Biomedical subjects

J E Saffitz

Publications and source records attributed to J E Saffitz.

At least 55 records · Page 3Linked to original sources

PubMed1997

Reversible myocardial ischemic injury is not associated with increased creatine kinase activity in plasma.

Creatine kinase (CK) isoenzymes MM, MB, and BB are located primarily in the cell cytosol, and increased CKMB in plasma is the hallmark of myocardial infarction. However, whether CK is released with reversible ischemic injury remains controversial. Here, we assessed plasma CK activity--cytosolic and mitochondrial CK--in serial samples (every 10 min for 60 min, then hourly or every 4 h for 48 h) from 46 conscious dogs after transient or sustained coronary occlusion. Four dogs were sham-operated (controls); four underwent sustained coronary occlusion (96 h); and 38 underwent transient coronary occlusion (10-40 min) followed by 48 h of reperfusion. In postmortem histological examination of the dogs' hearts by light and electron microscopy, we looked for ischemia or necrosis. The presence of cell swelling and glycogen depletion was indicative of ischemia, whereas the added presence of cell disruption indicated necrosis. Coronary occlusion for > or = 20 min consistently increased plasma mitochondrial and total CK activity and produced histologically evident myocardial necrosis. In contrast, after 10 to 15 min of coronary occlusion, 12 of 14 animals, despite extensive severe reversible ischemia, showed no increase in plasma CK; the remaining 2, which had increased plasma CK, had subendocardial necrosis. Thus, cytosolic or mitochondrial CK is released from the heart only when there has been irreversible myocardial injury-a finding with significant diagnostic and therapeutic implications.

Animals↗

PubMed1996

Beta-adrenergic and muscarinic cholinergic receptor densities in the human sinoatrial node: identification of a high beta 2-adrenergic receptor density.

The objective of this study was to measure autonomic receptor densities in the human sinoatrial node and adjacent atrial myocardium to gain further insights into autonomic regulation of sinoatrial node function in the human heart. Sinoatrial nodes (n = 9) were acquired from human donors. Quantitative light microscopic autoradiography of radioligand binding sites in tissue sections was used to compare beta-adrenergic and muscarinic cholinergic receptor densities within specific tissue compartments of the sinoatrial node and adjacent myocardium. Total beta-adrenergic receptors were measured with the nonsubtype selective radioligand [125I]iodocyanopindolol. beta 2-Adrenergic receptors were determined by measuring the amount of radioactivity bound to sections incubated with radioligand in the presence of the highly beta 1-selective antagonist CGP-20712A. Specific autoradiographic grain densities were normalized to myocyte area/unit tissue area. Myocytes in the sinoatrial node occupied 47.7% +/- 0.1% of the total tissue area compared with 92.8% +/- 0.1% in myocardium (P < 0.001). Total specific beta-adrenergic receptor density per unit myocyte area was 3.5 +/- 0.9 times greater in the sinoatrial node than in myocardium (P < 0.001). The relative densities of beta 1-(4.2, P < 0.002), beta 2-(2.6, P < 0.002), and muscarinic (3.3, P < 0.001) receptors were significantly greater in the sinoatrial node than in the atrium. Thus, total beta-adrenergic and muscarinic cholinergic receptor densities are > 3-fold higher in the sinoatrial node than adjacent atrial myocardium, reflecting their specialized roles in regulating cardiac rate and rhythm. The beta 1-subtype is predominant in both regions. The beta 2-subtype, however, is > 2.5-fold more abundant in the sinoatrial node than in atrial myocardium. The relatively high beta 2-receptor density in the human sinoatrial node is consistent with physiologic studies that implicate this receptor in regulating cardiac chronotropism.

Adolescent↗

PubMed1996

Subcellular compartmentalization of Gs alpha in cardiac myocytes and its redistribution in heart failure.

