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Biomedical subjects

J E Saffitz

Publications and source records attributed to J E Saffitz.

At least 73 records · Page 4Linked to original sources
PubMed1994

Inositol polyphosphate 1-phosphatase is present in the nucleus and inhibits DNA synthesis.

Inositol polyphosphate 1-phosphatase, an enzyme of the phosphatidylinositol signaling pathway, hydrolyzes the 1-phosphate from inositol 1,4-bisphosphate and inositol 1,3,4-trisphosphate. We have used indirect immunofluorescence microscopy, Western blot analysis, and enzyme assays to determine the cellular localization of the enzyme. We find that the enzyme is present, but not exclusively, in the nucleus of Madin-Darby bovine kidney cells, and also in COS-7 and HeLa cells that were transiently transfected with a cDNA encoding bovine inositol polyphosphate 1-phosphatase. DNA synthesis, as measured in COS-7 and HeLa cells transiently over-expressing enzyme, was reduced 50% in cells transfected with wild-type enzyme compared with nontransfected cells or cells transfected with an inactive mutant form of the enzyme. These data demonstrate that this response is mediated by one of the substrates or products of inositol polyphosphate 1-phosphatase. We propose that overexpressed inositol polyphosphate 1-phosphatase degrades a stimulatory inositol phosphate(s) and thereby inhibits DNA synthesis.

Amino Acid Sequence↗
PubMed1994

Transmural heterogeneity of norepinephrine uptake in failing human hearts.

OBJECTIVES: The aim of this study was to determine whether regional heterogeneity in myocardial sympathetic neural function measured by the uptake of norepinephrine could account for the spatial heterogeneity of beta-adrenergic receptor down-regulation that occurs in the failing human heart. BACKGROUND: Myocardial beta-adrenergic receptor density and function are diminished in patients with chronic heart failure. Down-regulation occurs predominantly in the subendocardium, suggesting that local rather than systemic alterations in sympathetic neural function may be responsible. Although some studies have implicated hypofunction of cardiac sympathetic nerves with defective norepinephrine uptake, others suggest increased cardiac sympathetic nerve activity with unimpaired uptake. METHODS: We measured norepinephrine uptake by incubating transmural slices of the left ventricle from 19 patients who had chronic heart failure and three nonfailing control hearts with [3H]norepinephrine with or without desipramine, a neuronal uptake blocker. The density of uptake sites was measured in subepicardial and subendocardial myocyte regions with light microscopic autoradiography. RESULTS: Although the amount of [3H]norepinephrine uptake varied considerably in failing ventricles, uptake was directly proportional (r = 0.46, p < 0.05) to beta 1-adrenergic receptor density measured in additional slices with radioligand binding assays. In addition, marked transmural heterogeneity in [3H] norepinephrine uptake was consistently observed in failing ventricles. Uptake in subendocardial myocyte regions was significantly less than in subepicardial regions (mean [ +/- SD] subepicardial/subendocardial uptake ratio 4.7 +/- 4.8, p < 0.01). The extent of transmural heterogeneity in norepinephrine uptake was similar in patients with idiopathic and ischemic cardiomyopathy. In contrast, nonfailing hearts exhibited more uniform transmural [3H]norepinephrine uptake (subepicardial/subendocardial uptake ratio 1.8 +/- 1.2, p = NS). CONCLUSIONS: Specific [3H]norepinephrine accumulation is approximately fivefold lower in subendocardial regions of failing left ventricles than in subepicardial regions. These findings support the hypothesis that a subendocardial defect in norepinephrine uptake may chronically elevate local interstitial catecholamine levels and thereby down-regulate beta-adrenergic receptors in a spatially heterogeneous distribution.

Adult↗
PubMed1994

Molecular cloning of two human cardiac gap junction proteins, connexin40 and connexin45.

