PRECLINICAL STUDY: EDITORIAL COMMENT
Hydrogen Peroxide and Metabolic Coronary Flow Regulation*
John M. Canty, Jr, MD, FACC , , ,* and
Vijay S. Iyer, MD, PhD,
Department of Medicine, Veterans Administration Western New York Health Care System, Buffalo, New York
Department of Physiology and Biophysics, Veterans Administration Western New York Health Care System, Buffalo, New York
Center for Research in Cardiovascular Medicine, State University of New York, Buffalo, New York.
* Reprint requests and correspondence: Dr. John M. Canty, Jr., Division of Cardiovascular Medicine, State University of New York at Buffalo, Biomedical Research Building, Room 347, 3435 Main Street, Buffalo, New York 14214. (Email: canty{at}buffalo.edu).
Myocardial perfusion is closely matched to increases in myocardial metabolism, because the extraction of oxygen is near maximal at rest (1). The net vascular resistance to coronary blood flow represents the integration of systolic compressive effects that vary throughout the cardiac cycle, with microvascular resistance networks that adjust tone in response to local physical factors (intraluminal pressure and luminal shear stress), vasodilator metabolites, autocoids, and adrenergic tone. The local microcirculatory resistance adjustments overcome compressive forces to match transmural variations in myocardial oxygen supply to demand in the steady state. Studies in conscious animals have demonstrated considerable redundancy in the mediators responsible for integrative coronary flow regulation such that multiple pathways need to be impaired before flow is altered at normal coronary pressures (2). In contrast, the importance of individual pathways (e.g., endothelial nitric oxide) can be unmasked by evaluating coronary regulation distal to a severe stenosis (3,4). Despite intensive study, however, the precise mediators responsible for initiating metabolic coronary vasodilation remain unknown (1).
Flow-mediated vasodilation in resistance arteries and arterioles is a generalized mechanism that can match segmental resistance of the local coronary microcirculation to meet changes in downstream demand that occur during increased myocardial metabolism. Although this is governed by local shear stress and is endothelium dependent, the mechanisms responsible for flow-mediated vasodilation vary among different organs. Even within the heart, mediators vary in different vascular segments as well as with the magnitude and pulsatile characteristics of flow (5–7). Whereas initial studies focused upon the role of nitric oxide as an endothelium-dependent vasodilator, there is increasing evidence that endothelium-dependent hyperpolarizing factors (EDHFs) are important in some microcirculatory segments. Hyperpolarizing mechanisms vary with age, species, and coexisting disease processes that up-regulate this pathway when nitric oxide-dependent mechanisms are deficient and are particularly important in coronary arterioles isolated from patients (8). A number of different mediators have been identified as candidate EDHFs, including epoxyeicososatreinoic acid metabolites and hydrogen peroxide (H2O2) formed from the dismutation of superoxide anion.
Reactive oxygen species have traditionally been viewed as deleterious byproducts of metabolism, but there is accumulating evidence that at lower concentrations, they also serve as important cellular signaling molecules in the coronary endothelium as well as in cardiac myocytes (9). Superoxide anion is continually released from mitochondria at a number of points in the electron transport chain as a byproduct of oxidative metabolism (Fig. 1). This unstable radical is converted to H2O2 via superoxide dismutase (SOD) localized within the mitochondria (MnSOD) or the extracellular space (CuZnSOD). Matoba et al. (10) were the first to demonstrate that H2O2 is an endothelium-derived hyperpolarizing factor in mice. Subsequent studies by Miura et al. (8,11) demonstrated that H2O2 was an endothelium-dependent hyperpolarizing factor that was responsible for flow-mediated vasodilation in isolated human coronary arterioles and could be blocked by the scavenger catalase. Liu et al. (12) demonstrated that H2O2 produced in response to flow was a byproduct of complex I and complex III of the endothelial mitochondrial electron transport chain.
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Figure 1 Production and Actions of H2O2 in the Heart
(A) Sources of superoxide anion and hydrogen peroxide (H2O2) during normal oxidative metabolism. Low levels of superoxide anion arise from complex I and complex III as byproducts of the mitochondrial electron transport chain. Superoxide anion is dismutated to H2O2 by manganese superoxide dismutase (MnSOD, mitochondrial matrix) or copper zinc superoxide dismutase (CuZnSOD). The H2O2 diffuses across the mitochondrial membrane to act on vascular smooth muscle. Modified and republished from Zhang and Gutterman (9) with permission from the American Physiological Society. (B) Potential cellular sources of H2O2 in the myocardium. In the endothelium, mitochondrial superoxide anion production is coupled to local luminal shear stress through mechanisms that are coupled to mechanical deformation of the cytoskeleton. The H2O2 diffuses abluminally to relax smooth muscle by activating calcium- (KCa) and/or voltage-dependent (Kv) potassium channels. Flow-dependent vasodilation from the H2O2 pathway appears to predominate in coronary resistance arterioles (<100 µm), whereas nitric oxide-dependent mechanisms mediate flow-dependent vasodilation in coronary resistance arteries (>100 µm). The H2O2 can also be produced in cardiac myocytes and is released in proportion to metabolism as superoxide anion is produced from the mitochondrial electron transport chain. See text for further discussion. GPX = glutathione peroxidase; MVO2 = myocardial oxygen consumption.
