The xanthine oxidase inhibitor oxypurinol reduces cancer cachexia-induced cardiomyopathy

Background

Cachexia is a common complication of cancer and may be responsible for 22% of all cancer-related deaths. The exact cause of death in cancer cachexia patients is unknown. Recently, atrophy of the heart has been described in cancer cachexia animal models, which resulted in impaired cardiac function and is likely to contribute to mortality. In cancer patients hyperuricaemia independent of tumour lysis syndrome is often associated with a worse prognosis. Xanthine oxidase (XO) metabolizes purines to uric acid and its inhibition has been shown to improve clinical outcome in patients with chronic heart failure.

Methods

The rat Yoshida AH-130 hepatoma cancer cachexia model was used in this study. Rats were treated with 4 or 40 mg/kg/d oxypurinol or placebo starting one day after tumour-inoculation for maximal 15 days. Cardiac function was analyzed by echocardiography on day 11.

Results

Here we show that inhibition of XO by oxypurinol significantly reduces wasting of the heart and preserves cardiac function. LVEF was higher in tumour-bearing rats treated with 4 mg/kg/d (61 ± 4%) or 40 mg/kg/d (64 ± 5%) oxypurinol vs placebo (51 ± 3%, both p < 0.05). Fractional shortening was improved by 4 mg/kg/d (43 ± 3%) oxypurinol vs placebo (30 ± 2, p < 0.05), while 40 mg/kg/d oxypurinol (41 ± 5%) did not reach statistical significance. Cardiac output was increased in the 4 mg/kg/d dose only (71 ± 11 mL/min vs placebo 38 ± 4 mL/min, p < 0.01).

Conclusion

Inhibition of XO with oxypurinol has beneficial effects on cardiac mass and function in a rat model of severe cancer cachexia, suggesting that XO might be a viable drug target in cancer cachexia.     

Absence of detectable xanthine oxidase in human myocardium

The enzyme xanthine oxidase has been implicated as a generator of toxic oxygen metabolites that contribute to ischemic injury. Because substantial species variability has been demonstrated and because there are minimal human data available, the relevance of xanthine oxidase to human heart damage has been in doubt. We report the absence of xanthine oxidase activity in nine human heart biopsy specimens obtained during cardiac surgery, and in two larger samples obtained during heart transplantation. A sensitive radiochemical assay was used to assess enzyme activity. Our findings imply that oxygen free radicals generated by xanthine oxidase are not relevant in terms of human myocardial injury.     

The xanthine oxidase inhibitor oxypurinol does not limit infarct size in a canine model of 40 minutes of ischemia with reperfusion

Free radicals such as superoxide (.O2-) produced by xanthine oxidase might cause cell death during reperfusion after myocardial ischemia. The effect of the xanthine oxidase inhibitor allopurinol on infarct size in ischemia-reperfusion models has been variable, possibly because of differences in treatment duration. Adequate inhibition of xanthine oxidase may require a sufficient pretreatment period to permit conversion of allopurinol to oxypurinol, the actual inhibitor of superoxide production. To test more definitively whether xanthine oxidase-derived free radicals cause cell death during reperfusion, the effect of oxypurinol on infarct size was evaluated in an ischemia-reperfusion model. Open chest dogs underwent 40 min of circumflex coronary artery occlusion followed by reperfusion for 4 days. Twelve dogs were treated with oxypurinol (10 mg/kg body weight intravenously 10 min before occlusion and 10 mg/kg intravenously 10 min before reperfusion) and 11 control dogs received drug vehicle alone (pH 10 normal saline solution). Nine control dogs from a concurrent study also were included. Infarct size was measured histologically and analyzed with respect to its major baseline predictors, including anatomic area at risk and collateral blood flow (measured with radioactive microspheres). Infarct size as a percent of the area at risk averaged 23.8 +/- 2.7% (mean +/- SEM) in the oxypurinol group (n = 10) and 23.1 +/- 4.2% in the control group (n = 17) (p = NS). Collateral blood flow to the inner two thirds of the ischemic wall averaged 0.08 +/- 0.01 ml/min per g in the oxypurinol group and 0.09 +/- 0.02 ml/min per g in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered myocardial contractility and energetics in hypertrophied myocardium