Intercellular coupling of cardiac myocytes through gap junction channels facilitates normal cardiac impulse conduction. Multiple gap junction sequences (connexins) have been previously identified in mammalian and avian heart, but only one, connexin43 (Cx43), has been identified in the human heart. We used the polymerase chain reaction and genomic cloning to isolate DNA encoding the gap junction proteins human connexin40 (Cx40) and connexin45 (Cx45). Northern blots showed that specific probes for Cx43, Cx40 and Cx45 all hybridize to distinct mRNAs in human ventricular RNA. Immunohistochemistry with connexin-specific antibodies confirmed that Cx40, Cx43, and Cx45 all localized to intercalated disk regions in frozen sections of human left ventricle. The presence of multiple connexins in human ventricle may contribute to divergent mechanisms of regulation of cardiac conduction.

Amino Acid Sequence↗
PubMed1994

Potential ablation of accessory atrioventricular pathways: injection of alcohol into the atrioventricular groove.

Accessory atrioventricular (AV) pathways responsible for the Wolff-Parkinson-White syndrome have been treated successfully by surgical and radiofrequency catheter ablation techniques. In this study, we explored the feasibility of permanent chemical ablation of accessory pathways by direct injection of 100% ethanol into the canine AV groove. Right coronary artery blood flow and AV compartment pressure were measured in 5 adult mongrel dogs (part I) during injection of saline solution (10 to 15 mL) into the right AV groove. Atrioventricular groove compartment pressure increased from 1 +/- 1 to 31 +/- 12 mm Hg (p < 0.0001) and right coronary artery blood flow increased slightly from 117 +/- 17 to 138 +/- 44 mL/min (p = not significant). In 8 additional dogs (part II), 100% ethanol (10 mL) was injected into the left AV groove. After 6 weeks (n = 6) or 12 weeks (n = 2), histologic examination of the left AV groove demonstrated localized fibrous replacement of injured epicardial muscle, with only scant inflammatory infiltrate. Mild intimal fibrosis and intimal proliferation were present in the circumflex artery and its larger branches, but no significant luminal narrowing was evident. Angiograms at 12 weeks (n = 2) demonstrated no significant narrowing of the circumflex artery. In summary, injection of 100% ethanol into the AV groove is capable of locally ablating myocardial tissue, presumably including conductive tissues, while sparing the coronary arteries.

Animals↗
PubMed1994

Distinct gap junction protein phenotypes in cardiac tissues with disparate conduction properties.

OBJECTIVES: We sought to characterize the connexin phenotypes of selected regions of the canine heart with different conduction properties to determine whether variations in connexin expression might contribute to the differences in intercellular resistance and conduction velocity that occur in different cardiac tissues. BACKGROUND: Gap junctions connect cardiac myocytes, allowing propagation of action potentials. Intercellular channels with different electrophysiologic properties are formed by different connexin proteins. METHODS: To determine which connexins were likely to be expressed in the sinus node, atrioventricular (AV) node and atrial and ventricular myocardium, messenger ribonucleic acids (RNAs) from each of these sites were hybridized with probes for connexin26, connexin31, connexin32, connexin37, connexin40, connexin43, connexin45, connexin46 and connexin50. Immunostaining with monospecific antibodies to connexin40, connexin43 and connexin45 was used to delineate the distribution of connexins in frozen sections of these different cardiac tissues. RESULTS: Only messenger RNAs coding for connexin40, connexin43 and connexin45 were detected by Northern blot analysis. By immunohistochemical staining, junctions in the sinus and AV nodes and proximal His bundle were virtually devoid of connexin43 but contained both connexin40 and connexin45. Gap junctions in the distal His bundle and the proximal bundle branches stained intensely for connexin40 and connexin43 and to a lesser extent for connexin45. Atrial gap junctions showed abundant staining of connexin43, connexin40 and connexin45. Ventricular gap junctions were characterized by abundant staining of connexin43 and connexin45 and much less staining of connexin40. CONCLUSIONS: Although most cardiac gap junctions contain connexin40, connexin43 and connexin45, the relative amounts of each of these connexins vary considerably in cardiac tissues with different conduction properties.

Animals↗
PubMed1994

Immunoelectron microscopic identification of cytoplasmic and nuclear Gs alpha in S49 lymphoma cells.

