Larry R. Bush

Effects of Diltiazem on Extent of Ultimate Myocardial Injury Resulting from Temporary Coronary Artery Occlusion in Dogs

Summary The calcium antagonist, diltiazem, was evaluated for its ability to reduce the extent of myocardial injury resulting from 90 min of left circumflex (LCX) coronary artery occlusion in anesthetized dogs. Administration of diltiazem (0.75 mg/kg over 10 min, followed by 600 μg/kg/h for 4 h) was initiated 30 min prior to LCX occlusion. Regional myocardial blood flow (RMBF) was measured with radioactive microspheres 30 min after LCX occlusion, and at 45 min and 24 h after reperfusion. At 24 h, after obtaining hemodynamic and RMBF measurements, excised hearts were processed by perfusion staining to determine the percent of left ventricle (LV) perfused by LCX (area at risk) and infarct size, with triphenyltetrazolium chloride. Infarct size, expressed as a percentage of the area at risk, was significantly lower in the diltiazem-treated group compared to the control group (27 ± 4 vs. 42 ± 5%, respectively). The area at risk, expressed as a percentage of left ventricular mass, was similar in both groups [41 ± 2 and 44 ± 3% (area at risk—LV)]. In addition, the marked elevation of tissue Ca2+ content in noninfarcted and infarcted myocardium within the area at risk (18 ± 2 and 42 ± 8 μmol Ca2+/g) in control animals was attenuated by diltiazem (6 ± 3 and 18 ± 8 μmol Ca2+/g). Diltiazem did not increase blood flow to ischemic myocardium during LCX occlusion. However, reflow to the inner layers of formerly ischemic myocardium during reperfusion was significantly greater in diltiazem-treated dogs. Both arterial blood pressure and heart rate were significantly lower in the diltiazem-treated group. In addition, mortality (1 vs. 4) and occurrence of ventricular arrhythmias during reperfusion were lower in diltiazem-treated dogs. The data suggest that diltiazem reduces myocardial ischemic injury by lowering myocardial oxygen demands indirectly via favorable hemodynamic alterations, and directly by limiting transmembrane Ca2+ fluxes during ischemia and reperfusion.

Cardioprotective effects of ibuprofen in experimental regional and global myocardial ischemia

Summary The effects of the nonsteroidal antiinflammatory agent, ibuprofen, were evaluated in two animal models of myocardial ischemia: regional myocardial ischemia produced by temporary occlusion followed by reperfusion of the canine left circumflex coronary artery (LCX), and global ischemia in the isolated, blood-perfused feline heart. In control dogs (n = 12), 60 min of LCX occlusion followed by 24 h of reperfusion resulted in 51 ± 4% of the area at risk, and 20 ± 2% of the total left ventricle becoming infarcted. The average infarct size of the group of dogs receiving oral ibuprofen (n = 12: dose of 12.5 mg/kg, q4h) was 33 ± 5% of the area at risk, and 12 ± 2% of the total left ventricle, a reduction in infarct size as compared to the controls of 35% (p > 0.02) and 39% (p < 0.02), respectively. The mechanism by which ibuprofen provided these beneficial effects could not be attributed to a reduction in myocardial oxygen consumption (M&OV0312;O2), estimated by rate–pressure products, or redistribution of regional myocardial blood flow, assessed by radioactive microspheres. The direct effect of ibuprofen on ischemic myocardium was assessed in an isolated, blood-perfused cat heart model of global ischemia. After 60 min of normothermic global ischemia, left ventricular pulse pressure of control hearts (n = 8) at 5 and 10 mm Hg end-diastolic pressure was reduced by 56 ± 97% and 60 ± 12%, respectively, relative to preischemic values. Administration of ibuprofen (12.5 mg/kg, i.v., to the blood donor cat, n = 6) did not significantly prevent the ischemia-induced deterioration of left ventricular function. Normothermic ischemia for 60 min resulted in a significant reduction in M&OV0312;O2 in both the control and ibuprofen-treated hearts. However, after 1 h of reperfusion, the average M&OV0312;O2 for the ibuprofen-treated hearts was 96% greater (4.5 ± 0.4 versus 2.3 ± 0.5 ml/min/100 g) than the average M&OV0312;O2 of the control hearts. While conventional thinking contends that myocardial infarct size may be limited by a reduction in M&OV0312;O2 or a redistribution of blood flow, the data from this study indicate that these two mechanisms are not relevant in the case of ibuprofen.