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In this issue of the Journal, Yada et al. (13) present an extensive series of in vivo experiments that demonstrate the importance of H2O2 in adjusting coronary resistance vessel tone during pacing-induced increases in myocardial oxygen consumption in open-chest anesthetized dogs. Measurements of coronary flow and myocardial oxygen consumption were coupled with microcirculatory measurements of arteriolar diameter during pacing. As previously demonstrated (5), the vasodilation to pacing was not affected by inhibiting nitric oxide synthase with L-nitro monomethyl arginine. Both catalase, which inactivates H2O2 by converting it to water, and inhibiting adenosine with 8-sulfophenyltheophyline (8-SPT) blunted the arteriolar vasodilation to pacing (arterioles <100 µm) but did not affect larger resistance arteries (>100 µm). Collectively, the results extend previous observations during autoregulation (14) and suggest a differential mechanism for flow-induced vasodilation with nitric oxide predominating in large resistance arteries and H2O2-mediated hyperpolarization predominating in small arterioles.
How does H2O2 cause vasodilation? A number of studies have demonstrated that H2O2 opens calcium-activated potassium channels (KCa) to cause smooth muscle relaxation. Activation of KCa channels causes efflux of potassium from within vascular smooth muscle, resulting in membrane hyperpolarization and closure of voltage-dependent Ca2+ channels. The reduction in Ca2+ influx leads to vascular relaxation. Experimental support implicating KCa channels include in vivo as well as in vitro studies using pharmacologic blockade, where relaxation and smooth muscle hyperpolarization can be blocked by antagonists such as tetraethylammonium (TEA). Merkus et al. (15) demonstrated that blocking KCa channels with TEA attenuates metabolic coronary vasodilation during exercise in conscious pigs (15). It is also possible that other potassium channel subtypes contribute to this response. For example, Rogers et al. (16) demonstrated that H2O2 activates voltage-dependent potassium channels which can be selectively blocked using 4-aminopyridine to inhibit the vasodilation to pacing in vivo. Whereas pharmacologic approaches are limited by drug specificity for certain channel subtypes, the available evidence supports the notion that both subtypes of potassium channels may be involved in mediating the effects of H2O2. The molecular interactions between H2O2 and potassium channels leading to vasodilation are currently under investigation.
The major limitation of the study of Yada et al. (13) is that the specific cellular source of H2O2 cannot be clearly identified. This is admittedly difficult in vivo, but the in vitro assessment of free radical production provided suggests that this arises in the arteriolar endothelium. Because hyperpolarization can occur in isolated arterioles removed from the myocardium, endothelial shear stress may be the primary stimulus (Fig. 1). The precise mechanism coupling shear stress to increased superoxide production and H2O2 in endothelial cells has not been established, but it may arise from mechanical deformation of the cytoskeleton. If the actions of H2O2 arise solely from shear stress, a different downstream metabolic mediator would still be needed to trigger the initial release of H2O2 in response to increases in tissue perfusion. The present data suggest that this could be adenosine, because arteriolar dilation during increased metabolism was blocked by 8-SPT. Smaller arterioles, beyond digital camera resolution, could also be the segment responsive to initial metabolic stimuli.
An alternative explanation for the link between H2O2 and metabolic flow regulation, proposed by Saitoh et al. (17), is that H2O2 arises from cardiac myocytes rather than the endothelium. This is attractive in coupling the production of this mediator to myocardial metabolism, because production of H2O2 is directly proportional to the production of superoxide anion as energy is produced via the mitochondrial electron transport chain. Saitoh et al. (17) demonstrated that superoxide anion production is increased in proportion to increases in cardiac metabolism using electron paramagnetic spectroscopy in isolated cardiac myocytes, leading to a concomitant increase in H2O2 production in vitro. In vivo, increases in metabolism elicited by pacing or catecholamine infusion were accompanied by increases in myocardial H2O2 levels and perfusion. Blocking the effects of H2O2 using 4-aminopyridine to block voltage-dependent potassium channels shifted relationships among oxygen consumption, coronary flow, and coronary venous pO2, confirming physiologically relevant effects on the regulation of myocardial perfusion.
A cardiomyocyte origin of H2O2 could also explain why metabolic flow regulation continues in disease states associated with intrinsically reduced myocardial metabolism and coronary flow at rest, such as heart failure (18) and hibernating myocardium (19). The lower metabolism and set point for superoxide release would attenuate baseline H2O2 production yet preserve the relation between relative increases in metabolism and H2O2. These in vivo observations, in conjunction with the results of Yada et al. (13), strengthen support for a broad role for H2O2 in metabolic coronary flow recruitment. Although additional studies are needed to clarify the independent role that H2O2 produced from endothelial cells (coupled to shear stress) versus myocytes (coupled to metabolism) plays in normal and pathophysiologic states, we now appear closer to identifying the critical link between myocardial metabolism and coronary blood flow.
