Dean English

Glycogen Metabolism in the Aerobic Hypertrophied Rat Heart

BACKGROUND Rates of glycolysis from exogenous glucose are accelerated in hypertrophied hearts. In this study, we determined whether alterations in the metabolism of glycogen, an endogenous storage form of glucose, also occur in hypertrophied hearts. METHODS AND RESULTS Rates of glycolysis ([3H]H2O production) and oxidation ([14C]CO2 production) from exogenous glucose and glycogen were measured in isolated working hearts from control and aortic-banded rats. Hearts in which glycogen was prelabeled with [5-(3)H]- or [U-(14)C]glucose were perfused with buffer containing 11 mmol/L [5-(3)H]- or [U-(14)C]glucose (different from the isotope used to prelabel glycogen), 0.4 mmol/L palmitate, 0.5 mmol/L lactate, and 100 microU/mL insulin. Rates of glycolysis from exogenous glucose were greater (3471+/-114 versus 2665+/-194 nmol glucose x min(-1) x g dry wt(-1), P<.05, n=4 to 6, mean+/-SEM) and rates of exogenous glucose oxidation (445+/-36 versus 619+/-16 nmol glucose x min(-1) x g dry wt(-1), P<.05, n=4 to 6) were lower in hypertrophied hearts than in control hearts. Rates of glycolysis and oxidation from glycogen were not different between hypertrophied and control hearts. A greater proportion of glycogen was oxidized (80% to 100%) than the proportion of exogenous glucose oxidized (13% to 24%) in both groups. Additionally, 10.5+/-1.4 and 12.3+/-1.0 micromol/g dry wt of glycogen was synthesized in hypertrophied and control hearts, respectively, indicating that simultaneous synthesis and degradation (ie, glycogen turnover) occurred in both groups. CONCLUSIONS Thus, aerobic myocardial glycogen metabolism in the hypertrophied heart is similar to that observed in the normal heart even though exogenous glucose metabolism is altered in the hypertrophied heart.

Ultrastructural evidence of early endothelial damage in coronary arteries of rat cardiac allografts.

BACKGROUND Events that occur early after transplantation, particularly immune recognition of allo-endothelium, initiate transplant vascular disease (TVD). Previous work suggests an important compromise of endothelial integrity as the allo-immune milieu evolves, although mechanisms by which integrity is altered remain unclear. Increased vascular permeability caused by endothelial damage may allow inflammatory cells, lipoproteins, other proteins, and plasma fluid to enter the sub-endothelial space, thereby contributing to the initiation of atherosclerosis. In this study, we examined endothelial integrity in coronary arteries and the proximal aorta after cardiac transplantation in rats. METHODS We used Lewis-to-Lewis and Lewis-to-F344 rat heterotopic cardiac transplant models. We studied the effects of cyclosporine (5mg/kg/day) therapy compared with saline-treated controls. En face silver nitrate staining was performed to demonstrate endothelial cell borders and gaps. We used scanning electron microscopy to extend silver nitrate findings and to further define the presence and nature of endothelial disruptions. We used transmission electron microscopy to further characterize immune cell identity and interaction with endothelium. RESULTS Syngrafts and cyclosporine-treated allografts showed normal-looking endothelium similar to that observed in arteries from native hearts. However, saline-treated allografts displayed progressive endothelial destruction, including large intercellular gaps, missing cells, and areas of bare extracellular matrix. Exfoliated surfaces were covered by platelets at various stages of adhesion, activation, and spreading. Similarly, we observed numerous leukocytes as either adherent to the endothelial lining or transmigrating into the sub-endothelial space. Cessation of cyclosporine therapy was associated with the development of similar abnormalities. CONCLUSIONS Our findings indicate that, especially when immunosuppression is insufficient, early endothelial damage may promote vascular permeability and thereby initiate TVD.