The subcellular distribution of Gs alpha was characterized in S49 lymphoma cells with two polyclonal antisera directed against specific COOH- and NH2-terminal epitopes. Nonspecific binding was determined in each subcellular compartment by incubating cyc- S49 cells, known to be deficient in Gs alpha and its mRNA, with primary and secondary antisera. Small proportions of total specific binding sites were localized to the plasmalemma as well as the nuclear envelope. Because of their small size, these compartments contained a high concentration of Gs alpha. However, most of the specific binding sites were found in nonstructured cytoplasm and within the nucleus. Specific binding was abolished or significantly reduced by preincubating primary antisera with their peptide immunogens but not with an irrelevant peptide. Intracellular Gs alpha immunoreactive binding sites did not colocalize with gold-conjugated transferrin in cells preincubated with this ligand to mark a classical endocytotic pathway. The intracellular and intranuclear location of Gs alpha was confirmed with confocal microscopy of S49 cells immunostained with specific primary and fluorescently labeled secondary antibodies. Gs alpha was also detected with immunoblots of proteins extracted from purified S49 cell nuclei. Thus, Gs alpha is abundantly distributed in intracellular and intranuclear sites in S49 cells and occurs in loci distinct from organelles of the transferrin pathway. The substantial intracellular distribution of Gs alpha suggests that Gs may subserve intracellular and, perhaps, intranuclear functions that may be important in proliferating cells.

Animals↗
PubMed1994

Effects of reperfusion on ischemic right ventricular dysfunction. Disparate mechanisms of benefit related to duration of ischemia.

BACKGROUND: Right ventricular free wall (RVFW) ischemia impairs global RV performance and may result in acute hemodynamic compromise. However, RV function and hemodynamic performance typically improve spontaneously over time. This study was designed to determine whether reperfusion facilitates recovery of function in the ischemic right ventricle. METHODS AND RESULTS: Closed chest dogs underwent right coronary balloon occlusion for 1 hour (n = 9), 4 hours (n = 6), or 8 hours (n = 7). In all animals, occlusion depressed RVFW function and global RV performance. After 1 hour of ischemia, reperfusion led to immediate improvement in RVFW function and consequently global RV performance, with complete recovery over 4 weeks and scar in < 1% of total RVFW area. Reperfusion after 4- and 8-hour occlusions resulted in acute improvement in global RV performance but to a lesser extent and by different mechanisms, since RVFW contraction remained severely impaired. This disproportionate recovery of global RV function was attributable to diminished RVFW dyskinesis associated with reperfusion-induced increments in RVFW diastolic thickness (characterized histopathologically in 6 additional animals subjected to 4-hour occlusions but killed 1 hour after reperfusion by interstitial edema, contraction band necrosis, and hemorrhage). Although later reperfusion was associated with a slower pace and lesser extent of recovery, RVFW contraction improved markedly over time. At 4 weeks, there was trivial RVFW scar in 4-hour animals (2% of total RVFW area), and, although fibrosis was significantly greater in 8-hour animals (7% of RVFW area), infarction was minimal relative to the extent of jeopardized myocardium. CONCLUSIONS: The responses of ischemic RV myocardium to reperfusion are complex, with disparate effects according to the duration of preceding ischemia. Early reperfusion results in prompt improvement in and subsequent complete recovery of RVFW contraction and global RV performance, with trivial or no RVFW scar. Late reperfusion leads to little acute recovery of RVFW function, but global performance improves owing to diminished RVFW dyskinesis associated with reperfusion-induced increments in RVFW diastolic thickness. Nevertheless, RVFW function improves over time, with minimal evidence of infarction. Therefore, reperfusion facilitates recovery of RV function and minimizes the extent of infarction even after prolonged ischemia.

Acute Disease↗
PubMed1994

Tissue-specific determinants of anisotropic conduction velocity in canine atrial and ventricular myocardium.