Alterations of myocardial contractility and energetics were examined in cardiac hypertrophy induced by different types of cardiac overload. Myocardial contractility was estimated by isometric contraction of isolated left ventricular papillary muscles. Myocardial energetics were assessed from ventricular myosin isoenzyme patterns obtained by pyrophosphate gel electrophoresis. Cardiac hypertrophy was induced by endurance swim-training and sustained pressure-overload by abdominal aortic constriction or volume-overload created by an arteriovenous shunt. In swim-trained hypertrophied myocardium, isometric developed tension (T) and dT/dtmax showed a tendency to increase and response of dT/dtmax to isoproterenol increased significantly as compared with sedentary rats. Training shifted the left ventricular myosin isoenzyme pattern toward VM-1, which has the highest ATPase activity. In pressure- or volume-overloaded myocardium, dT/dtmax decreased significantly and mechanical response to isoproterenol also decreased (or tended to decrease in volume-overloaded hearts) as compared with the respective sham-operated controls. In pressure- or volume-overloaded hearts, left ventricular myosin isoenzyme pattern shifted toward VM-3, which has the lowest ATPase activity. These results indicate that alterations in myocardial contractility, mechanical catecholamine responsiveness and myocardial energetics in hypertrophied myocardium do not always display the same trend, but are greatly influenced by the causes or duration of cardiac overload.     

Abnormalities of calcium metabolism and myocardial contractility depression in the failing heart

Heart failure (HF) is characterized by molecular and cellular defects which jointly contribute to decreased cardiac pump function. During the development of the initial cardiac damage which leads to HF, adaptive responses activate physiological countermeasures to overcome depressed cardiac function and to maintain blood supply to vital organs in demand of nutrients. However, during the chronic course of most HF syndromes, these compensatory mechanisms are sustained beyond months and contribute to progressive maladaptive remodeling of the heart which is associated with a worse outcome. Of pathophysiological significance are mechanisms which directly control cardiac contractile function including ion- and receptor-mediated intracellular signaling pathways. Importantly, signaling cascades of stress adaptation such as intracellular calcium (Ca²⁺) and 3′-5′-cyclic adenosine monophosphate (cAMP) become dysregulated in HF directly contributing to adverse cardiac remodeling and depression of systolic and diastolic function. Here, we provide an update about Ca²⁺ and cAMP dependent signaling changes in HF, how these changes affect cardiac function, and novel therapeutic strategies which directly address the signaling defects.     

Cross-bridge dynamics in human ventricular myocardium. Regulation of contractility in the failing heart.

BACKGROUND. To investigate whether altered cross-bridge kinetics contribute to the contractile abnormalities observed in heart failure, we determined the mechanical properties of cardiac muscles from control and myopathic hearts. METHODS AND RESULTS. Muscle fibers from normal (n = 5) and dilated cardiomyopathy (n = 6) hearts were obtained and chemically skinned with saponin. The muscles were then maximally activated at saturating calcium concentrations. Unloaded shortening velocities (V0) were determined in both groups. V0 in control was 0.72 +/- 0.09 Lmax/sec, whereas in myopathic muscles, V0 was 0.41 +/- 0.06 Lmax/sec at 22 degrees C. The muscles were also sinusoidally oscillated at frequencies ranging between 0.01 and 100 Hz. The dynamic stiffness of the muscles was calculated from the ratio of force response amplitude to length oscillation amplitude. At low frequencies (< 0.2 Hz) the stiffness was constant but was larger in myopathic muscles. In the range of 0.2-1 Hz, there was a drop in the magnitude of dynamic stiffness to approximately one quarter of the low-frequency baseline. This range reflects cross-bridge turnover kinetics. In control muscles, the frequency of minimum stiffness was 0.78 +/- 0.06 Hz, whereas it was 0.42 +/- 0.07 Hz in myopathic muscles. At higher frequencies, the dynamic stiffness increased and reached a plateau at 30 Hz. There were no differences in the plateau reached between control and myopathic muscles. CONCLUSIONS. Because myopathic hearts have a markedly diminished energy reserve, the slowing of the cross-bridge cycling rate plays an important adaptational role in the observed contractility changes in human heart failure. Although the potential to generate maximal Ca(2+)-activated force is similar in normal and myopathic hearts, alterations in contractile protein composition could explain the diminished cross-bridge cycling rate in failing hearts.     