Intracellular compartmentalization of G proteins may contribute to regulating signal transduction pathways in normal and failing myocardium. To test this hypothesis, we used postembedment immunogold electron microscopy to characterize the subcellular distribution of Gs alpha in normal canine and porcine left ventricular myocytes and in myocytes from a pacing-induced heart failure model in pigs in which beta-adrenergic signaling is impaired. Gs alpha was highly compartmentalized in normal canine myocytes and was localized specifically to the sarcolemma, intercalated disks, T-tubule and sarcoplasmic reticulum (SR) triads, and myoplasm. The highest Gs alpha concentration was observed in the intercalated disks. Only 20 +/- 5% of total cellular Gs alpha was localized to the sarcolemma. The triads and myoplasm compartments contained 45 +/- 13 and 27 +/- 8% of total cellular Gs alpha, respectively. The distribution of Gs alpha in normal porcine and canine myocytes was similar. However, in failing porcine myocytes Gs alpha was redistributed from the sarcolemma and T-tubule and SR triads to the myoplasm. The proportion of total cellular Gs alpha in the sarcolemma fell from 22 +/- 5 in normal to 11 +/- 4% in failing myocytes (P < 0.005), and the proportion in T-tubule and SR triads fell from 55 +/- 5 to 40 +/- 5% (P < 0.01), with a quantitatively corresponding increase in the proportion in the myoplasm from 19 +/- 3 to 43 +/- 4% (P < 0.0001). Thus redistribution of Gs alpha from the sarcolemma and the T-tubule and SR triads, where it may transduce beta-adrenergic signals, to internal sites where such actions may be precluded, might contribute to the pathophysiology of heart failure.

Animals↗

PubMed1996

Anisotropic activation spread in heart cell monolayers assessed by high-resolution optical mapping. Role of tissue discontinuities.

The role of tissue discontinuities in anisotropic impulse propagation was assessed in two-dimensional anisotropic monolayers of neonatal rat myocytes cultured on a growth-directing substrate of collagen. Activation spread and distribution of maximal upstroke rate of rise (Vmax) of the action potential were measured with an optical system using a voltage-sensitive fluorescent dye (RH-327) and a 10x10 photodiode array with a spatial resolution ranging from 7 to 15 microns. Activation maps were compared with the cellular architecture and the distribution of gap junctions obtained from immunostaining the gap junction protein connexin43 (Cx43). Four types of structures were studied: (1) dense cell cultures, (2) cultures with anisotropic intercellular clefts of variable size, (3) discontinuities created by inclusion of nonmyocyte cells, and (4) discontinuities resulting from nonuniform expression of gap junctions. In dense monolayers, activation spread was continuous with microinhomogeneities in both longitudinal and transverse directions. The average cell dimensions in such monolayers were smaller than in adult canine myocardium. However, the degree of cellular anisotropy (length-to-width ratio of 5.3 +/- 1.4) and connectivity were similar. The presence of small intercellular clefts (less than one cell in length) did not disturb the general pattern of transverse or longitudinal activation spread, but it was associated with wave front microcollisions during transverse propagation and a concomitant increase of Vmax beyond the cleft. Long intercellular clefts caused discontinuous transverse propagation. Conduction velocity and Vmax decreased significantly at narrow isthmuses formed by closely apposed clefts, rendering such sites susceptible for conduction block. In contrast Vmax increased when the wave front faced the borders of the clefts. Nonmyocyte cells were electrically connected to myocytes and served as sinks for electrotonic currents, thereby producing localized conduction slowing and a decrease in Vmax. Localized inhomogeneity in Cx43 distribution correlated accurately with circumscribed conduction block and changes in Vmax. Our results provide direct experimental evidence that the cellular structure and gap junction distribution correlate with action potential propagation and distribution of Vmax. We suggest that in tissue with a nonuniform anisotropy, connective tissue separating fiber bundles or sites of inhomogeneous connexin distribution may represent predilective sites for block in transverse direction.

Animals↗

PubMed1996

Functional and structural assessment of intercellular communication. Increased conduction velocity and enhanced connexin expression in dibutyryl cAMP-treated cultured cardiac myocytes.

Remodeling of conduction pathways in the hypertrophic response to myocardial injury is a potential mechanism leading to the development of anatomic substrates of lethal arrhythmias. To delineate the responsible mechanisms and to directly relate changes in intercellular coupling at gap junctions with electrophysiological alterations, we studied the effects of cAMP, a mediator of cardiac hypertrophy, on action potential conduction velocity and connexin expression in neonatal rat ventricular myocyte cultures. Conduction velocity was measured with an optical activation mapping technique in cells loaded with the voltage-sensitive dye RH-237. Action potentials were conducted 24% to 29% more rapidly (P < .005) after incubating cultures for 24 hours with the cAMP analogue dibutyryl cAMP (db-cAMP, 1 mmol/L). However, db-cAMP caused no change in the maximum rate of rise of the action potential upstroke, Vmax. Electron and immunofluorescence microscopy revealed a significant increase in the number and size of gap junctions in db-cAMP-treated cells. Immunoblotting showed that the total amounts of the ventricular gap junction proteins connexin43 and connexin45 (Cx43 and Cx45, respectively) increased 2- to 4-fold. Immuno-precipitation of metabolically labeled connexin proteins revealed a dose-dependent increase in the rate of Cx45 protein synthesis in myocytes exposed to db-cAMP ( > 2-fold after a 4-hour exposure) but no change in the Cx43 synthesis rate. Northern blot analysis demonstrated a time-dependent increase in the amount of Cx43 mRNA, with a maximum 3.3-fold increase after 4 hours of exposure to 1 mmol/L db-cAMP; cycloheximide did not block this effect. In contrast, Cx45 mRNA levels were not altered significantly after db-cAMP treatment. Thus, cAMP causes a significant increase in conduction velocity that appears to be attributable largely to enhanced expression of proteins responsible for intercellular communication. Cx43 and Cx45 levels appear to be upregulated by cAMP by disparate molecular mechanisms.

