RESEARCH

A  Guide to Cardiovascular Issues in Chronic Fatigue Syndrome Part IVa: Superoxide and the Heart by Cort Johnson

Background: free radicals - The organic molecules found in the body are normally stable; that is, their orbitals possess two electrons that spin in opposite directions. Free radicals, on the other hand, are molecules that for short periods of time, contain unpaired electrons. This imbalance causes them, in their search for balance, to either grab onto or give away electrons to other molecules. Some free radicals are so unbalanced that they are able to rip an electron from almost any molecule found nearby. These very volatile free radicals are very short-lived, lasting sometimes only as long as a 1000^th of a second. Others may persist for several minutes. The loss of a single election can potentially alter the shape of protein in such a way that it becomes inactive. Free radicals can poke holes in cellular membranes, degrade and alter DNA and alter enzyme functioning.

Free radicals and the heart - The discovery of superoxide in the late 1960 opened the door on what would eventually become a floodgate of research activity on the role reactive oxygen species (ROS – superoxide, hydrogen peroxide, hydroxyl ion) and reactive nitrogen species (RNS – nitric oxide, peroxynitrite) play in disease and aging. Since that time studies have indicated these oxidative agents play a role in many chronic diseases through their ability to damage lipids (in the membranes surrounding and in cells), DNA and proteins, (Hare and Stamler 2005). The finding that high free radical production occurs in several facets of heart disease including ischemia-reperfusion and atherosclerosis has sparked an enormous amount of research into the oxidative processes underlying heart disease.

Since the peroxynitrite was first observed fifteen years ago much research has indicated it plays a role in several processes (atherosclerosis, ischemia-reperfusion) implicated in heart attack and heart disease. While peroxynitrite appears to play a mostly (but not completely) negative role in the heart, NO plays a mostly (not completely) positive role.

While free radical damage occurs in many chronic diseases it has been less than clear, however, what role free radicals play in the pathogenesis or etiology of these diseases. Because the markers of oxidative damage are quite similar from system to system in different diseases it has been suggested that while increased oxidative stress accompanies many diseases and facilitates the disease process, it plays a role in the initiation of few, if any, of them (Hare and Stamler 2005). Recent studies indicate oxidative stress plays a key role in the progression of heart failure.

Both nitric oxide and peroxynitrite have been proposed to play a critical role in CFS as well. Pall’s compelling theory of sustained peroxynitrite production in CFS (Pall 2000, Pall 2001, Pall and Satterlee 2001) has prompted an examination of the role nitric oxide plays in CFS. Several independent studies suggest increased peroxynitrite production, increased oxidative stress and decreased antioxidant levels in CFS (James et. al 2005, Richards et. al. 2000, Keenoy et. al. 2001) It has become clearer and clearer overtime that oxidative stressors play an important role in CFS.

The Sieverling paper reports Dr. Cheney believes that either mercury or increased hydrogen peroxide levels could inactivate superoxide dismutase (SOD) the main scavenger of superoxide in CFS patients. Dr. Cheney asserts that (a) superoxide mediated mitochondrial membrane damage lies at the heart of the reduced ATP production in CFS, (b) superoxide production is the limiting factor for peroxynitrite formation and (c) peroxynitrite is largely responsible for the damage seen in heart failure. Superoxide, then, is a key element in Dr. Cheney’s theory.

Superoxide and the vascular endothelium – Proper functioning of the vascular endothelium is critical to heart health. When the heart needs more blood nitric oxide (NO) secretion by the endothelial cells lining the blood vessels prompts the smooth muscles to dilate those blood vessels and increase blood flows to the heart. Reduced NO induced vasodilation is in fact a major contributor to the damage caused by reduced blood flows to the heart found during heart failure.

Since heart failure is by definition a disease of diminished circulation, why blood vessel dilation is impaired (instead of enhanced) in heart failure is of the utmost concern. Two causes of impaired vasodilation have been found, both of which appear to involve increased superoxide production. (1) Superoxide or peroxynitrite can impair eNOS activity and (2) superoxide levels can rise so high that it binds with NO before NO can signal the smooth muscles to dilate the blood vessels. In addition if peroxynitrite is produced it may, by oxidizing one of the co-factors (BH4) for NO production, prompt NOS to produce superoxide instead of NO.

