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J E Saffitz

Publications and source records attributed to J E Saffitz.

At least 145 records · Page 8Linked to original sources
PubMed1985

Temporal changes in 12-HETE formation in two models of canine myocardial infarction.

Arachidonic acid (AA) metabolism by infarcted canine myocardium was studied and correlated with matched histologic analyses following permanently occluded or reperfused infarction. Histologic analysis of tissues from reperfused infarcts showed a marked acceleration of inflammatory cell invasion and of granulation tissue formation when compared to the occlusive infarct. In the reperfused infarct, polymorphonuclear leukocytes (PMNs) were very prominent at one day after infarction while in the occlusive infarcts the neutrophilic invasion was less intense but more sustained. At one day following reperfused infarction the major arachidonate product, which co-migrated by thin layer chromatography with the mono-hydroxyeicosatetraenoic acids (HETEs), was significantly elevated (254 +/- 49 pmoles/gm wet weight, n = 3) when compared to normal tissue (48 +/- 6 pmoles/gm n = 19). This occurred at a time when the number of PMNs was maximal in the infarcted tissue. Addition of the calcium ionophore A23187 caused a further marked stimulation in HETE production in the one day reperfused infarct but not at the other time points studied. The production of HETE was not significantly different in the infarcted tissue than in the normal tissue at three and seven days following reperfused infarction or at one, three, or seven days after occlusive infarction. The identity of this HETE product was investigated using reverse phase high performance liquid chromatography (RP-HPLC) and gas chromatography-mass spectrometry (GC-MS) and found to be predominantly 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) with a small amount of 15-HETE. Thus the production of 12-HETE parallels the number of neutrophils invading the infarcted area of the heart.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1985

Uptake and subcellular distribution of [3H]arachidonic acid in murine fibrosarcoma cells measured by electron microscope autoradiography.

We have used quantitative electron microscope autoradiography to study uptake and distribution of arachidonate in HSDM1C1 murine fibrosarcoma cells and in EPU-1B, a mutant HSDM1C1 line defective in high affinity arachidonate uptake. Cells were labeled with [3H]arachidonate for 15 min, 40 min, 2 h, or 24 h. Label was found almost exclusively in cellular phospholipids; 92-96% of incorporated radioactivity was retained in cells during fixation and tissue processing. All incorporated radioactivity was found to be associated with cellular membranes. Endoplasmic reticulum (ER) contained the bulk of [3H]arachidonate at all time points in both cell types, while mitochondria, which contain a large portion of cellular membrane, were labeled slowly and to substantially lower specific activity. Plasma membrane (PM) also labeled slowly, achieving a specific activity only one-sixth that of ER at 15 min in HSDM1C1 cells (6% of total label) and one-third of ER in EPU-1B (10% of total label). Nuclear membrane (NM) exhibited the highest specific activity of labeling at 15 min in HSDM1C1 cells (twice that of ER) but was not preferentially labeled in the mutant. Over 24 h, PM label intensity increased to that of ER in both cell lines. However, NM activity diminished in HSDM1C1 cells by 24 h to a small fraction of that in ER. In response to agonists, HSDM1C1 cells release labeled arachidonate for eicosanoid synthesis most readily when they have been labeled for short times. Our results therefore suggest that NM and ER, sites of cyclooxygenase in murine fibroblasts, are probably sources for release of [3H]arachidonate, whereas PM and mitochondria are unlikely to be major sources of eicosanoid precursors.

Animals↗
PubMed1985

Intramural reentry as a mechanism of ventricular tachycardia during evolving canine myocardial infarction.