Neutrophil Activation and Lung Injury Associated with Chronic Endotoxemia in Rabbits

Chronic endotoxemia produces emphysematous lung destruction in several animal models. The present study was designed to examine changes in the polymorphonuclear leukocytes (PMN) and the lung parenchyma of rabbits that received either saline (control, n = 6) or Escherichia coli endotoxin (LPS, n = 6) 2-3 times weekly for 15 to 28 weeks. Peripheral blood was collected just before and after each intravenous injection and lung tissue was processed at the end of the experiment. PMN myeloperoxidase was stained with diaminobenzidine tetrahydrochloride (DAB)-H2O2, and CD11/CD18 was detected with immunogold. The changes in the PMN and the lung parenchyma were quantitatively analyzed. The results show that each dose of LPS produced an initial fall, followed by a rise in the circulating mature and immature PMN cell counts. Repeated doses of LPS induced PMN activation, degranulation, and an increase in the mean thickness of the alveolar wall (control, 4.1 +/- 0.2; LPS, 5.1 +/- 0.5; p < .05) at 28 weeks without evidence of alveolar septa destruction. Morphometric analysis of intravascular PMN showed an increase in the volume (V) of myeloperoxidase-containing azurophil granules (control, 6.1 +/- 1.3 microns3; LPS, 13.1 +/- 2.8 microns3; p < .05); a trend for a decrease in the V of specific granules (control, 15.8 +/- 3.4 microns3; LPS, 10.2 +/- 1.5 microns3; p = .09); an increase in the V of the cytoplasm (control, 37.3 +/- 6.4 microns3; LPS, 54.5 +/- 7.1 microns3; p < .05); and an increase in CD11/CD18 expressed as the number of gold particles per micrometer of cell surface membrane (G/micron) (control, 7.1 +/- 1.4 G/micron; LPS, 18.1 +/- 7.8 G/micron; p < .05). The results indicate that chronic endoxemia in rabbits, accelerates the release of PMN from the bone marrow, enhances the retention of both mature and immature activated PMN in the pulmonary microvessels, and causes alveolar wall thickening rather than emphysematous lung destruction.

Regional myocardial capillary erythrocyte transit time in the normal resting heart.

A major determinant of oxygen transport to the myocardium is the time spent by the erythrocytes (red blood cells [RBCs]) traversing the microcirculation. Although it has been shown that the myocardium has regional differences in blood volume, blood flow, metabolism, and sensitivity to ischemic injury, the regional distribution of RBC transit times through the myocardial capillaries has not been previously measured. The present study was designed to measure the regional myocardial capillary RBC transit time by a new technique to determine whether there are regional differences in the capillary RBC transit time in the normal resting heart. Anesthetized open-chest male New Zealand White rabbits (3.0-3.7 kg, n = 8) were studied. Regional myocardial blood volume was determined using chromium-51-labeled RBCs, and regional blood flow was measured using a reference flow technique and a left atrial injection of 15-microns-radiolabeled (gadolinium-153, 10-20 muCi) microspheres. Capillary blood volume was determined by multiplying the regional blood volume by the histologically determined fraction of the total blood volume that was in the capillaries. Capillary RBC transit time was calculated as the quotient of capillary blood volume and blood flow. The myocardial capillary blood volume was the same in the endocardium and the epicardium (4.67 +/- 0.67 ml/100 g for endocardium versus 4.52 +/- 0.70 ml/100 g for epicardium, p = NS), whereas myocardial blood flow tended to be greater in the endocardium (6.09 +/- 0.73 ml/sec per 100 g for endocardium versus 5.47 +/- 0.75 ml/sec per 100 g for epicardium), although this was not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

The nature of leukocyte shape changes in the pulmonary capillaries

The size discrepancy between leukocytes [white blood cells (WBCs)] and pulmonary capillaries requires WBCs to deform. We investigated the persistence of this deformation on cells leaving the capillary bed and the role played by the cytoskeleton. Isolated rabbit lungs were perfused in situ via the pulmonary artery with effluent fractions collected from the left ventricle. Washout curves from cell counts in each fraction confirmed that WBCs are preferentially retained over erythrocytes. WBC deformation present on exit from the circulation was compared with that present after recovery in paired fractions, fixed either immediately or 60 min later. These cells were compared with cells recovered from the capillary in perfused fixative or fixed in peripheral blood. Our results show that leukocyte deformation persisted after the cells exited the pulmonary circulation. This deformation was associated with minimal submembranous F-actin staining, and microtubule distribution and cell polarization were unchanged. We conclude that cytoskeletal changes that occur during WBC deformation in the pulmonary capillaries are minimal and differ from those known to occur in actively migrating cells during chemotaxis.