The Superoxide/Angiotensin II Connection in Heart Failure – Superoxide does not only disrupt endothelial vasodilation, it also teams up with angiotensin II (Ang II) to induce heart remodeling (increased heart stiffness, hypertrophy (heart enlargement).

One of the really bad players in heart failure, Ang II is a peptide produced by the kidney that appears to become involved in heart failure through its attempts to maintain circulation in face of reduced cardiac output. Ang II does this by prompting aldosterone to increase blood volume and by increasing the sympathetic tone. By causing the blood vessels to constrict increased SNS activity does increase tissue perfusion to the central organs such as the heart and the brain but the increased resistance in the circulation places an additional load on the heart. Paradoxically increased plasma norepinephrine (NE) levels in heart failure are accompanied by reduced norepinephrine levels in the heart cells themselves. It appears the high plasma NE levels compensate for a time for the reduced heart cell NE levels but are ultimately insufficient and heart contraction is reduced. High NE levels over time can also damage heart cells. Why heart cells become unable to synthesize NE in heart failure is unclear.

As noted earlier Ang II is released into the general circulation by the kidney. From there it binds to SNS receptors on the circumventricular organs (CVO’s) in the brain found outside of the blood brain barrier. The CVO’s appear are a way for the brain to communicate with the outside world. i.e. the body, by interacting with signaling elements found in the general circulation (Zimmerman and Davisson 2004)

The CVO’s are also the center of sympathetic nervous system activity in the brain. That chronic activation of the SNS neurons in the CVO’s after a heart attack precedes heart failure by several weeks suggests this process plays a major role in the pathogenesis of heart failure. Not surprisingly a great deal of research is being devoted to teasing out just how Ang II activates these neurons.

A recent study suggests superoxide plays a key role in that activation (Lindley et. al. 2004). This study found that both cardiac SNS activity and a marker of neuronal activation (the fos gene) were reduced when either type of SOD (CuZnSOD - cytosolic SOD, MnSOD - mitochondrial SOD), the main superoxide scavenger, were injected into the brains of rodents (Lindley et. al. 2004).

Just how superoxide assists Ang II in jump starting SNS activity is not entirely clear but it appears to involve the signaling system (Lindley et. al. 2004, Zimmerman and Davisson 2004). Its now becoming apparent that reactive oxygen species such as superoxide play a role in a wide array of Ang II mediated effects including the inflammation of the vascular endothelium, impairment of endothelial relaxation and hypertrophy of the heart.

But what causes the increased superoxide production in this area of the brain? The ability of Ang II and aldosterone to trigger superoxide production by NAD(P)H oxidase (remember that name) suggests NAD(P)H oxidase activity is critical to the increased SNS activity in heart failure. (By stimulating TNF-a production aldosterone could also increase reactive oxygen species (ROS) such as superoxide in the CVO). A recent study was able to abolish (renal) sympathetic activity by inhibiting NAD(P)H oxidase (Gao et. al. 2004). Alternately, since NO inhibits neuronal activity, superoxide could promote SNS activity simply by scavenging an important neuronal inhibitor.

Speculation - A Connection to CFS? – While this system is a critical component of the progression of heart failure preliminary reports indicate it is not operating in CFS. It appears that postural tachycardia (POTS) patients and by extension some CFS patients do not have increased angiotensin levels – indeed they appear to have reduced activation of the renin-aldosterone-angiotensin (RAA) system (Raj et. al. 2005). If heart failure does turn out to be a prominent feature of CFS, one wonders though if deactivation of this key system is partially responsible for their lack of progression?

Superoxide and heart cell activity – Superoxide does not just affect the endothelium, it can also affect the heart cells themselves. One study that examined the effects of superoxide dismutase (SOD) inhibition on heart cells found that rats engineered to have increased superoxide production did not, as Dr. Cheney suggests, develop mitochondrial membrane damage (Siwik et.al. 1999). Nor were there indications of the increased cell death usually associated with increased free radical production. Instead these cells exhibited increased rates of apoptosis (cell suicide) and cardiac cell enlargement (hypertrophy), a commonly occurring feature in heart failure (Mungrue et. al. 2002). Preliminary reports from the 2005 Cheney presentation in Dallas suggest, however, that Dr. Cheney is not commonly finding hypertrophy in CFS patients

Intriguingly the authors noted that by chelating (removing) the copper ion in CuZnSOD, the particular SOD antagonist used in this study could also lead to decreased heart copper levels. Rats feed a copper deficient diet developed a similar pathology (dilated cardiomyopathy, hypertrophy) to that noted in the Siwik study.