We evaluated the contribution of intramural electrical events in initiation and maintenance of ventricular tachycardia in 15 dogs 3-8 days after either permanent (n = 2) or transient (n = 13) coronary artery occlusion. Seven of the dogs (47%) demonstrated eight distinct monomorphic ventricular tachycardia patterns which were mapped by means of a recently designed computerized system capable of simultaneously detecting, storing, and assessing information from 232 individual cardiac sites. Using both epicardial and intramural electrodes, we found definitive evidence for intramural reentry in seven of the eight monomorphic tachycardias analyzed. Furthermore, five of these animals (71%) demonstrated microreentry, in which small epicardial conduction loops exited intermittently into nonrefractory subendocardium to initiate succeeding beats, while, in the remaining two dogs, ventricular tachycardia was due to macroreentry, during which the broad subendocardial wavefronts depolarizing the ventricle constituted the proximal (fast) reentry limbs. Detailed anatomical analysis of the resultant infarcts demonstrated the thin surviving epicardial tissue rim to be the site of conduction delay necessary for reentry, whereas "preferred pathways" of exit into the subendocardial plane occurred at the infarct borders and were of variable configuration. Successful interruption of these rhythms should accompany interference with the process of exit into nonrefractory subendocardial tissue.

Animals↗
PubMed1985

Effects of endogenously produced leukotrienes, thromboxane, and prostaglandins on coronary vascular resistance in rabbit myocardial infarction.

In an effort to evaluate the synthesis and function of eicosanoids in myocardial infarction, we have developed a technique of in vivo myocardial infarction in rabbits followed by ex vivo cardiac perfusion. Isolated Langendorff perfused infarcted hearts (removed 1 or 4 d after infarction) responded to the inflammatory cell agonist N-formylmethionyl-leucyl-phenylalanine (fMLP) with (a) the release of leukotrienes B4, C4, and D4; (b) the release of large amounts of thromboxane (235 +/- 66 ng/5 min), prostacyclin (714 +/- 285 ng/5 min), and prostaglandin E2 (PGE2) (330 +/- 108 ng/5 min); and (c) a coronary vasoconstriction (21.1 +/- 2.5% increase in coronary perfusion pressure) that was specifically inhibited by the peptidoleukotriene receptor antagonist FPL-55712. While noninfarcted hearts challenged with fMLP also released leukotrienes B4, C4, and D4, they released only small amounts of the cyclooxygenase products (thromboxane, 30 +/- 9 ng/5 min; prostacyclin, 120 +/- 54 ng/5 min; PGE2, 27 +/- 10 ng/5 min) and showed minimal vasoconstriction (5.6 +/- 2.1% increase in perfusion pressure). Similarly, hearts challenged with fMLP 30 d following infarction released only small amounts of the cyclooxygenase products (thromboxane, 42 +/- 8 ng/5 min; prostacyclin, 386 +/- 31 ng/5 min; PGE2, 79 +/- 25 ng/5 min). When bradykinin was administered, no leukotrienes were produced, but acutely infarcted hearts released 10 times more thromboxane, prostacyclin, and PGE2 than normal hearts and significantly larger amounts of these products than 30-d infarcted hearts. Histologic analysis showed no inflammatory cells in normal hearts, a prominent polymorphonuclear leukocyte infiltration in 1-d infarcted tissue, fibroblast proliferation with mononuclear cell invasion in 4-d infarcted tissue, and a fibrotic scar with scanty mononuclear cell infiltrate in 30-d infarcted tissue. Inflammatory cell invasion was temporarily associated with augmented cyclooxygenase metabolism, suggesting that infiltrating leukocytes may be responsible for production of thromboxane, prostacyclin, and PGE2 in acutely infarcted hearts. The finding that endogenously produced peptidoleukotrienes are potent coronary vasoconstrictors in infarcted rabbit hearts suggests that these products may contribute to tissue injury in myocardial infarction.

Animals↗
PubMed1985

Preservation of arachidonoyl phospholipids during tissue processing for electron microscopic autoradiography.

