reperfused rat myocardium

J Mol Cell Cardiol23

(Supplement

III) (1991)

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IN WV0 PROTEIN OXIDATION IN IS~~ERFUSED RAT MYOCARDJUM Henry Bliss, Micheline Theriault, Department of Physiology, University of Ottawa, Ottawa, Canada, KlH 8M5 Reperfusion of ischemic myocardium can exacerbate injury to the heart. Some of this injury apparently results from the production of oxidants by neutrophils (PMN) which accumulate in the affected tissues. We have shown previously that PMN oxidants can cause the in vitro oxidation of proteins in cardiac tissues. The present investigations were designed to determine the extent of in vivo PMN accumulation and protein oxidation in ischemic and subsequently reperfused rat myocardium. Male rats were anesthetized, the chest wall was opened, and the heart was exteriorized briefly. The left main coronary artery was occluded with a suture, the heart was returned to the thoracic cavity, and the chest wall was closed. After 30 min, the coronary artery was reperfused by removing the suture. At 24 hr of reperfusion, the rats were anesthetized and were infused intravenously with Evans Blue dye to outline ischemic regions in the heart. The ischemic regions (observed in 50% of the rats undergoing this procedure) were excised and analyzed for PMN accumulation (myeloperoxidase (MPO) concentration), and protein oxidation (percent methionine oxidized, met(o)). MPO content increased 3-fold in the ischemic zone vs. non-ischemic right ventricular muscle (71.6k9.3 vs. 23223.9 U/g protein, respectively, n = 12, pcO.01). In hearts which did not contain an ischemic zone, the MPO content in the left ventriclar wall (19.4+2.9 U/g protein, n = 12) was not significantly different from right ventricular muscle. Met(o), as assayedby previously established protocols (PNA8 i30:7160,1983),was also elevated in the ischemic zone vs. non-ischemic right-ventricular muscle (19.7+1.2% vs. 14.8?0.8%, respectively, n = 11, peO.Ol), bsut no significant increase in met(o) was observed in left ventricular muscle of hearts not containing an ischemic zone (14.3x0.9%, n = 9). Our data show for the first time that PMN influx into reperfused myocardium is associate’dwith increased protein oxidation in viva.

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ROLE OF EYDROXYL RADICALS THE PERFUSED RAT HEART

IN EARLY

REPERFUSION

INJURY

AITER

GRADED

ISCHEMIA

IN

Geuzeu Take-, Tomoya Gnodera, MuhammadAshraf. Depaament of Pathology and Laboratory Medicine, University of Cincinnati Medical Center, Cm&m&i, OH 45261. Hydroxyl radicals (OH) were measuredin the coronary effluent collected from the perked rat hearts during reperfusion after global ischemia of 5, 10, 15, 20, 30 or 60 min. Salicyhc acid was used as a happing agent for CIH and its derivative, 2,5dihydroxybenzoic acid @HBA), was quantified using HPLC. The amount of 2,5-DHBA was negligible in the non-ischemic group, but significautly increasedin the hearts which were ischemic for more than 10 min. During the 15 min reperfnsion Period, the peak of 2,5-DHBA was seenwitbin 30 to 60 sec.after the onset of repzfusion. The accumulatedamount of 2,5-DHBA was maximal in the 15 ruin ischemic group (3.14 + 0.66 nmol/l5 midg), but it deer-easedas the ischemic period increased. There was no correlation between the amount of 2,5-DHBA and function recovery (dpldt) and LDH release. The results suggest that a maximum amount of ,OH is formed when reperfusion is initiated after shorter Period of ischemia and they do not appear to causecritical cell dnmaQeat the time of their peak formation.

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HYDROGENPEROXIDE-INDUCED DEPRESSION OF CARDIAC PHOSPHOLIPASE D Jian Dai, Vincenzo Panagia. Division of Cardiovascular Sciences, St. Boniface C.H. Research Centre and Departments of Physiology and Anatomy, University of Manitoba, Winnipeg, R2H 2A6, CANADA The effects of partially reduced forms of oxygen on phosphatidylcholine-specific phospholipase D (PC-PL D) were examined in sarcolemmal (SL) and sarcoplasmic reticular (SR) membranes isolated from rat heart. This phospholipase catalyzes the formation of phosphatidic acid (PA) and seems to be associated with a PA phosphohydrolase for the subsequent production of a PC-derived diacylglycerol pool. Membranal PL D hydrolytic activity was assayed by measuring the formation of labeled PA in the presence of [14C] PC as an exogenous substrate. Preexposure of SL to xanthine-xanthine oxidase (superoxide anion radical-generating system) for 20 min at 37’C did not alter PL D activity. Conversely, preexposure to hydrogen peroxide (H208) resulted in a significant decrease of SL PL D, which was prevented by catalase as well as by the physiological reducing thiol. glutathione. Oxidized glutathione significantly depressed the SL PL D activity. Similar results were obtained when SR PC-PL D was examined under the above conditions. The results indicate that H202 depresses the PC-hydrolyzing PL D activity in SL and SR membranes, and suggest that this enzyme may be controlled by the glutathione redox state of the cardiac cells. (Supported by Heart and Stroke Foundation of Manitoba.) s.41