Electrical conduction is very rapid and highly anisotropic in atrial fiber bundles, such as the crista terminalis. In contrast to left ventricular myocardium in which the ratio of longitudinal to transverse conduction velocities is approximately 3, propagation velocity in the crista terminalis is approximately 10 times greater in the longitudinal than in the transverse direction. To elucidate potential determinants of these distinct conduction properties, we characterized structural and molecular features of intercellular coupling in the crista terminalis and left ventricular myocardium of the canine heart. Analysis of the number and spatial orientation of myocyte interconnections at gap junctions revealed that a typical left ventricular myocyte was connected to 11.3 +/- 2.2 other myocytes. Approximately equal numbers of connections occurred between ventricular myocytes juxtaposed in side-to-side and end-to-end orientation. In contrast, a typical myocyte of the crista terminalis was connected to only 6.4 +/- 1.7 other cells (P < .05), but nearly 80% of these connections occurred between cells oriented in an end-to-end configuration. In comparison with the ventricular pattern, this spatial distribution of connections would limit intercellular current transfer between laterally apposed cells and thereby enhance anisotropy of conduction velocity in the longitudinal and transverse directions. Ultrastructural analysis showed that crista terminalis myocytes were connected by numerous small gap junctions that occurred in relatively simple, straight intercalated disks. Northern blot analysis showed approximately equivalent amounts of mRNAs encoding the gap junction channel proteins connexin43 and connexin45 but approximately four times more connexin40 mRNA in crista terminalis than in the left ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Expression of multiple gap junction proteins in human fetal and infant hearts.

Mammalian cardiac myocytes express multiple gap junction channel proteins or connexins. Expression patterns of the avian homologues of the mammalian cardiac connexins change during cardiac morphogenesis in association with changes in the electrophysiologic properties of intercellular junctions in chick cardiac myocytes. To determine whether expression of cardiac connexins is developmentally regulated in humans, we characterized connexin mRNA and protein content and distribution in hearts of 11 human fetuses (74 to 122 d gestational age), seven children (0.5 mo to 3 y of age), and two adults. Northern blot analysis identified transcripts of connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45) genes in all hearts analyzed. Cx40 mRNA was approximately 5-fold more abundant in samples from fetal hearts than in hearts of children or adults. However, fetal samples used for RNA extraction included atrial as well as ventricular myocardium, whereas samples from children and adults were exclusively ventricular. Northern analysis of adult human right atrial appendages revealed abundant Cx40 mRNA, thus suggesting that the greater amount of Cx40 signal seen on Northern blots from fetal hearts could have been attributable to atrial contributions. Neither Cx43 nor Cx45 mRNA varied significantly in amount in samples from the different developmental stages analyzed. Immunofluorescence identified abundant Cx43 in the known distribution of gap junctions in myocytes in sections of all hearts. Cx45 staining was inconspicuous in fetal hearts but was readily apparent in cardiac myocytes in hearts of older subjects. In contrast, Cx40 staining in the ventricle was confined to mural coronary arteries, apparently in endothelial cells, whereas in the atrium Cx40 staining at myocyte junctions was abundant.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Northern↗
PubMed1993

Physiologic relevance of the membrane attack complex inhibitory protein CD59 in human seminal plasma: CD59 is present on extracellular organelles (prostasomes), binds cell membranes, and inhibits complement-mediated lysis.

We demonstrate here that CD59, an inhibitor of the membrane attack complex (MAC) of the complement system, is present in cell-free seminal plasma (SP) at a concentration of at least 20 micrograms/ml. Analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and Edman degradation indicated that this protein, SP CD59, was similar, if not identical, to CD59 isolated from erythrocyte (E) membranes (E CD59). Like purified E CD59, SP CD59 also possesses a glycosyl phosphatidyl inositol (GPI) anchor and incorporates into the membranes of heterologous cells where it inhibits lysis by the human MAC. This phenomenon could be demonstrated not only if cells were incubated with purified SP CD59 but also if unfractionated SP were used. Further, CD59 in unfractionated SP bound to washed spermatozoa, increasing their membrane content of the protein. The mechanism by which this protein retains its GPI anchor while apparently present in the fluid phase is of interest and was further investigated. Using the techniques of high-speed centrifugation, fast performance liquid chromatography fractionation, and electron microscopy, we found that all detectable SP CD59 was associated with vesicular extracellular organelles. These organelles, named "prostasomes," were previously known to be present in SP and to interact with spermatozoa, although their function was uncertain. Interaction of heterologous E with prostasomes rendered the cells more resistant to lysis by human MACs. We propose that these organelles represent a pool of CD59 from which protein lost from spermatozoa, perhaps as a result of low level complement attack or of normal membrane turnover, can be replenished.