Time-dependent changes in canine cardiac mitochondrial function and ultrastructure resulting from coronary occlusion and reperfusion

SummaryTime-dependent changes in mitochondrial function and structure resulting from 1 hr of left circumflex coronary artery occlusion followed by 2 to 24 hrs of reperfusion were examined. These changes were correlated with changes in myocardial ultrastructure, tissue water content, infarct size and mitochondrial calcium content. The heart was removed after different periods of reperfusion, and mitochondria were isolated from ischemic and nonischemic regions of the left ventricle. Tissue samples from ischemic and nonischemic myocardium also were taken for electron microscopy and tissue water content determinations. Infarct size was measured by the nitroblue tetrazolium staining method. Oxygen consumption by mitochondria isolated from ischemic and nonischemic myocardium was measuredin vitro. Mitochondria from ischemic myocardium showed time-dependent decreases in rates of oxygen consumption and tightness of coupling. Electron microscopy revealed progressive ultrastructural deterioration in ischemic myocardium, including accumulation of calcium deposits within mitochondria, a finding corroborated by elevated concentrations of calcium in mitochondria isolated from the same area. Tissue wet-to-dry weight ratios were increased significantly in ischemic myocardium. A small, but significant, decrease in respiratory function was observed in mitochondria isolated from nonischemic myocardium several hrs after reperfusion; however, nomal respiration was observed 24 hrs after release of occlusion. This latter observation indicates that the nonischemic zone also is affected by regional ischemia. The results obtained indicate that temporary left circumflex artery occlusion and reperfusion result in progressively decreasing mitochondrial function and structure within the ischemic myocardium, and that these changes are accompanied by cellular electrolyte alterations.ZusammenfassungUntersucht wurden zeitabhängige Veränderungen in Struktur und Funktion der Mitochondrien, die durch einstündigen Verschluß und 2- bis 24stündige Reperfusion des Ramus circumflexus der linken Koronararterie erzeugt wurden. Diese Veränderungen wurden mit Veränderungen der myokardialen Ultrastruktur, dem Wassergehalt des Gewebes, der Infarktgröße und dem mitochondrialen Calciumgehalt korreliert. Das Herz wurde nach verschiedenen Reperfusionszeiten entnommen und die Mitochondrien aus ischämischen und nichtischämischen Gebieten des linken Ventrikels isoliert. Ebenso wurden Gewebeproben von ischämischem und nichtischämischem Myokard für Elektronenmikroskopie und Bestimmung des Wassergehaltes des Gewebes entnommen. Die Infarktgröße wurde durch die Anfärbung mit Nitroblau-Tetrazolium bestimmt. Der Sauerstoffverbrauch der Mitochondrien aus ischämischem und nichtischämischem Myokard wurdein vitro gemessen. Mitochondrien aus ischämischem Myokard zeigten eine zeitabhängige Abnahme des Sauerstoffverbrauchs und seiner Bindung an die Phosphorylierung von ADP. Die Elektronenmikroskopie zeigte eine fortschreitende Zerstörung der Ultrastruktur im ischämischen Myokard, einschließlich einer Zunahme der Calciumablagerungen in Mitochondrien, was mit erhöhten Calciumkonzentrationen in Mitochondrien aus dem gleichen Gebiet übereinstimmte. Im ischämischen Myokard war die Relation Feuchtgewicht/Trockengewicht signifikant erhöht. Eine geringe, aber signifikante Abnahme der Atmung wurde in Mitochondrien, die nach einigen Stunden Reperfusion aus nichtischämischem Myokard isoliert worden waren, beobachtet; aber nach 24 h Reperfusion fand sich normale Atmung. Letzteres weist darauf hin, daß auch das nichtischämische Gebiet von der regionalen Ischämie betroffen ist. Die Ergebnisse zeigen, daß vorübergehender Verschluß des Ramus circumflexus der linken Koronararterie und Reperfusion zu fortschreitender Zerstörung mitochondrialer Funktion und Struktur führen und daß diese Veränderungen von Änderungen des Electrolytstatus der Zelle begleitet werden.