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Footnotes
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Supported by the Veterans Administration, the National Heart, Lung, and Blood Institute (HL-55324, HL-61610), and the Albert and Elizabeth Rekate Fund.
* Editorials published in the Journal of the American College of Cardiology reflect the views of the authors and do not necessarily represent the views of JACC or the American College of Cardiology. 
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References
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1. Canty Jr. JM. Coronary blood flow and myocardial ischemiaIn: Libby P, Bonow RO, Mann DL, et al. editors. Braunwald’s Heart Disease. 8th edition. Philadelphia, PA: Elsevier; 2007. pp. 1167-1194.2. Duncker DJ, Bache RJ. Regulation of coronary vasomotor tone under normal conditions and during acute myocardial hypoperfusion Pharmacol Ther 2000;86:87-110.[CrossRef][Web of Science][Medline] 3. Smith Jr. TP, Canty Jr. JM. Modulation of coronary autoregulatory responses by nitric oxide: evidence for flow-dependent resistance adjustments in conscious dogs Circ Res 1993;73:232-240.[Abstract/Free Full Text] 4. Duncker DJ, Bache RJ. Inhibition of nitric oxide production aggravates myocardial hypoperfusion during exercise in the presence of a coronary artery stenosis Circ Res 1994;74:629-640.[Abstract/Free Full Text] 5. Canty Jr. JM, Schwartz JS. Nitric oxide mediates flow-dependent epicardial coronary vasodilation to changes in pulse frequency but not mean flow in conscious dogs Circulation 1994;89:375-384.[Abstract/Free Full Text] 6. Kuo L, Davis MJ, Chilian WM. Longitudinal gradients for endothelium-dependent and -independent vascular responses in the coronary microcirculation Circulation 1995;92:518-525.[Abstract/Free Full Text] 7. Dube S, Canty Jr. JM. Shear-stress induced vasodilation in porcine coronary conduit arteries is independent of nitric oxide release Am J Physiol 2001;280:H2581-H2590.[Web of Science] 8. Miura H, Wachtel RE, Liu Y, et al. Flow-induced dilation of human coronary arterioles: important role of Ca2+-activated K+ channels Circulation 2001;103:1992-1998.[Abstract/Free Full Text] 9. Zhang DX, Gutterman DD. Mitochondrial reactive oxygen species-mediated signaling in endothelial cells Am J Physiol Heart Circ Physiol 2007;292:H2023-H2031.[Abstract/Free Full Text] 10. Matoba T, Shimokawa H, Nakashima M, et al. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice J Clin Invest 2000;106:1521-1530.[Web of Science][Medline] 11. Miura H, Bosnjak JJ, Ning G, et al. Role for hydrogen peroxide in flow-induced dilation of human coronary arterioles Circ Res 2003;92:e31-e40.[Abstract/Free Full Text] 12. Liu Y, Zhao H, Li H, et al. Mitochondrial sources of H2O2 generation play a key role in flow-mediated dilation in human coronary resistance arteries Circ Res 2003;93:573-580.[Abstract/Free Full Text] 13. Yada T, Shimokawa H, Hiramatsu O, et al. Important role of endogenous hydrogen peroxide in pacing-induced metabolic coronary vasodilatation in dogs in vivo J Am Coll Cardiol 2007;50:1272-1278.[Abstract/Free Full Text] 14. Yada T, Shimokawa H, Hiramatsu O, et al. Hydrogen peroxide, an endogenous endothelium-derived hyperpolarizing factor, plays an important role in coronary autoregulation in vivo Circulation 2003;107:1040-1045.[Abstract/Free Full Text] 15. Merkus D, Sorop O, Houweling B, et al. KCa+ channels contribute to exercise-induced coronary vasodilation in swine Am J Physiol Heart Circ Physiol 2006;291:H2090-H2097.[Abstract/Free Full Text] 16. Rogers PA, Dick GM, Knudson JD, et al. H2O2-induced redox-sensitive coronary vasodilation is mediated by 4-aminopyridine–sensitive K+ channels Am J Physiol Heart Circ Physiol 2006;291:H2473-H2482.[Abstract/Free Full Text] 17. Saitoh S, Zhang C, Tune JD, et al. Hydrogen peroxide: a feed-forward dilator that couples myocardial metabolism to coronary blood flow Arterioscler Thromb Vasc Biol 2006;26:2614-2621.[Abstract/Free Full Text] 18. Traverse JH, Melchert P, Pierpont GL, et al. Regulation of myocardial blood flow by oxygen consumption is maintained in the failing heart during exercise Circ Res 1999;84:401-408.[Abstract/Free Full Text] 19. Fallavollita JA, Malm BJ, Canty Jr. JM. Hibernating myocardium retains metabolic and contractile reserve despite regional reductions in flow, function, and oxygen consumption at rest Circ Res 2003;92:48-55.[Abstract/Free Full Text]
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