A Guide to Cardiovascular Issues in CFS IVc: Peroxynitrite and the Heart

The Sieverling paper reports Dr. Cheney believes peroxynitrite is the central agent of damage of heart failure both in the idiopathic cardiomyopathy Dr. Cheney experienced and in CFS patients. Most of the treatments Dr. Cheney recommends are aimed at reducing nitric oxide, superoxide or peroxynitrite levels.

For such a well researched subject it is surprising that peroxynitrite was only discovered about 15 years ago. Peroxynitrite is formed and vanishes so quickly it is almost impossible to measure in the body. Really potent free radicals are so imbalanced that they often quickly revert to a more balanced state. Instead of measuring peroxynitrite directly, researchers usually determine its presence by measuring one of its by-products, 3-nitrotyrosine, which is formed when peroxynitrite nitrates the tyrosine amino acids in proteins. Thus while it is possible to determine the positive or negative aspects of peroxynitrite, it is difficult to tell exactly how it achieves them.

The Bad In vitro tests of peroxynitrite’s effect on the animal hearts indicate it increases lipid peroxidation (cell membrane damage), depletes antioxidant levels (leaving cells vulnerable to free radicals), disrupts intracellular signaling (throws a monkey wrench into the machinery), dysregulates intracellular calcium levels (causing cell suicide among other things), inhibits contractile protein activity (reduces heart pumping), injures DNA and increases cell adhesion to the vascular wall (increasing inflammation, atherosclerosis) (Pacher et al. 2005, Hare and Stamler 2005) (!!!!). By oxidizing BH4 peroxynitrite may also play a role in both reduced NO production and increased superoxide production.

Both nitric oxide and peroxynitrite can inhibit mitochondrial electron transport (ATP production) through inactivation of complexes I, II, III in the electron transport chain. Protein nitration (nitrotyrosine formation) also knocks out or alters the activity of the main superoxide scavenger in the mitochondria MnSOD. The position of the tyrosine residues near cytochrome C’s catalytic region renders it particularly susceptible to peroxynitrite damage (Cassina et. al. 2000, Reiter et. al. 2000). Peroxynitrite does its damage by oxidizing the lipids (fats) in cell membranes (they are very fatty), by fragmenting DNA and by damaging (nitrating) proteins.

During pathologic conditions such as hypertension (high blood pressure), high cholesterol, and during the early moments of the reperfusion process, both NO and superoxide and therefore peroxynitrite are produced in large amounts (Ronson et. al. 1999 ). Besides its direct negative effects peroxynitrite can potentially have indirect negative effects simply because so much NO is used up making it. This results in low levels of NO, which we have seen is an important anti-oxidant, vasodilator and mitochondrial regulator.

Peroxynitrite appears to be particularly involved in two aspects of cardiovascular health; ischemia-reperfusion and coronary artery disease.

Peroxynitrite and ischemia-reperfusion  As noted earlier ischemia occurs when low blood flows cause tissue damage. After ischemic tissues are reperfused with blood high levels of free radicals, including peroxynitrite, superoxide and the hydroxyl radical are formed. As neutrophils and macrophages ‘clean up’ cells that have been killed by low blood flows they release large amounts of reactive oxygen species that can cause further tissue damage. Interestingly, macrophages in reperfused tissues often have become reduced in L-arg, a condition which causes them to produce both superoxide and nitric oxide and thus peroxynitrite (Ronson et. al. 1999)

Ischemic injury to the heart muscle most often occurs through blockage of the coronary arteries but it can also occur through blockage of the smaller blood vessels that feed the heart itself. Since CFS patients do not appear overly susceptible to heart attack (or small vessel blockage?) it us unclear how much they have to worry about peroxynitrite formed through ischemia-reperfusion (?).

Ischemic episodes can, however, chronically occur when increased demand occurs in the context of chronically low cardiac blood flows (Dewald et. al. 2003). The diversion of blood to one tissue during conditions of low blood flows apparently results in the pathologically low blood flows to other tissues. One wonders if this occurs systemically in people with low blood volume; a subset of CFS patients have low blood volume. Ischemic episodes in mice result in chemokine production, ROS formation and inflammation. This results in macrophage infiltration, fibrosis (fiber deposition in the heart) and contributes to diastolic restriction (i.e. left ventricular stiffening).

Peroxynitrite and atherosclerosis - The discovery almost 25 years ago that oxidatively damaged LDL cholesterol particles were attacked by macrophages gave rise to several decades of intense research to determine the effects this had on the process of atherosclerosis. Peroxynitrite production in atherosclerotic lesions is strikingly high and exceeds that found in non-pathologic LDL particles by 90x’s (!) While much research has indicated that oxidation of the lipid membranes surrounding LDL cholesterol particles plays a key role in atherosclerosis, it is still unclear whether it is the cause of the disease (Rubbo and O’Donnell 2005).

Peroxynitrite appears to be the main agent of LDL cholesterol oxidation. Exactly how peroxynitrite oxidizes LDL particle, however, is still not clear. A macrophage attack on these particles produces a cholesteryl ester which promotes the formation of the ‘fatty streaks’ on the artery wall that eventuate in the production of plaque. Since peroxynitrite very rapidly ‘protonates’ to form peroxynitrous acid (ONOOH) or via H+ or CO2 a number of other compounds (nitrogen dioxide (NO2), hydroxyl radical (OH-) or the carbonate anion radical (CO3-), each of which can have negative effects, it is difficult to know if its peroxynitrite or one of its by-products that does the actual damage in atherosclerosis.