Microvascular display of xanthine oxidase and NADPH oxidase in the spontaneously hypertensive rat

OBJECTIVE: Oxygen free radical production in hypertension may be associated with elevated arteriolar tone and organ injury. Previous results suggest an enhanced level of oxygen free radical formation in microvascular endothelium and in circulating neutrophils associated with xanthine oxidase activity in the spontaneously hypertensive rats (SHR) compared with their normotensive controls, the Wistar Kyoto rats (WKY). The aim of this study was to gain more detailed understanding of where oxidative enzymes are located in the microcirculation. METHODS: An approach was developed to delineate the cellular distribution of two selected oxidative enzymes, xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH) dependent oxidase (protein 67-kDa fraction). Immunolabeling with peroxidase substrate was utilized, which permits full delineation of the primary antibody in all microvascular structures of the mesentery. RESULTS: Xanthine oxidase is present in the endothelium of all segments of the microcirculation, in mast cells, and in parenchymal cells of the mesentery. NADPH oxidase can be detected in the endothelium, leukocytes, and mast cells and with lower levels in parenchymal cells. The mesentery of WKY and SHR has similar enzyme distributions with enhancements on the arteriolar and venular side of the microcirculation that coincide with the sites of enhanced free radical production recently reported. Immune label measurements under standardized conditions indicate that both enzymes are significantly enhanced in the SHR. Adrenalectomy, which serves to reduce the blood pressure and free radical production of the SHR to normotensive levels, leads to a reduction of NADPH and xanthine oxidase to normotensive levels, while supplementation of adrenalectomized SHR with dexamethasone significantly increases the oxidase expression in several parts of the microcirculation to levels above the WKY rats. CONCLUSION: The results indicate that enhanced expression of NADPH and xanthine oxidase in the SHR depends on an adrenal pathway that is detectable in the arteriolar and venular network at high and low pressure regions of the circulation.     

Sex differences in myocardial contractility in the rat

Summary We examined the intrinsic contractile performance of papillary muscles removed from the left ventricle of male and female Wistar rats. Muscles were studied isometrically and isotonically, stimulated at 0.1 Hz, perfused with Tyrode's solution having an external calcium concentration=2.4 and maintained at 30°C. In addition, we examined muscle response to changes in external calcium, added norepinephrine or verapamil and alterations in contraction frequency. No significant change in peak isometric development tension was observed between male and female preparations. However, muscles from male rats showed a significantly greater isometric time-to-peak tension and time to 1/2 relaxation with a depression of both the maximum rate of tension rise and maximum rate of tension decay. Isotonically, although peak shortening showed no difference between male and female preparations, the maximum velocities of shortening and relaxation were significantly depressed in muscles from male rats. Muscles from male animals also displayed significant prolongation of the time-to-peak shortening and time-to-peak velocity of shortening. These differences in papillary muscle performance were found over a wide range of muscle lengths, stimulus frequencies and bath concentrations of calcium, norepinephrine and verapamil. Thus differences in intrinsic contractile performance between papillary muscle from male and female rats have been characterized.Supported in part by NIH grants #HL 21933-02, #HL 07071-05 and a Herman Raucher Investigatorship Award of the New York Heart Association to Dr. J. M. Capasso.     

