Nitric Oxide, Myocardial Oxygen Consumption, and ATP Synthesis
NO is a free radical gas, NO•, in equilibrium with the closely related redox forms NO− and NO+. With a molecular weight of only 30 Da/mol (for reference, molecular oxygen is 32 Da/mol), NO is readily diffusible. The half-life of NO is 0.1 to 5 seconds. NO synthesis is catalyzed by a family of proteins, NO synthases (NOSs). The NOS originally described as the source of NO in conduit vessel endothelial cells (eNOS or NOS3) has been found in the endothelium of the endocardium and the coronary vasculature, in atrial and ventricular myocytes, and in conduction cells of the heart.1 2 3
Because NO is readily diffusible, it can affect many molecular targets. Molecular oxygen is itself a target, forming NO2, which in turn forms NO2−, and NO3−. NO reacts with the free radical superoxide O2− to form the highly reactive species peroxynitrite ONOO−. NO also reacts with iron to form Fe2+NO complexes and with amino, thiol, diazo, and tyrosyl groups in proteins. On the basis of this chemistry, the macromolecular targets of NO and its derivatives in cells can be understood. For example, activation of the signal transduction protein guanylyl cyclase occurs via NO binding to its heme. Inhibition of creatine kinase and some of the glycolytic proteins, including GAPDH, by NO occurs by binding to labile reactive −SH groups. Thus, NO functions to alter macromolecular function in 2 distinct ways: by direct chemical modification and by activation of the guanylyl cyclase signaling pathway.
NO and ATP Synthesis
NO plays a role in regulating the activity of proteins essential for maintaining normal ATP synthesis rates. Although the identity of the inhibitor was not known at the time, the first report showing that NO could alter the primary pathway for ATP synthesis in cells was published by Granger and Lehninger in 1982.4 These investigators showed that mitochondrial respiration was inhibited in a leukemia cell line injured by exposure to cytotoxic macrophages, which are now known to express inducible NOS (iNOS or NOS2). Moreover, they identified the precise molecular targets responsible for the inhibition: complex I (NADH-coenzyme Q reductase) and complex II (succinate-coenzyme Q reductase) in the electron transport chain. We now know that it was NO produced by the toxic macrophages that inhibited respiration by binding to the Fe-S target in these 2 protein complexes. The Fe-S center of aconitase in the Krebs cycle and the heme of cytochrome oxidase in the electron transport chain are also targets of NO. These discoveries illustrate the important principle that the targets of NO are specific, not ubiquitous. Specificity is due to availability (NO must diffuse to the target) and, given equal availability of NO, to the relative binding affinity of the various candidate target moieties for NO and its related molecules.
The experiments described above were performed using permeabilized cells and thus ensure that NO was near to the target proteins in the mitochondria. Could respiration be controlled by NO in this way in vivo? Previous work of Hintze and colleagues5 6 7 studying whole body oxygen consumption in the dog has provided good evidence that NO produced in physiological amounts by eNOS in the capillary endothelium most likely contributes to the regulation of oxygen consumption in skeletal muscle. Shen et al5 first showed that supplying an NOS inhibitor to resting dogs increased both total body oxygen extraction and total body oxygen consumption. This unexpected result suggested that NO produced by the capillary endothelium partially inhibits oxygen consumption. If so, constitutive NO production could serve as a brake for oxygen consumption, much like phospholamban serves as a brake for Ca2+ release by the sarcoplasmic reticulum in the heart. In this way, there would always be a reserve for new ATP synthesis when needed. Subsequent reports from many laboratories support a physiological role of NO linking the circulation and the target parenchymal cell in both skeletal and cardiac muscle.
NO and Heart Failure
iNOS can be induced in cardiomyocytes and endothelial cells under pathophysiological conditions. Cationic amino acid transporters and enzymes responsible for synthesizing NOS cofactors needed to ensure maximal NO production are coexpressed.8 Given its high chemical reactivity, it is likely that when high concentrations of NO are produced by iNOS, a host of direct chemical modifications and changes downstream from guanylyl cyclase signaling pathway ensues that could contribute to impaired contractile function and even to increased susceptibility to cell injury. In 1996, Haywood et al9 studied biopsy specimens obtained from ventricles of failing human hearts and showed that iNOS is induced in heart failure. Using immunohistochemistry, they found diffuse cytosolic staining of iNOS in hearts failing as a result of a variety of causes. This and several subsequent studies also showing increased capacity for NO production are consistent with the notion that NO production was increased in heart failure and that this could contribute to pump failure.
In a study published in this issue of Circulation Research by Recchia et al,10 NO production in the heart was directly measured in the pacing-induced heart failure in the dog by measuring net efflux of NO2− and NO3− across the coronary circulation (note the potential limitation of the technique discussed in the article). NO production estimated in this way in this model of heart failure decreased, not increased. As would be predicted from the working hypothesis that less NO would relieve the inhibition for O2 consumption, MVO2 increased. The study went further. By directly measuring cardiac extraction of the various carbon-based substrates for ATP synthesis, it was found that, in this model of heart failure, metabolism switched from fatty acids utilization to glucose utilization.
As with all good research, this study raises more questions than it addresses. Some of these are as follows:
Is decreased production of NO a common characteristic of heart failure of all causes? If so, how does one explain the upregulation of iNOS in human heart failure with the progressive decrease of NO production in this model of heart failure?
What are the consequences of the switch from fatty acid utilization to glucose utilization on ATP synthesis? Although the P:O ratio for oxidation of glucose is higher than for fatty acids (ie, more ATP is produced per mole of O2 consumed), it is nonetheless the case that the yield of ATP per mole of carbon-based substrate is very much less for glucose than for fatty acids. Can glucose utilization alone maintain normal ATP levels in the failing heart?
How does the substrate selection switch occur? What enzymes are involved? Are they up- or downregulated? Is the action of NO direct and/or via the cyclase signaling pathway?
A major strength of the study presented by Recchia et al10 is that the metabolites of interest—NO derivatives and O2 and the primary carbon-based substrates for ATP synthesis—were directly measured. We have been living in an era of extrapolating the chemistry of the cell from the levels of mRNAs and protein. But the amount of mRNA or even of the protein tells us only the capacity of the reaction, not flux through that chemical reaction. It is rare that flux through any step in a metabolic pathway is near its capacity. Instead, flux is usually only a small fraction of capacity, thereby allowing up and down “regulation” to accommodate the metabolic demands of the cell. A good example of this is the unchanged glycolytic flux measured in hearts with only 30% of normal GAPDH activity.11 This plasticity is a major mechanism for cell survival.
The results of this study10 will direct future research in this area: we must determine the mechanisms for decreased NO production in the failing heart and identify not only its molecular causes but also its consequences on the myriad of possible targets for NO within the cell.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
- © 1998 American Heart Association, Inc.
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