Myoglobin: A scavenger of bioactive NO

Abstract

The present study explored the role of myoglobin (Mb) in cardiac NO homeostasis and its functional relevance by employing isolated hearts of wild-type (WT) and myoglobin knockout mice. (1)H NMR spectroscopy was used to measure directly the conversion of oxygenated Mb (MbO(2)) to metmyoglobin (metMb) by reaction with NO. NO was applied intracoronarily (5 nM to 25 microM), or its endogenous production was stimulated with bradykinin (Bk; 10 nM to 2 microM). We found that infusion of authentic NO solutions dose-dependently (>/= 2.5 microM NO) increased metMb formation in WT hearts that was rapidly reversible on cessation of NO infusion. Likewise, Bk-induced release of NO was associated with significant metMb formation in the WT (>/=1 microM Bk). Hearts lacking Mb reacted more sensitively to infused NO in that vasodilatation and the cardiodepressant actions of NO were more pronounced. Similar results were obtained with Bk. The lower sensitivity of WT hearts to changes in NO concentration fits well with the hypothesis that in the presence of Mb, a continuous degradation of NO takes place by reaction of MbO(2) + NO to metMb + NO(3)(-), thereby effectively reducing cytosolic NO concentration. This breakdown protects myocytic cytochromes against transient rises in cytosolic NO. Regeneration of metMb by metMb reductase to Mb and subsequent association with O(2) leads to reformation of MbO(2) available for another NO degradation cycle. Our data indicate that this cycle is crucial in the breakdown of NO and substantially determines the dose-response curve of the NO effects on coronary blood flow and cardiac contractility.

Figure 1

¹H (Left) and ³¹P NMR (Right) spectra of WT hearts showing the effect of increasing NO concentration on cardiac Mb and energy status. Assignments: ATP, adenosine triphosphate (γ-, α-, and β-phosphorus); MbO₂, oxygenated myoglobin; metMb, metmyoglobin; PCr, phosphocreatine; P_i (ext.) and (int.), extracellular and intracellular inorganic phosphate, respectively.

Figure 2

Analysis of cardiac Mb (Top), function (Middle), and energetics (Bottom) of isolated perfused hearts with increasing NO concentration. Symbols show means ± SD for n = 8 hearts; * = P < 0.05; ** = P < 0.01; For abbreviations see Fig. 1. The shaded area emphasizes the association of metMb formation in WT hearts and the more pronounced impairment of cardiac function and energy status in Mb-deficient as compared with WT hearts.

Figure 3

Effect of the NO synthase inhibitors ETU (50 μM) and l-NMMA (100 μM) on coronary perfusion pressure of constant-flow-perfused hearts of WT and myo^−/− mice. Symbols show means ± SD for n = 6 hearts; ** = P < 0.01.

Figure 4

Analysis of coronary perfusion pressure (Upper) and cardiac function (Lower) of constant flow perfused hearts under Bk stimulation. Symbols show means ± SD for n = 8 hearts; * = P < 0.05; ** = P < 0.01.

Figure 5

¹H NMR spectra (Left, 4 min accumulation time) of WT hearts showing the effect of Bk stimulation on cardiac Mb. Symbols on the Right show means ± SD for n = 6 hearts, * = P < 0.05.

Figure 6

Schematic drawing summarizing possible interactions of the different Mb compounds with O₂ and NO. For a detailed discussion refer to the text. Cap., capillary; Endo., endothelium; Mito., mitochondrion.