Action Potentials↗

PubMed1996

Echocardiographic characteristics of biopsy-proven cellular rejection in infant heart transplant recipients.

BACKGROUND: Echocardiography has been used as a primary means to detect cellular rejection in infant heart transplant recipients. There is, however, limited information correlating echocardiography and biopsy-proven rejection in this age group. METHODS: Between September 1989 and July 1994, 32 consecutive heart transplantations were done in infants younger than 20 months old, who were followed up for 2 to 58 months (mean 28 months) with concurrent endomyocardial biopsy and M-mode echocardiography with digitization. M-mode data from all 16 episodes of rejection (International Society for Heart and Lung Transplantation grade 3A or greater) that occurred in 12 grafts were compared with data from the same grafts with histologic resolution of rejection 2 weeks after treatment and with data from biopsy-proven nonrejecting control grafts matched for sex, time after transplantation, donor weight, and donor age. RESULTS: Left ventricular mass index increased in rejection (86 +/- 9 gm/m2) versus resolution (64 +/- 6 gm/m2) and versus that in nonrejecting control grafts (59 +/- 8 gm/m2). Left ventricular shortening fraction increased in rejection (40% +/- 2%) versus resolution (38% +/- 10%). Septal thickening fraction decreased in rejection (33% +/- 9%) versus nonrejection (68% +/- 16%). These changes became significant only in grafts transplanted more than 1 month before study. Substantial overlap of measurements prevented identification of threshold values. Intraobserver and interobserver variabilities for standard M-mode data were 7% to 8% and 12% to 22%, respectively, whereas those for digitized parameters were markedly elevated at 37% to 71% and 51% to 81%, respectively. CONCLUSIONS: We found (1) left ventricular mass index increases in cellular rejection but may be unreliable less than 1 month after transplantation and (2) significant interobserver and intraobserver variability may limit the applicability of digitized echo parameters to the detection of rejection in infant heart transplant recipients.

Acute Disease↗

PubMed1995

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

Animals↗

PubMed1995

Structural and molecular determinants of intercellular coupling in cardiac myocytes.

Electrical activation of the heart requires intercellular transfer of current through gap junctions connecting individual cardiac myocytes. Using a combination of light and electron microscopic techniques and molecular approaches, we have characterized the number, size, and spatial distribution of intercellular connections at gap junctions in cardiac myocytes and have also cloned, sequenced, and elucidated the subcellular distribution of three physiologically distinct gap junction channel proteins. In this review, we present evidence to suggest that the spatial distribution of myocyte interconnections and the molecular composition of gap junction channels may confer distinct conduction properties on specific tissues of the mammalian heart such as atrial and ventricular myocardium, and the nodes and bundles of the cardiac conduction system.

Animals↗

PubMed1995

Deleterious effects of a systemic lytic state on reperfused myocardium. Minimization of reperfusion injury and enhanced recovery of myocardial function by direct angioplasty.