Protective effects of β-adrenergic blockade in isolated ischemic hearts

Abstract The protective effects of the β-adrenergic blocking drugs, propranolol and atenolol, were tested in a model of global ischemia and assessed electron microscopically. Cat isolated hearts were perfused retrogradely with arterial blood drawn from donor cats. After a period of equilibration, isolated hearts rendered globally ischemic for 1 h and subsequently reperfused for another hour. Hearts were then flushed with physiological salt solution followed by perfusion-fixation with cacodylate-buffered glutaraldehyde, containing ionic lanthanum. Lanthanum was included as a probe of myocardial membrane integrity. Left ventricular subendocardial samples were processed and examined electron microscopically. Nontreated hearts, which underwent normothermic ischemic and reperfusion, displayed extensive ultrastructural damage. Nonischemic and donor cat control myocardial tissue appeared normal in all respects. Hearts that received either propranolol or atenol maintained their ultrastructural integrity, resembling controls. Ionic lanthanum proved to be reliable as a marker of membrane integrity and permeability, as nontreated hearts displayed intracellular deposition of the marker, indicating that deteriorations of membrane integrity occurred. The results suggest that β-adrenergic blockade may be valuable in preserving myocardium subjected to ischemia and reperfusion.

Lanthanum staining of coronary microvascular endothelium: Effects of ischemia reperfusion, propranolol, and atenolol

Abstract Cat isolated hearts were perfused via the aorta with normothermic arterial blood from donor cats. After 1 hr of equilibration, dl-propranolol (1.9 mg/kg), atenolol (1.65 mg/kg), or physiological saline solution was infused via the aortic cannula. The hearts were made globally ischemic for 1 hr and reperfused for 1 hr. Hearts given saline but not made ischemic, and hearts from blood-donor cats served as controls. The hearts were flushed with physiological saline for 2 min, then perfused with cacodylate-buffered glutaraldehyde containing 1% LaCl3. Samples of left ventricle were postfixed in osmium and prepared for electron microscopy. Microvessels in nonischemic tissues had heavy La3+ staining on luminal surfaces of endothelial cells. Many plasmalemmal vesicles along luminal surfaces of endothelial cells were filled with La3+. Several vesicles appeared to open onto both surfaces thus forming channels through the endothelium. Lanthanum penetrated into, and occasionally through, interendothelial junctions. Endothelial cells lining vessels in ischemic myocardium were swollen, had pale cytoplasm, and showed little La3+ on the luminal surfaces. Few plasmalemmal vesicles were present and the mitochondria contained deposits of La3+. Extravascular spaces were distended but interendothelial junctions seemed to be intact. Lanthanum staining and morphology of endothelial cells in hearts treated with propranolol or atenolol were very similar to nonischemic myocardium. The data suggest that the β-blocking agents, propranolol and atenolol, maintain the integrity of coronary vascular endothelium during ischemia.

Comparative Effects of Aspartate and Glutamate during Myocardial Ischemia

The effects of amino acids aspartate (Asp) and glutamate (Glu) on recovery of contractile function and preservation of compliance were studied in globally ischemic, isolated, blood-perfused cat hearts. Ischemia-induced declines in contractility and compliance were measured with an intraventricular fluid-filled balloon. Asp and Glu were delivered to isolated hearts in physiological salt solution (PSS) containing 10 mM glucose, just prior to, and intermittently during (every 15 min for 1 min) 1 h of normothermic ischemia. Isolated hearts which received Asp and Glu showed recoveries of left ventricular (LV) developed pressure of 79 +/- 8 and 50 +/- 7% of their preischemic values, respectively, compared to 34 +/- 7% in hearts perfused only with PSS. These alterations of contractile function were paralleled by changes in LV compliance. The addition of amino-oxyacetate, and aminotransferase inhibitor, to Asp- containing PSS markedly attenuated the beneficial effects of this amino acid. The results indicate that certain amino acids can protect the ischemic myocardium, presumably through effects on intermediary metabolism.

The effect of diltiazem on coronary thrombosis in the conscious canine.

The effect of diltiazem vs. saline was studied in a conscious canine model of coronary thrombosis. Diltiazem given as a 0.75 mg/kg loading dose intravenously followed by 0.4 mg/kg intravenously every 4 h for 24 h had no significant effect on thrombus wet weight, left ventricular infarct size, frequency of ventricular arrhythmias or ex vivo platelet aggregation. The search for antithrombotic agents using in vitro or ex vivo platelet aggregation studies should include concomitant in vivo thrombosis studies using therapeutic dosages of the drug in question.