range of 3–100 µM.

2. Lack of Mb leads to a delayed postischemic recovery of cardiac function paralleled by an enhanced release of ROS during reperfusion
To assess whether the greater sensitivity of Mb-deficient hearts to oxidative stress is of functional significance, we applied an ischemia-reperfusion (IR) protocol to enhance the endogenous release of ROS. Hearts were subjected to a 12 min period of a global no-flow ischemia and subsequently reperfused for 1 h. Cardiac contractility together with ³¹P NMR spectroscopy was used to monitor function and energy state of the hearts. In parallel experiments, hearts were perfused via a home-built adaptor inside a Berthold LB 500 luminometer (Fig. 1 A) under corresponding conditions and cardiac ROS release was continuously monitored by lucigenin-enhanced chemiluminescence (measured as relative light units (RLU; Fig. 1B ). Cardiac function and energetic parameters of WT and myo^–/– hearts were not different under control conditions or during ischemia. In reperfusion, however, WT hearts showed a significantly faster recovery of the postischemic function compared with myo^–/– hearts: LVDP was 35.6 ± 7.5 mmHg in WT vs. 22.4 ± 5.3 mmHg in myo^–/– after 10 min of reperfusion (P<0.01, n=8). ³¹P NMR revealed that, concomitantly, a substantially larger overshoot of the phosphocreatine (PCr) signal occurred in myo^–/– hearts (125±5% of control in myo^–/– vs. 110±7% of control in WT after 10 min of reperfusion, P<0.05, n=8). As shown in Fig. 1C , the retarded restoration of functional and metabolic parameters in myo^–/– compared with WT hearts was accompanied by increased release of ROS in myo^–/– hearts (465±87 RLU in myo^–/– vs. 287±73 RLU in WT after 2.5 min of reperfusion, P<0.05, n=8). Additional experiments in presence of the NO synthase inhibitor N^G-monomethyl-L-arginine methyl ester (L-NMMA, 100 µM) confirmed the findings: again, the retarded recovery of cardiac contractility and energetics in Mb-deficient hearts was accompanied by a significantly higher release of ROS in myo^–/– than in WT hearts.

View larger version (38K): [in this window] [in a new window]   Figure 1. Analysis of cardiac ROS release of isolated perfused hearts. A) Langendorff setup for chemiluminescence measurements of ROS release. Mouse hearts were perfused via a home-built adaptor inside the luminometer and paced at 500 bpm. B) Typical recording: 1) start of the lucigenin infusion, 2) begin ischemia, 3) reperfusion. Perfusion pressure (top), coronary flow (middle), and lucigenin-enhanced chemiluminescence (bottom) were continuously acquired. C) Cardiac ROS release of perfused WT and myo–/– hearts during 12 min of global ischemia and subsequent reperfusion for 1 h. Symbols show means ± SD for n = 8 hearts; P<0.05 vs. WT; RLU, relative light units. Shaded area emphasizes the ischemic period.

3. Oxidative stressors lead to the formation of globin radical intermediates
We further examined whether intermediates of ROS-consuming reactions by Mb could be identified in control hearts. It was recently shown that the reaction of Mb with H₂O₂ results in covalent binding of the heme prosthetic group, most likely via formation of a tyrosyl radical at position 103 of the apo-myoglobin chain. Thus, these protein-bound heme adducts are indicators of Mb’s peroxidase activity. Heme-Mb adducts retain their redox activity even after SDS-PAGE and electroblotting, and can be detected by the use of enhanced chemiluminescence detection reagents. Extracts from WT hearts perfused with 30 µM H₂O₂, the concentration where the most pronounced differences in cardiac contractility between WT and myo^–/– were observed, revealed three major signals, including a band of 17,000 Da. No signal corresponding to Mb was detected in extracts from myo^–/– mice or in hearts under basal conditions.

CONCLUSIONS

This study describes a novel in vivo function of Mb: the attenuation of oxidative stress in cardiac muscle. Hearts from myo^–/– mice were found to be more sensitive to the infusion of ROS (e.g.,. H₂O₂ and superoxide) in that depression of myocardial contractile force was more pronounced than in WT controls. Mb-deficient hearts released significantly more ROS during IR; this was accompanied by a delayed functional and metabolic recovery after the ischemic insult.

The reaction of H₂O₂ with hemoglobin was described for the first time more than 100 years ago; and in the meantime the interactions of ROS with hemoproteins have been object of numerous in vitro investigations. From these studies it became evident that ROS formed by the endothelial xanthine oxidase or incomplete reduction of O₂ within the mitochondria during IR are likely to interact with Mb, as hypothesized in Fig. 2 . Reaction of peroxides with both Mb (Fe^II) and metMb (Fe^III) yields the ferryl derivative (Fe^IV) of Mb, characterized by a very high oxidative capacity. Comproportionation of ferryl Mb with Mb leads to formation of metMb; the reduction of metMb by cardiac metMb reductase results in regeneration of Mb (Fe^II) available for initiation of another breakdown cycle. Although the individual steps are known to occur in vitro, the present study indicates that together they can constitute in vivo a metabolic cycle that contributes to the attenuation of oxidative stress in the heart.

View larger version (31K): [in this window] [in a new window]   Figure 2. Schematic drawing summarizing possible interactions of the different Mb compounds with reactive oxygen species. Mb2+, Mb3+, and Mb4+ stand for Mb with a heme iron in the oxidation state FeII, FeIII, and FeIV, respectively. See text for details.