3T3 Cells↗
PubMed1993

Early results after pediatric cardiac transplantation with triple immunosuppression therapy.

Pediatric heart transplant recipients were previously reported to have higher early mortality and morbidity than do adult patients treated with triple immunosuppression therapy (steroids, azathioprine and cyclosporine). Nineteen patients (11 infants and 8 older children) underwent orthotopic transplantation using triple immunosuppression therapy. Surveillance for cellular rejection and coronary arteriopathy was performed with endomyocardial biopsy and selective coronary angiography in all patients, with continuous monitoring for hypertension and serious infection. Seventeen of 19 patients (89%; 10 infants and 7 older children) are current survivors, with a median follow-up of 29 months (range 17 to 94). There were 5 and 7 episodes of rejection in the first 12 months after transplantation in the infant and older groups, respectively, for actuarial freedom-from-rejection rates of 65% at 3 months and 54% at 12 months. Severe coronary arteriopathy was detected in 1 infant 11 months after transplantation. In the first 12 months after transplantation, there were 3 hospitalizations for infection, and 2 patients needed treatment for hypertension in the infant group, compared with 1 hospitalization for infection, and 4 patients on antihypertensives in the older group. An increased prevalence of noninfectious complications in the infant group led to significantly longer postoperative stays than in the older group (mean 27.3 vs 19.4 days; p < 0.05). The results indicate that cardiac transplantation using triple immunosuppression therapy in infants, children and adolescents is associated with a high survival rate, and low rates of rejection and serious infection.

Actuarial Analysis↗
PubMed1993

Renal dysfunction and intravascular coagulation with aprotinin and hypothermic circulatory arrest.

High-dose aprotinin was used in 20 patients undergoing primary or repeat operations on the thoracic or thoracoabdominal aorta using cardiopulmonary bypass and hypothermic circulatory arrest. The activated clotting times immediately before the establishment of hypothermic circulatory arrest exceeded 700 seconds in all but 1 patient. Three patients (15%) required reoperation for bleeding. Seven patients died during hospitalization, and 5 had postmortem examination. Platelet-fibrin thrombi were present in multiple organs including the coronary arteries of 4 patients with myocardial infarction or failure, the pulmonary arteries of 2 patients, 1 of whom died of acute right ventricular failure, the brains of 2 patients who sustained a stroke, and the kidneys of 4 patients, 3 of whom had development of renal dysfunction. Renal dysfunction occurred in 13 patients (65%), and all were 65 years of age or older. Five of these patients required hemodialysis. Among 20 age-matched patients who had similar operations without aprotinin, there was one hospital death (5%) from myocardial infarction, and renal dysfunction developed in 1 patient (5%), who did not require dialysis. None of these 20 patients required reoperation for bleeding. Although aprotinin has been shown to reduce blood loss in patients having cardiac operations employing cardiopulmonary bypass, this benefit was not attained in this group of patients with thoracic aortic disease in whom hypothermic circulatory arrest was used. Use of aprotinin in elderly patients undergoing these procedures was associated with an increased risk of renal dysfunction and failure, and of myocardial infarction and death.

Aged↗
PubMed1993

Subcellular distribution of endogenous long chain acylcarnitines during hypoxia in adult canine myocytes.