To facilitate autoradiographic subcellular localization of arachidonoyl phospholipids, the retention of radioactivity during tissue processing of murine fibrosarcoma cells labeled in vitro with 3H-arachidonate was assessed. Approximately 94% of cell radioactivity was incorporated into phospholipids. During tissue processing, extraction of radioactivity was monitored by liquid scintillation spectrometry. Fixation of cells in glutaraldehyde-tannic acid, postfixation in osmium tetroxide, en bloc staining in uranyl magnesium acetate, dehydration in ethanol, and embedding in Epon resulted in preservation of 93.5% of total tissue radioactivity. Analysis of extracted radioactivity by thin layer chromatography revealed that no specific class of phospholipids was selectively extracted. Fixation with osmium tetroxide alone was nearly as effective as the complete fixation protocol and resulted in retention of 90.0% of radioactivity. However, fixation with glutaraldehyde-tannic acid alone without osmium tetroxide post-fixation led to extraction of 69.8% of total cell radioactivity. Thus, osmium tetroxide is crucial in the preservation of arachidonoyl phospholipids and presumably forms extensive cross-links between polyunsaturated acyl residues. This degree of preservation of arachidonoyl phospholipids is indicative of spatial fixation of the radiolabeled moieties and will permit quantitative studies of subcellular loci of eicosanoid metabolism by electron microscopic autoradiography.

Animals↗
PubMed1985

A fixation procedure suitable for autoradiography of endogenous long-chain acyl carnitine.

A tissue processing procedure was evaluated for fixation of endogenous long-chain acyl carnitine (LCA) to facilitate autoradiographic subcellular localization of this amphiphile. Suspensions of neonatal rat myocytes labeled with exogenous 14C-palmitoyl carnitine retained 85.2% of the radiolabel after tissue processing. Autoradiography demonstrated no significant translocation of radiolabeled LCA from myocytes to unlabeled sheep erythrocytes mixed in equal proportions and processed together. To evaluate endogenous LCA fixation, cultured myocytes were incubated for 3 days with 3H-carnitine. Radioactivity was distributed in LCA, short-chain acyl carnitine, and free carnitine pools in proportion to the physiological concentrations of the metabolites traced. Before tissue processing, LCA contained 4.5% of total radioactivity. After tissue processing, labeled water-soluble components were lost and 88% of the retained radioactivity was in the LCA pool. The enrichment of endogenous LCA radioactivity was attributable to the selective extraction of endogenous short-chain and free carnitine. Nearly 75% of endogenous LCA was preserved. In contrast, 99.5% of both endogenous short-chain and free carnitine were extracted. Thus, endogenous LCA can be selectively preserved, permitting quantitative subcellular localization of this amphiphile with ultrastructural autoradiography.

Animals↗
PubMed1985

Retrograde axonal transport of 125I-nerve growth factor in rat ileal mesenteric nerves. Effect of streptozocin diabetes.

The retrograde axonal transport of intravenously (i.v.) administered 125I-nerve growth factor (125I-NGF) was examined in mesenteric nerves innervating the small bowel of rats with streptozocin (STZ) diabetes using methods described in detail in the companion article. The accumulation of 125I-NGF distal to a ligature on the ileal mesenteric nerves of diabetic animals was 30-40% less than in control animals. The inhibition of accumulation of 125I-NGF in diabetic animals was greater at a ligature tied 2 h after i.v. administration than at a ligature tied after 14 h, which suggests that the diabetic animals may have a lag in initiation of NGF transport in the terminal axon or retardation of transport at some site along the axon. The 125I-NGF transport defect was observed as early as 3 days after the induction of diabetes, a time before the development of structural axonal lesions, and did not worsen at later times when dystrophic axonopathy is present. Both the ileal mesenteric nerves, which eventually develop dystrophic axonopathy in experimental diabetes, and the jejunal mesenteric nerves, which never develop comparable structural alterations, showed similar 125I-NGF transport deficits, suggesting that the existence of the transport abnormality does not predict the eventual development of dystrophic axonal lesions. Autoradiographic localization of 125I-NGF in the ileal mesenteric nerves of animals that had been diabetic for 11-13 mo demonstrated decreased amounts of 125I-NGF in transit in unligated paravascular nerve fascicles. There was, however, no evidence for focal retardation of transported 125I-NGF at the sites of dystrophic axonal lesions.

Animals↗
PubMed1984

Applicability of ultrasonic tissue characterization for longitudinal assessment and differentiation of calcification and fibrosis in cardiomyopathy.