Influence of oxygen radicals generated by xanthine oxidase in the isolated perfused rat heart

To investigate the cardiac effects of enzymatically generated oxygen radicals isolated Langendorff rat heart preparations were perfused with hypoxanthine (0.96 mmol X litre-1) plus xanthine oxidase (0.025 U X ml-1). Oxygen radicals produced an immediate increase in coronary flow. After 10 min a pronounced reduction in contractile performance, as well as in concentrations of high energy phosphates, was seen. Electron microscopical examination showed damage with cellular oedema as a prominent finding. These effects were all effectively reversed by the specific enzymes, superoxide dismutase and catalase, proving that they were due to oxygen radicals.     

Intracellular calcium and myocardial contractility. IV. Distribution of calcium in the failing heart

Myocardial calcium and its distribution in intracellular fractions were measured in chronic heart failure occurring in rabbits with experimental aortic insufficiency. In these animals heart failure was characterized by the presence of pulmonary congestion and edema and the development of diminished myocardial contractility. Hearts were analyzed for calcium contents either after perfusion in vitro or immediately after removal from the animals. Total myocardial calcium was similar in control and failing hearts after in vitro perfusion and was only minimally increased in those failing hearts which were not perfused. However, calcium was elevated in intracellular fractions prepared from failing hearts. An increase of calcium was found in mitochondria of hearts perfused in vitro from a control of 10.9 ± 0.5 to 16.3 ± 1.6 nmol/mg. Similar increments in mitochondrial calcium were seen in the failing hearts which were not perfused but analyzed immediately after removal from the animal. Changes of calcium in the microsomal fractions were more difficult to interpret because of the greater heterogeneity of these fractions.     

Imbalance between xanthine oxidase and nitric oxide synthase signaling pathways underlies mechanoenergetic uncoupling in the failing heart

Inhibition of xanthine oxidase (XO) in failing hearts improves cardiac efficiency by an unknown mechanism. We hypothesized that this energetic effect is due to reduced oxidative stress and critically depends on nitric oxide synthase (NOS) activity, reflecting a balance between generation of nitric oxide (NO) and reactive oxygen species. In dogs with pacing-induced heart failure (HF), ascorbate (1000 mg) mimicked the beneficial energetic effects of allopurinol, increasing both contractility and efficiency, suggesting an antioxidant mechanism. Allopurinol had no additive effect beyond that of ascorbate. Crosstalk between XO and NOS signaling was assessed. NOS inhibition with N(G)-monomethyl-L-arginine (L-NMMA; 20 mg/kg) had no effect on basal contractility or efficiency in HF, but prevented the +26.2+/-3.5% and +66.5+/-17% enhancements of contractility and efficiency, respectively, observed with allopurinol alone. Similarly, improvements in contractility and energetics due to ascorbate were also inhibited by L-NMMA. Because of the observed NOS-XO crosstalk, we predicted that in normal hearts NOS inhibition would uncover a depression of energetics caused by XO activity. In normal conscious dogs, L-NMMA increased myocardial oxygen consumption (MVO2) while lowering left ventricular external work, reducing efficiency by 31.1+/-3.8% (P<0.005). Lowered efficiency was reversed by XO inhibition (allopurinol, 200 mg) or by ascorbate without affecting cardiac load or systemic hemodynamics. Single-cell immunofluorescence detected XO protein in cardiac myocytes that was enhanced in HF, consistent with autocrine signaling. These data show that both NOS and XO signaling systems participate in the regulation of myocardial mechanical efficiency and that upregulation of XO relative to NOS contributes to mechanoenergetic uncoupling in heart failure.     