Since mitochondrial superoxide production is an important feature of Dr. Cheney’s theory the ways superoxide is produced in the heart is examined in some detail. A dearth of studies on the effects of mitochondrial superoxide production in heart failure as well as the scanty mention given it by most reviews of the subject suggest it is not a major concern for the heart research establishment as a whole. It is perhaps not surprising that this aspect of Dr. Cheney’s theory would apply only to CFS patients.

While most reviews do acknowledge mitochondrial oxidases as potential sources of superoxide, it is three other enzymes, NAD(P)H oxidase, xanthine oxidase and nitric oxide synthase, that get the lions share of attention with regard to superoxide production in heart failure. Oxidase’s add electrons to oxygen, i.e. they turn oxygen into superoxide. Oxygen is a key element in oxidation reactions because with two unpaired electrons in its outer shell it can easily accept other electrons.

NAD(P)H oxidase - Found in both endothelial cells and smooth muscles in the vasculature, as well as the heart muscle itself, NAD(P)H oxidase is the superoxide producer par excellence during heart failure. While some hormones and cytokines can activate NAD(P)H oxidase it is primarily activated in heart failure by Ang II.

NAD(P)H oxidase activity is increased in the two of the major processes causing heart damage, ischemia/reperfusion and atherosclerosis (Hare and Stamler 2005). Ischemia reperfusion occurs when cells are first deprived of blood (ischemia) and then perfused with it (reperfusion).The ischemia/reperfusion process produces large amounts of free radicals. NAD(P)H oxidase also plays a role in promoting the conversion of xanthine dehydrogenase, a non reactive oxygen species producing enzyme, into xanthine oxidase – an enzyme that pumps out superoxide. NAD(P)H oxidase also plays a role in NOS activity.

Xanthine oxidase (XO) - The xanthine degrading enzymes exist in two forms; the benign form, xanthine dehydrogenase, which breaks down xanthines without creating free radicals and the destructive form, xanthine oxidase, which produces superoxide as it degrades xanthines. Both xanthines catalyze the reaction O2, and H2O to produce urate.

Xanthines are a consequence of ADP buildup during exercise or ischemia or through high intracellular calcium levels. The body uses ADP during exercise to regenerate ATP but as it does so it leaves behind a substance, inosine monophosphate (IMP) that cannot pass through the cellular membranes. Inosine degredation leaves behind small amounts of inosine that are eventually converted to xanthine (and finally urate).

Upregulated in the heart and blood vessels in heart failure, XO produces either superoxide or hydrogen peroxide as by-products of xanthine metabolism.

Could the low uric acid levels in CFS patients be due to their low activity levels? Do the low uric acid levels reported by Dr.Cheney indicate that ischemia and XO activity are low in CFS.

Nitric oxide synthase - Another important source of superoxide may be NOS itself. The NOS’s use electrons provided by NAD(P)H oxidase (NAD(P)H oxidase again!) to transform oxygen and arginine into NO and L-citrulline. When essential co-factors such as L-arginine and BH4 are missing, however, the NOS’s produce superoxide instead of NO (Dixon et. al. 2003, Kalinowski and Malinski 2004).

Superoxide production by NOS is believed to occur in several cardiovascular diseases including hypertension, diabetes, hypercholesterolemia and myocardial infarction (heart attack). Interestingly given the concerns of NO overproduction in CFS, L-arginine supplementation has been shown to increase (i.e. restore) NO levels and reduce superoxide levels in hypertension, hypercholesterolemia and diabetes. Thus while NO can under certain conditions contribute to peroxynitrite formation proper NO functioning can be important in preventing peroxynitrite production as well.

Hemoglobin - is the biggest reservoir of both oxygen and NO in the body and may play an important role in vascular dilation through NO release. Because heme, the oxygen carrying molecule in hemoglobin, releases superoxide when it is desaturated (i.e. not filled with oxygen), hemoglobin can be an important contributor to ROS production in conditions like heart failure with low oxygen levels.

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