Progress in tissue characterization of myocardium with ultrasound suggests that quantitative recognition of ischemic or scarred tissue will be achieved. Despite the increasing recognition and importance of cardiomyopathy, its diagnosis generally requires invasive procedures such as cardiac catheterization and biopsy. To investigate methods that permit the characterization of longitudinal cardiomyopathic changes that might ultimately be extended for noninvasive studies in patients, quantitative ultrasonic methods were utilized for in vitro tissue characterization of hearts from Syrian hamsters of selected age of either 2 to 3 or 5 to 7 months. Normal hamsters were used as controls. Myocardial sites (n = 600) from the young Syrian hamsters exhibited values (+/- standard error) of integrated ultrasonic backscatter averaging -53.87 +/- 0.26 dB, which were significantly different from values (n = 500) in age-matched control hamsters (-58.07 +/- 0.08 dB; p less than 0.001). Cardiomyopathic hearts from older animals exhibited backscatter values (n = 500 sites) averaging -50.87 +/- 0.22 dB, again significantly different from values (n = 300 sites) in age-matched control hamsters (-55.91 +/- 0.11 dB; p less than 0.001). In addition, ultrasonic attenuation was significantly different for hearts from the control and cardiomyopathic hamsters of both age ranges. The results correlated with sequential calcification and fibrosis characteristics assessed histopathologically. This study indicates that quantitative characterization of myocardium with ultrasound may permit longitudinal assessment of cardiomyopathic changes in diverse disease entities and their response to therapy.

Age Factors↗
PubMed1984

Fibronectin in rat heart: a link between cardiac myocytes and collagen.

Fibronectin, a glycoprotein that binds to collagen and modifies the adhesion properties and motility of cells in culture, is present in the interstitium of rat hearts. To localize fibronectin more precisely and to assess its relationship to the myocyte and to connective tissue elements, we employed a double antibody technique to label myocardial fibronectin with electron-dense ferritin to permit an ultrastructural analysis. Fibronectin was found to be associated with collagen, and in some cases appeared to link collagen fibers. Fibronectin was also found inserted along the surfaces of cardiac myocytes, connecting these cells to perimyocytic collagen. These ultrastructural relationships imply that fibronectin is a major component of the myocardial interstitium, and may affect myocardial compliance and control the motion of myocytes during the contraction and relaxation of the heart.

Animals↗
PubMed1984

Pathophysiologic concentrations of lysophosphoglycerides quantified by electron microscopic autoradiography.

Lysophosphoglycerides accumulate in ischemic myocardium and appear to contribute to malignant dysrhythmia. Exposure of normoxic isolated Purkinje fibers or ventricular muscle strips to exogenous lysophosphatidyl choline (LPC) results in rapid, yet reversible, electrophysiologic derangements analogous to changes associated with ischemia in vivo. However, subcellular, local concentrations required for induction of electrophysiologic effects have not yet been elucidated unambiguously. The present study was designed to delineate the subcellular distribution and the sarcolemmal concentration of LPC by quantitative electron microscopic autoradiography after exposure of canine ventricular muscle strips to 3H-methyl LPC (200 microM) for 10 minutes. Tissue was processed for electron microscopy with a new procedure developed specifically to spatially fix choline phosphatides and render them insoluble in lipid solvents. Grain distributions were analyzed in the same group of cells that had been monitored electrophysiologically while superfused with LPC. LPC significantly decreased the resting membrane potential, action potential amplitude, duration, Vmax of phase 0, and conduction time. Grains were concentrated in the membranous organelles of cardiac myocytes. Myocyte sarcolemma exhibited the highest grain density (129 grains/100 micron2 versus a background of 0.27 grains/100 micron2). Calculations based on grain densities, emulsion sensitivity, exposure time, and specific activity indicated that the sarcolemma incorporated 5.4 X 10(6) LPC molecules/micron3 of membrane volume corresponding to approximately 1% of total sarcolemmal phospholipid. Since incorporation of only this small amount of lysophosphoglyceride into the sarcolemma was sufficient to elicit electrophysiologic disturbances, the observed accumulation of lysophosphoglycerides induced by ischemia appears to be sufficient to contribute to malignant dysrhythmia in vivo.

Animals↗