Inotropic therapy of the failing myocardium

The clinical syndrome of congestive heart failure remains a therapeutic dilemma and challenge for the physician in 1992. This is a disease process that appears to be increasing in frequency and continues to carry an unacceptably high mortality rate. For years it has been well recognized that the combination of digoxin, Lasix and vasodilator therapy improved symptoms in these patients and decreased hospitalization, but did not increase survival. It was not until 1986 that the combination of digoxin, Lasix, Isordil, and hydralazine was shown to increase survival. Further significant improvement in quality of life and survival has recently been established in three large clinical trials, and it is now safe to say that the standard of care for symptomatic congestive heart failure in 1992 is digoxin, furosemide, and an ACE inhibitor, with the survival trials favoring the ACE inhibitor enalapril. The IV inotropic drug dobutamine remains the mainstay of pharmacological therapy for the treatment of severely refractory heart failure. Unfortunately, the phosphodiesterase inhibitors—amrinone, milrinone, and enoximone—have demonstrated unacceptable clinical side effects and have been withdrawn from further clinical study. In spite of these promising developments, the mortality and morbidity of congestive heart failure remains unacceptably high, and continued investigation in the new fields of pharmacology and the pathophysiology of congestive heart failure still must be aggressively pursued.     

Oxygen radicals generated by the enzyme xanthine oxidase lyse rat pancreatic islet cells in vitro

Summary The endothelium-associated enzyme xanthine oxidase is known to generate reactive oxygen intermediates which may damage the surrounding tissue. We investigated whether reactive oxygen intermediates released by xanthine oxidase exert a toxic effect on isolated rat islet cells. The xanthine oxidase (25 mU/ml)/hypoxanthine (0.5 mmol/1) system released reactive oxygen intermediates in vitro as detected by luminol in a chemiluminescence analysing system. The addition of nicotinamide inhibited the release of reactive oxygen intermediates in a dose-dependent manner (50 % inhibition at 20 mmol/1). Exposure of islet cells to enzyme generated reactive oxygen intermediates caused lysis of 39% of the cells within 15 h. Monitoring the mitochondrial function of islet cells by the conversion of tetrazolium bromide to its formazan product revealed a significant reduction of the respiratory activity down to 51 % of that of the controls by 30 min after the initiation of the xanthine oxidase reaction. Mitochondrial dysfunction preceded plasma membrane damage. The addition of nicotinamide, a radical scavenger and inhibitor of the DNA repair enzyme poly(ADP-ribose) synthetase protected the islet cells from lysis and partially preserved their mitochondrial activity in the presence of reactive oxygen intermediates. We conclude that activation of the endothelial enzyme xanthine oxidase, known to be induced by mediators of immune cells or by episodes of ischaemia and reperfusion causes islet cell damage with subsequent cell death in early phases of pancreatic islet cell destruction.     

Infarct size limitation by the xanthine oxidase inhibitor, allopurinol, in closed-chest dogs with small infarcts

The present study was designed to evaluate the ability of allopurinol to limit infarct size following permanent coronary occlusion in the greyhound. Coronary occlusion was produced by injecting 2.5 mm plastic beads into the coronary artery of the closed chest dog. Non-perfused myocardium, the area at risk, was visualised by autoradiography of 141Cerium labelled microspheres which were infused immediately following coronary embolization. The treated dogs (n = 12) received 400 mg of allopurinol orally one day before surgery. A 25 mg . kg-1 bolus was administered (iv) immediately before occlusion, and repeated every 8 h. 11 dogs served as controls. After 24 h, the dogs were killed and the hearts were sliced into 5.0 mm transverse sections. The infarcted myocardium was visualised by triphenyl tetrazolium chloride staining. The percentage of the risk zone which evolved to infarct was calculated. This percentage was 18.1 +/- 3.95% in the allopurinol group vs 58.4 +/- 2.81% in the control group (p less than 0.001). We conclude that allopurinol is a potent drug for the limitation of infarct size in the dog with permanent coronary occlusion.     