CFS patients are not dropping dead from heart attack nor do they appear susceptible to coronary artery disease.

And Good (yes, the GOOD) of Peroxynitrite – Several in vitro studies have indicated increased peroxynitrite levels in perfused rodent hearts negatively effect heart contraction; an outcome believed due to peroxynitrites ability to inhibit oxidative metabolism (energy production). Reperfusion occurs when hearts that have been deprived with blood are then re-perfused with it. In vitro tests usually use a buffering solution rather than actual blood. As we shall see below this may skew the test results.

In vivo studies (i.e. in the living body) however, have found that the same levels of peroxynitrite infusion that damaged hearts in vitro tests had protective effects on heart tissues. Accumulations of neutrophils (myeloperoxidase activity) were less in hearts perfused with peroxynitrite than in those without it and systolic functioning was improved (Ronson et. al. 1999). Peroxynitrite infusions at higher levels, however, (10-20x’s higher) had negative effects. A follow up study employing intermediate peroxynitrite levels found it to be cardioprotective at those levels. Bizarrely, extremely high levels of peroxynitrite infusion stopped endothelial dysfunction (increased vasodilation) and thus were protective. This paradoxical finding was believed due to sensitization of the endothelium to the ischemic-reperfusion process (?).

Nitrosothiols Depending on its concentration, then, peroxynitrite can either have positive or negative effects on the vascular endothelium, coronary arteries and/or the heart muscle. How could this incredibly reactive free radical be beneficial to the heart? It turns out that when glutathione or other thiol agents (albumin, cysteine) interact with peroxynitrite they produce an agent called a nitrosothiol which has anti-inflammatory and pro-vasodilatory effects. The cardioprotective effects of nitrosothiols could derive from three processes; they have the ability to (a) terminate the free radical chain reaction process, (b) lower intracellular calcium concentrations by stimulating cGMP production, (c) inhibit leukocyte adhesion through NO release. Glutathione has been shown to increase heart vasodilation; it is now thought its vasodilatory properties are due to its interactions with low levels of ONOO- present during basal (non-pathogenic) conditions (Cheung et. al. 2000). This, of course, suggests a) reduced glutathione levels could impair vascular functioning and b) glutathione supplementation may be helpful in heart failure.

It is possible the negative/positive effects of peroxynitrite depend on the peroxynitrite/thiol balance. As soon as the levels of peroxynitrite exceed the ability of thiols to transform it its negative effects may begin to occur. High rates of peroxynitrite induction during atherosclerosis, ischemia-reperfusion, etc. apparently exceed the bodies ability to transform it into a useful substance.

(A recent paper, however, that suggests nitrosothiols are not as beneficial as they may seem once again illustrates how complex oxidant:antioxidant interactions are (Kitagawa et. al. 2005.While nitrosothiols have beneficial properties they may degrade into substances that are anything but benign. The degree to which this occurs in the body is, however, unclear.)

Summary - Peroxynitrite production plays a significant role in the progression of one of the great killers of our time, atherosclerosis. It also appears to depress heart contractility, impair endothelial functioning and vasodilation, deplete antioxidant levels and contribute to the damage caused by the ischemia/reperfusion process. In non-pathological situations, however, peroxynitrite interacts with glutathione and other thiols to produce substances (nitrosothiols) that appear to have decidedly positive effects on the heart. Whether peroxynitrite has negative or positive effects is at least in part, therefore, dependent upon the whether sufficient thiol agents, in particular glutathione, are present to transform it into not just a benign but a helpful agent.

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Cassina, A., Hodara, R., Sousa, J., Thomson, L., Castro, L., Ischiropoulos, H., Freeman, B. and R. Radi. 2000. Cytochrome c nitration by peroxynitrite. The Journal of Biological Chemistry 275: 21409-21415.

Cheung, P., Wang, W. and R. Schulz. 2000. Glutathione protects against myocardial ischemia-reperfusion injury by detoxifying peroxynitrite. J. Mol. Cell. Cardio. 32: 1669-1678.

Dewald

Hare, J. and J. Stamler. 2005. No/redox disequilibrium in the failing heart and cardiovascular system. The Journal of Clinical Investigation 115: 509-517.

Kitigawa

Pacher, P., Schulz, R., Liaudet, L. and C. Szabo. 2005. Nitrosative stress and pharmacological modulation of heart failure. Trends in Pharmacological Sciences 26, 302-310.

Reiter, C., Teng, R. and J. Beckman. 2000. Superoxide reacts with nitric oxide to nitrate tyrosine at physiological pH via peroxynitrite. The Journal of Biological Chemistry 275: 32460-32466.

Ronson, R., Nakamura, M. and J. Vinten-Johansen. 1999. The cardiovascular effects and implications of peroxynitrite. Cardiovascular Research 44, 47-59.

Rubbo, H and V. O’Donnell. 2005. Nitric oxide, peroxynitrite and lipoxygenase in atherogenesis: mechanistic insights.

Siwik, D., Tzortzis, J., Pimental, D., Chang, D., Pagano, P., Singh, K., Sawyer, D. and W. Colucci. 1999. Circ. Res. 85: 147-153.

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