OBJECTIVE: Previous studies from our laboratory showed a pronounced increase in the sarcolemmal accumulation of long chain acylcarnitines in isolated neonatal rat myocytes after a prolonged (60 minute) hypoxic interval. Because much shorter intervals of hypoxia are associated with electrophysiological alterations in adult cells, the present study was performed to assess the extent of sarcolemmal accumulation of long chain acylcarnitines during hypoxia in adult canine myocytes. METHODS: Cells were incubated (for 24 hours) with 3H-carnitine and the uniformity of incorporation into carnitine fractions was verified biochemically. Cells were exposed to hypoxia (PO2 < 15 mm Hg) for 10 or 20 minutes in the presence or absence of sodium 2-[5-(4-chlorphenyl)-pentyl]-oxirane-2-carboxylate (POCA; 10 microM), an inhibitor of carnitine acyltransferase I. Cells were processed for electron microscopical autoradiography with a technique to spatially fix endogenous long chain acylcarnitines with selective and complete removal of short chain and free carnitine. Grain distributions were analysed by the maximum likelihood method from digitised micrographs. RESULTS: Total mass of long chain acylcarnitines increased ninefold [42.3(3.3) to 374(42) pmol.mg-1 protein] by 10 minutes of hypoxia and 15-fold [to 632(36) nmol.mg-1 protein] by 20 minutes of hypoxia. Normoxic cells exhibited little long chain acylcarnitines in the sarcolemma, and modest amounts in mitochondria and cytoplasm. In hypoxic cells, content of long chain acylcarnitines in mitochondria and cytoplasm increased by a maximum of twofold. By contrast, long chain acylcarnitines increased 100-fold in the sarcolemma to 4.18 x 10(6) molecules.microns-3 after 10 minutes of hypoxia. The increase in long chain acylcarnitines with hypoxia was completely prevented by pretreatment with POCA. CONCLUSION: Hypoxia in adult ventricular myocytes induces a rapid and preferential increase in endogenous long chain acylcarnitines within the sarcolemma.

Animals↗
PubMed1993

Heterogeneous transmural distribution of beta-adrenergic receptor subtypes in failing human hearts.

BACKGROUND: Downregulation of myocardial beta-adrenergic receptor density does not occur in a spatially uniform distribution in patients with congestive heart failure. Rather, it results primarily from loss of receptors in the subendocardium. In patients with dilated cardiomyopathy, beta 1-receptors have been found to be downregulated selectively. These observations suggest that considerable transmural heterogeneity in the distribution of beta-adrenergic receptor subtypes exists in the failing human heart. The present study was designed to test this hypothesis. METHODS AND RESULTS: We used quantitative autoradiography of radioligand binding sites to measure the distribution of beta-adrenergic receptor subtypes in transmural sections of left ventricular myocardium obtained from cardiac transplant patients with ischemic (n = 13) and idiopathic dilated (n = 12) cardiomyopathy and from 4 subjects with no history of cognitive heart failure. Analysis of radioligand binding isotherms revealed a significant reduction in total beta-adrenergic receptor density in hearts of patients with ischemic and idiopathic cardiomyopathy (20.3 +/- 1.9 and 18.2 +/- 2.0 fmol/mg protein, respectively, versus 40.0 +/- 11.4 in control subjects; P < .01 for both). Loss of the beta 1-subtype accounted for 86% of the total reduction in beta-receptor density in failing hearts. Despite the significant decreases in overall tissue receptor content, the densities of total beta-receptors and beta-receptor subtypes in subepicardial myocytes were equivalent in failing and control hearts. However, in contrast to control hearts, in which the transmural distribution of total and beta 1-receptors was uniform (endocardial: epicardial receptor density ratios, 0.97 +/- 0.14 and 1.0 +/- 0.2, respectively), hearts of patients with ischemic and idiopathic dilated cardiomyopathy had significantly lower total beta-receptor and beta 1-receptor densities in the subendocardium (ratios, 0.66 +/- 0.06 and 0.46 +/- 0.09 for total and beta 1-receptors, respectively, in ischemic cardiomyopathy and 0.60 +/- 0.08 and 0.52 +/- 0.11 in dilated cardiomyopathy; P < .001 for all values compared with a ratio of 1). Thus, beta 1: beta 2 receptor density ratios were markedly decreased in the subendocardium of ischemic and idiopathic dilated left ventricles compared with control hearts. CONCLUSIONS: A significant transmural gradient in the density of myocardial beta 1-adrenergic receptors exists in the hearts of patients with ischemic and dilated cardiomyopathy, resulting in a markedly altered beta 1: beta 2 receptor density ratio in the subendocardium.

Adrenergic beta-Antagonists↗