Jian Zhong Sun

Use of aromatic hydroxylation of phenylalanine to measure production of hydroxyl radicals after myocardial ischemia in vivo. Direct evidence for a pathogenetic role of the hydroxyl radical in myocardial stunning.

A pathogenetic role of .OH in myocardial stunning has been inferred from the protective effects of .OH scavengers and iron chelators. However, conclusive demonstration of the .OH radical hypothesis of myocardial stunning requires direct verification of three major, but still unproven, assumptions: (1) .OH is produced in the stunned myocardium in vivo; (2) antioxidant therapy inhibits .OH production; and (3) such inhibition results in enhanced recovery of contractility (ie, .OH is necessary for the development of myocardial stunning). Since phenylalanine (Phe) reacts with .OH to form the hydroxylated products ortho-, meta-, and para-tyrosines (o-, m-, and p-tyr), we used aromatic hydroxylation of Phe to detect .OH formation in the stunned myocardium. Open-chest dogs undergoing a 15-minute coronary occlusion followed by reperfusion received an intravenous infusion of Phe (54.3 mg/kg for 11.5 minutes beginning 90 seconds before reperfusion); these animals were given either no antioxidant therapy (group I, n = 15), N-2-mercaptopropionyl glycine (MPG) (group II, n = 11), or MPG combined with superoxide dismutase, catalase, and desferrioxamine (group III, n = 12). In addition, group IV (nonischemic control group, n = 6) received Phe but did not undergo coronary occlusion, whereas group V (ischemic control group, n = 16) underwent a 15-minute occlusion but did not receive Phe or antioxidants. The plasma concentrations of tyrosines in the local venous effluent and in the arterial blood were measured with high-performance liquid chromatography. In group I, production of o- and m-tyr, which are specific markers of .OH formation, began during coronary occlusion but increased dramatically immediately after reperfusion, peaking at 1 minute and continuing up to 10 minutes of reperfusion. In group II, the production of o- and m-tyr was markedly decreased throughout the first 10 minutes of reperfusion. In group III, the production of m-tyr was decreased to levels similar to those in group II, whereas the production of o-tyr was almost completely abolished. There was no appreciable production of o- or m-tyr in group IV. Recovery of contractile function (assessed as systolic wall thickening) was increased in group I vs group V. Recovery of function was further enhanced in group II, with only a slight additional improvement in group III.(ABSTRACT TRUNCATED AT 400 WORDS)

Augmentation of endogenous adenosine attenuates myocardial 'stunning' independently of coronary flow or hemodynamic effects.

BackgroundMounting evidence suggests a protective effect of exogenous adenosine in myocardial ischemia and reperfusion. We tested the hypothesis that augmentation of endogenous adenosine levels, achieved by inhibiting adenosine catabolism and washout, is beneficial in postischemic myocardial dysfunction (“stunning”). Methods and ResultsIn phase I of the study, open-chest dogs undergoing a 15-minute coronary artery occlusion and 4 hours of reperfusion received an intracoronary infusion of either saline (controls, n=23) or 6-(4-nitrobenzyl)-mercapto: purine ribonucleoside (NBMPR, a selective nucleoside transport inhibitor) combined with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, a potent adenosine deaminase inhibitor) (EHNA+NBMPR, n=15) starting 15 minutes before coronary occlusion and ending 15 minutes after the initiation of reflow. Regional myocardial function (assessed as systolic wall thickening) was similar in control and treated groups at baseline and during ischemia. After reperfusion, however, the dogs treated with EHNA+NBMPR exhibited a significant improvement in the recovery of function, which was evident as early as 30 minutes after restoration of flow and was sustained throughout the rest of the reperfusion phase. The enhanced recovery effected by EHNA+NBMPR could not be attributed to nonspecific factors such as differences in collateral flow during occlusion, coronary flow after reperfusion, arterial pressure, heart rate, or other hemodynamic variables. In phase II of the study, the myocardial content of adenine nucleotides and nucleosides was measured by high performance liquid chromatography in myocardial biopsies obtained serially from open-chest dogs undergoing the same protocol used in phase I. There were no significant differences between control (n=8) and treated (n=9) dogs with respect to myocardial levels of adenosine triphosphate (ATP) at 30 and 60 minutes after reperfusion, indicating that the beneficial effects of EHNA+NBMPR cannot be ascribed to repletion of ATP stores. Compared with controls, dogs treated with EHNA+NBMPR exhibited a much larger increase in myocardial adenosine (6.07±1.47 vs 1.03±0.16 nmol/mg protein, P<.05) and a much smaller increase in inosine (0.52±0.27 vs 3.04±0.54 nmol/mg protein, P<.05) at the end of ischemia, such that the inosine-to-adenosine ratio noted in controls was completely reversed (=6:1 vs =1:6, respectively). In the treated group, adenosine levels remained markedly increased compared with controls up to 1 hour after reperfusion. ConclusionsThis study demonstrates that (1) administration of an adenosine deaminase inhibitor plus a nucleoside transport blocker is remarkably effective in augmenting myocardial adenosine levels during regional ischemia and subsequent reperfusion in vivo, (2) this augmentation of adenosine results in a significant and sustained attenuation of myocardial stunning, and (3) the attenuation of stunning is not due to ATP repletion or to nonspecific actions on hemodynamic variables or coronary flow. These findings suggest that endogenous adenosine production during ischemia serves as an important pathophysiological mechanism that protects against myocardial stunning. The results also suggest that augmentation of endogenous adenosine (without exogenous adenosine administration) represents an effective therapeutic approach to the alleviation of reversible postischemic dysfunction.

Late preconditioning against stunning is not mediated by increased antioxidant defenses in conscious pigs.

Previous studies in conscious pigs have demonstrated that a sequence of ten 2-min coronary occlusion/2-min reperfusion cycles renders the heart relatively resistant to myocardial stunning 24 h later [late preconditioning (PC) against stunning] by an unknown mechanism. Since oxygen radicals contribute importantly to myocardial stunning and since antioxidant enzymes have been reported to be upregulated 24 h after PC in dogs and rabbits, we tested the hypothesis that late PC against stunning is related to an increase in endogenous antioxidant defenses. Chronically instrumented conscious pigs underwent a sequence of ten 2-min coronary occlusion/2-min reperfusion cycles (preconditioned group, n = 11) or received no intervention (control group, n = 5). Twenty-four hours later, pigs were killed and the myocardial levels of Mn superoxide dismutase (SOD), Cu-Zn SOD, catalase, glutathione (GSH) peroxidase, GSH reductase, GSH, GSH disulfide, α-tocopherol, and ascorbate were measured. There were no differences in any of the enzymatic or nonenzymatic antioxidants between the ischemic and nonischemic regions in the preconditioned group or between the control and the preconditioned group. Thus, when a marked protection against stunning was present (24 h after PC), no alteration in antioxidant defenses was observed. These results indicate that, in conscious pigs, late PC against myocardial stunning is not mediated by increased endogenous antioxidant defenses, thereby refuting one of the major current hypotheses regarding this phenomenon.