Effect of trimecaine on myocardial contractility and the rhythmoinotropic properties of the myocardium in hypoxia

In the papillary muscles from 14 adult rats and myocardial specimens from 14 newborn ones, trimecaine in doses of 1.10(-6) and 5.10(-6) g/ml produced a dose-dependent negative inotropic effect, reversed the rhythmoinotropic correlation into a negative one and minimized the efficiency of postextrasystolic potentiation. Trimecaine in the former dose reduced hypoxic damage of myocardial specimens in the both groups as reflected by a smaller amplitude of hypoxic contracture and better contractility recovery following a hour hypoxia. It is suggested that reduction in hypoxic contracture and damage to the myocardium occur in the presence of trimecaine from limited Ca2+ entry into myocytes via the Na-Ca turnover mechanism.     

Gastric mucosal injury in the rat. Role of iron and xanthine oxidase

Recent studies have implicated oxygen free radicals in ischemia-reperfusion injury to the gastric mucosa. The aims of the present study were to test the hypothesis that the enzyme xanthine oxidase is the source of the oxygen radicals in the ischemic stomach and determine the importance of the iron-catalyzed Haber-Weiss reaction in generating the cytotoxic oxygen radicals. Gastric mucosal clearance of 51Cr-labeled red blood cells was measured during a 30-min control period, a 30-min ischemic period (hemorrhage to 25 mmHg arterial pressure), and a 60-80-min reperfusion period (reinfusion of shed blood). In untreated (control) rats, a dramatic rise (100-fold) in the leakage of 51Cr-labeled red blood cells into the gastric lumen was observed only during the reperfusion period. After the reperfusion period, gastric mucosal damage was further assessed using gross lesion area and histology. Rats were placed on a sodium tungstate diet (to inactivate xanthine oxidase), or treated with either deferoxamine (an iron chelating agent) or superoxide dismutase (a superoxide scavenger). All three interventions substantially reduced 51Cr-labeled red blood cell clearance and gross lesion area relative to untreated rats. However, tissue injury assessed histologically was similar in both treated and untreated animals. The results of this study support the hypothesis that oxygen free radicals mediate the hemorrhagic shock-induced extravasation of red blood cells. The data also indicate that xanthine oxidase is the source of the oxy-radicals and that the iron-catalyzed Haber-Weiss reaction is largely responsible for hydroxyl radical generation in this model.     

Inhibition of gonadotropin action and progesterone synthesis by xanthine oxidase in rat luteal cells.

Hydrogen peroxide produces marked antigonadotropic and lytic actions in luteal cells, but the effects of superoxide, the archetypal oxygen radical, are unknown. Xanthine oxidase generates superoxide, and the activity of this enzyme, and purine substrate, are increased under ischemia, such as that seen at luteal regression. We therefore examined the actions of xanthine oxidase on luteal cells to assess the effects of this enzyme and the superoxide anion on luteal function. Xanthine oxidase, in the presence of hypoxanthine (50 microM), produced marked inhibition of LH-sensitive cAMP and progesterone production with complete inhibition at 25 mU/ml and half-maximal inhibition at about 5 mU/ml. These antigonadotropic actions of xanthine oxidase were rapid with maximal effects within 5 min, followed several minutes later by substantial depletion of ATP. Heat, superoxide dismutase, and catalase or catalase alone abolished the actions of xanthine oxidase. While depletion of ATP by xanthine oxidase was prevented by 3-amino-benzamide, an inhibitor of DNA repair, inhibition of cAMP and progesterone production was still evident. Xanthine oxidase also inhibited progesterone synthesis stimulated by 8-bromo-cAMP. Isobutylmethylxanthine, a cAMP phosphodiesterase inhibitor, did not reverse the inhibition of cAMP accumulation by xanthine oxidase, and the enzyme had no effect on LH receptor binding activity. Since catalase reversed the effects of xanthine oxidase, we conclude that superoxide was rapidly dismuted to hydrogen peroxide and mediated the antigonadotropic and antisteroidogenic actions of xanthine oxidase in luteal cells. The sensitivity of luteal cells to xanthine oxidase raises the possibility that this enzyme may serve as a significant source of hydrogen peroxide in the corpus luteum.