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Neonatal rat cardiac myocytes were subjected to 5h of hypoxia (PO₂<5 mmHg) in glucose-free, modified Tyrodes solution. Prior to hypoxic exposure, cells were pretreated for 90 min with media alone or containing the trolox analog, U-78517F, at 0.3, 1, or 3 µM. Hypoxic injury was characterized by a significant loss of sarcolemmal integrity [76±12% of the total cell fluorescence (mean±S.E., n=14 cultures)] quantified by nuclear staining with the membrane-impermeant fluorophore propidium iodide. In addition, hypoxia also induced significant decreases in the intracellular content of creatine kinase (43±7%, n=12), lactate dehydrogenase (25±5%, n=8), adenosine 5'-triphosphate (49±12%, n=6), and sulfhydryls (34±4%, n=6). Sarcolemmal integrity was significantly preserved by pretreatment with 1 µM and 3 µM U-78517F which reduced propidium iodine staining below the hypoxic control value by 51±9% (n=12) and 75±4% (n=14, P<0.01 v hypoxic control), respectively. Similar results were obtained in cells pretreated with α-tocopherol suggesting that these two compounds are equipotent at preserving the sarcolemmal integrity of cardiac myocytes exposed to hypoxia. U-78517F at 3 µM also significantly reduced the hypoxia-induced decreases in creatinine kinase and lactate dehydrogenase to 63±13% (n=12, P<0.05) and 50±14% (n=8, P<0.01) of hypoxic control values, respectively. U-78517F pretreatment had no effect on the hypoxia-induced loss of either cellular adenosine 5'-triphosphate or sulfhydryls. U-78517F was also ineffective at reducing lactate dehydrogenase leakage in myocyte cultures which were reoxygenated for 18 h following the 5-h hypoxic exposure. The results demonstrate that U-78517F pretreatment preserves the sarcolemmal integrity of isolated cardiac mycocytes subjected to hypoxic injury in vitro, but the drug is unable to significantly inhibit the limited amount of reoxygenation injury observed in neonatal myocytes. Copyright 1994, 1999 Academic Press

We wished to determine, using a novel specific antagonist of BK2, HOE 140, (a) if the angiotensin-converting enzyme (ACE) inhibitor, ramiprilat, reduces myocardial infarct size in a well-established animal model of ischemia/reperfusion with minimal coronary collater-alization, and (b) if the reduction in myocardial infarct size occurred through a bradykinin-dependent mechanism Saline vehicle, ramiprilat, HOE 140, or ramiprilat plus HOE 140 (n = 6 each group), was administered intravenously (i.v.) in intact animal preparations of experimentally induced acute myocardial ischemia. Anesthetized, open-chest rabbits were instrumented for measurement of systemic hemodynamics and left ventricular pressure (LVP), from which LV + dP/dtmaxwas derived. Animals were subjected to 30-min left main coronary artery occlusion (marginal branch) followed by 2-h reperfusion. Ramiprilat (50 μg/kg) or saline was administered before reperfusion, and rabbits receiving HOE 140 were pretreated before occlusion (1 μg/kg). In separate duration of action experiments (n = 6 each group), the above doses of ramiprilat or HOE 140 had significant vascular antagonism of sufficient duration against serial challenge with angiotensin I (AI) or bradykinin, respectively. After reperfusion, myocardial infarct size (IS) was determined by tetrazolium staining and expressed as a percentage of area at risk (AR). IS/AR was significantly reduced in rabbits that received ramiprilat (20 ± 6, p < 0.05) as compared with those that received saline (41 ± 6), ramiprilat plus HOE 140 (47 ± 2), or HOE 140 alone (43 ± 4, mean ± SEM). AR as a percentage of total LV mass was not different between any of the four treatment groups. Tachycardia was observed during early reperfusion in each group treated with ramiprilat. Sustained hypotension (15–20 mm Hg) and a significant reduction in LV + dP/dtmaxwere evident in all groups throughout reperfusion, except in rabbits treated only with HOE 140, in which hemodynamics were maintained. The effect of ramiprilat to reduce infarct size is abolished by pretreatment with the specific bradykinin-2 antagonist, HOE 140, suggesting that bradykinin plays an important role in the myocardial protective action of ramiprilat.

The objective of this investigation was to examine the ability of two diphosphonate compounds, ethylenediaminetetraacetic acid (EDTA), and D-mannitol to specifically target ischemic/infarcted myocardium. Dichloromethylenephosphonic tetraester or tetraacid, EDTA, or D-mannitol (n=6 each group) was administered iv in intact animal preparations of experimentally induced acute myocardial ischemia. All four of the above compounds were radioactively labeled with 14C. Anesthetized, open-chest rabbits were instrumented for measurement of systemic hemodynamics and left ventricular pressure. Animals were subjected to 30 min of left main coronary artery occlusion (marginal branch) followed by 2 h of reperfusion. All test agents (25 μCi each) were administered 5 min prior to reperfusion via a previously implanted left atrial catheter. Following reperfusion, samples of infarcted myocardium and adjacent ischemic but still viable myocardium were identified for comparison to the normal zone. Among the four drug treatments, diphosphonate acid was highly deposited in infarcted versus normal myocardium (31.5 ± 10.1,* mean ± SEM of individual experimental ratios, infarct dpm/normal dpm) compared to this ratio calculated for diphosphonate ester (1.5 ± 0.2), EDTA (1.4 ± 0.1), and D-mannitol (2.0 ± 0.2, *P < 0.05). Systemic hemodynamics and left ventricular pressures were not significantly altered by ischemia, reperfusion, or administration of any test agents. The results indicate that diphosphonate acid preferentially targets infarcted myocardium.

The angiotensin-converting enzyme inhibitor ramiprilat has been previously demonstrated to protect myocardium from ischemia/reperfusion injury. The objective of these investigations was to examine the roles of bradykinin, angiotensin II, and nitric oxide in the cardioprotective effects of ramiprilat.