Protection against oxidant injury is particularly meaningful during the early reperfusion period after an ischemic insult, which is characterized by the sudden formation of large amounts of ROS. The chemiluminescence approach presented in this study enabled us via intracoronary infusion of low nontoxic lucigenin doses to measure online the kinetics of ROS release into the venous effluent perfusate, yielding similar time courses for the initial oxygen radical burst and the subsequent slow decrement when compared with spin trap and ESR measurements or by determination of peroxide production. The 60% increased chemiluminescence level in Mb-deficient hearts during early reperfusion indicates that redox cycling of Mb contributes significantly to ROS breakdown under these conditions. Therefore, Mb can be regarded as an additional oxygen radical scavenger among the various enzymatic and nonenzymatic antioxidant defenses including superoxide dismutase (SOD), glutathione peroxidase, catalase, etc. Since the high amounts of cytosolic Mb (200 µmol/kg wet weight) can account for a substantial ROS breakdown, our results may explain why cytosolic Cu/Zn-SOD knockout mice appeared to be almost normal whereas knockout of mitochondrial Mn-SOD resulted in a dilated cardiomyopathy and lethality.

The functional significance of ROS scavenging by Mb is apparent when comparing the data for cardiac contractility and energetics during IR in WT and myo^–/– hearts. The more pronounced PCr overshoot during initial reperfusion in Mb-deficient hearts is consistent with the retarded restoration of cardiac contractility in these hearts, since the degree of PCr overshoot has been related to the extent of mechanical dysfunction during reperfusion. The PCr overshoot in IR has been reported to be caused by an inactivation of cytosolic creatine kinase (CK_cyto) due to H₂O₂ oxidation of methionine-bound sulfhydryl groups of this enzyme: The impaired CK_cyto activity prevents cytoplasmic conversion of PCr to ATP, impeding the efficient delivery of energy to utilization sites with resultant depression of cardiac contractility. In our experiments it is most likely that sustained inhibition of CK_cyto by elevated levels of ROS during reperfusion caused the more pronounced overshoot of PCr in myo^–/– hearts. Aside from CK_cyto inactivation, ROS may exert further detrimental effects on cardiac function by the initiation of peroxidation of cell membranes, unspecific destruction of enzymes, and cleavage of DNA strands.

Several lines of evidence suggest that during IR there is an enhanced production of NO by endothelial NO synthase that results in a NO burst in the initial phase of reperfusion. Since we have shown that Mb contributes to NO breakdown in the heart, it is conceivable that a diminished NO degradation plays a role in the delayed recovery of myo^–/– hearts during IR. However, inhibition of endogenous NO release by L-NMMA did not alter the retarded restoration of functional and metabolic parameters in Mb-deficient hearts, indicating that NO does not account for the differences observed. Comparing the time course of ROS and NO production during reperfusion, it seems likely that the prolonged release of ROS observed in this and other studies outweighs the NO burst, which lasts only 1–2 min. Our findings can explain the recently described (but mechanistically unresolved) protection of neurons from hypoxic-ischemic injury by the homologous neuroglobin, a phenomenon that has been shown to be independent of NO or oxygen storage.

The data of the present study show that lack of Mb leads to increased vulnerability of the heart when challenged by oxidative stress. However, previous investigations by us and others provided no evidence for an unbalanced oxidative status in Mb-deficient mice under basal conditions. Similarly, genomic and proteomic analysis did not reveal the induction of potential regulatory mechanisms to encounter an increased oxidative stress or reprogramming of antioxidant-related genes in myo^–/– mice. Deoxygenated Mb has been described to be the species oxidized by H₂O₂ (cf. Fig. 2 ), so that myocardial PO₂ appears to be a major determinant in this context. It has been shown that the reaction of H₂O₂ with the ferric form of Mb (metMb) is almost 10-fold faster than the rate of H₂O₂ reaction with the ferrous deoxygenated form of Mb. Thus, the reaction of Mb with ROS is likely to be functionally relevant under conditions of reduced oxygen supply associated with enhanced deoxygenation of Mb, and even more so during oxidative stress when increased amounts of metMb are formed, but not in the well-oxygenated heart where MbO₂ dominates. This view is supported by the detection of globin radical intermediates in the presence of oxidative stressors, suggesting a significant contribution of the Mb-peroxidase reaction to H₂O₂ turnover especially during oxidative challenge. This may be particularly important in diving mammals, whose Mb concentrations exceed those of terrestrial mammals by up to 10-fold. Here, the high Mb content is likely not only to extend diving time by O₂ storage when pulmonary ventilation ceases, but also to attenuate cytosolic oxidative stress during reoxygenation after surfacing.

In summary, our data indicate that Mb contributes to the attenuation of increased oxidant stress and substantially determines the dose-dependent effects of ROS on cardiac contractility and energetics. Thus, Mb may be regarded as a molecular radical scavenger protecting other targets (such as creatine kinase) against transient rises of cytosolic ROS brought about after short periods of ischemia, thereby complementing the known muscular antioxidant defense mechanisms. Taking into account Mb’s NO scavenging properties, we propose that Mb is not only important in intracellular oxygen supply but also constitutes a key element to influence various redox pathways in the muscle cell, supporting the recent suggestion to classify Mb as a multifunctional allosteric enzyme.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1382fje; doi: 10.1096/fj.03-1382.fje

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