BACKGROUND: The beneficial effects of flow restoration and the deleterious impact of reperfusion injury on ischemic myocardium are well known. However, most experimental studies have induced reperfusion by mechanical release of nonthrombotic occlusions, only occasionally in the presence of a systemic lytic state. Conditions differ markedly in patients undergoing pharmacological or mechanical recanalization of thrombotically occluded coronary arteries. Accordingly, this study was designed to determine whether the method of coronary occlusion and mode of recanalization influence the response of the heart to reperfusion. METHODS AND RESULTS: The acute effects of reperfusion on right ventricular (RV) function and histology were studied in open-chest dogs subjected to right coronary artery (RCA) balloon occlusion and deflation alone (group 1), pharmacological lysis of thrombotic occlusions (group 2), balloon occlusion with reperfusion induced by balloon deflation in the presence of a systemic lytic state (group 3), and recanalization of thrombotically occluded vessels by direct angioplasty (group 4). In all groups, 1 hour of RCA occlusion led to RV free wall (FW) dyskinesis. In group 1, reperfusion promptly improved RVFW function, with normal RVFW thickness and only minimal edema by microscopy. In contrast, in group 2, clot lysis led to acute RVFW swelling and impaired recovery of RVFW contraction associated with striking interstitial edema, contraction band necrosis, and hemorrhage by microscopy. In group 3, balloon deflation in the presence of a lytic state led to a similar but less severe pattern of abrupt RVFW swelling and impaired recovery of RVFW function but lesser histological alterations than in group 2. However, mechanical recanalization of thrombotically occluded vessels (group 4) led to prompt recovery of RVFW function without significant RVFW swelling or histological abnormalities. CONCLUSIONS: Our observations indicate that the responses of ischemic myocardium to reperfusion are influenced by factors beyond those effects attributable to ischemia and reperfusion per se. Pharmacological lysis of coronary thrombi results in alterations characteristic of reperfusion injury and associated with impaired functional recovery. Such changes are also evident, although to a lesser extent, when reperfusion of northrombotic occlusions is induced by mechanical recanalization in the presence of a systemic lytic state but not in its absence. However, such effects were not seen with direct mechanical recanalization of thrombotically occluded vessels. In aggregate, these findings indicate that induction of a systemic lytic state, together with products released by lysis of intracoronary thrombi, generates an inurious milieu that exerts adverse effects on reperfused myocardium.

Angioplasty, Balloon, Coronary↗

PubMed1995

Gap junction protein phenotypes of the human heart and conduction system.

INTRODUCTION: Gap junction channels are major determinants of intercellular resistance to current flow between cardiac myocytes. Alterations in gap junctions may contribute to development of arrhythmia substrates in patients. However, there is significant interspecies variation in the types and amounts of gap junction subunit proteins (connexins) expressed in disparate regions of mammalian hearts. To elucidate determinants of conduction properties in the human heart, we characterized connexin phenotypes of specific human cardiac tissues with different conduction properties. METHODS AND RESULTS: The distribution and relative abundance of Cx37, Cx40, Cx43, Cx45, and Cx46 were studied immunohistochemically using monospecific antibodies and frozen sections of the sinoatrial node and adjacent atria. AV node and His bundle, the bundle branches, and the left and right ventricular walls. Patterns of expression of these connexins in the human heart differed from those in previous animal studies. Sinus node gap junctions were small and sparse and contained Cx45 and apparently smaller amounts of Cx40 but no Cx43. AV node gap junctions were also small and contained mainly Cx45 and Cx40 but, unlike the sinus node, also expressed Cx43. Atrial gap junctions were larger than nodal junctions and contained moderate amounts of Cx40, Cx43, and Cx45. Junctions in the bundle branches were the largest in size and contained abundant amounts of Cx40, Cx43, and Cx45. Gap junctions in ventricular myocardium contained mainly Cx43 and Cx45; only a very small and amount of ventricular Cx40 was detected in subendocardial myocyte junctions and endothelial cells of small to medium sized intramural coronary arteries. Minimal Cx37 and Cx46 immunoreactivity was detected between occasional atrial or ventricular myocytes. CONCLUSIONS: The relative amounts of individual connexins and the number and size of gap junctions vary greatly in specific regions of the human heart with different conduction properties. These differences likely play a role in regulating cardiac conduction velocity. Differences in the connexin phenotypes of specific regions of the human heart and experimental animal hearts must be considered in future experimental or modeling studies of cardiac conduction.

Adult↗

PubMed1995

The molecular basis of anisotropy: role of gap junctions.

Electrical activation of the heart requires transfer of current from one discrete cardiac myocyte to another, a process that occurs at gap junctions. Recent advances in knowledge have established that, like most differentiated cells, individual cardiac myocytes express multiple gap junction channel proteins that are members of a multigene family of channel proteins called connexins. These proteins form channels with unique biophysical properties. Furthermore, functionally distinct cardiac tissues such as the nodes and bundles of the conduction system and atrial and ventricular muscle express different combinations of connexins. Myocytes in these tissues are interconnected by gap junctions that differ in tissue-specific manner in terms of their number, size, and three-dimensional distribution. These observations suggest that both molecular and structural aspects of gap junctions are critical determinants of the anisotropic conduction properties of different cardiac tissues. Expression of multiple connexins also creates the possibility that "hybrid" channels composed of more than one connexin protein type can form, thus greatly increasing the potential for fine control of intercellular ion flow and communication within the heart.

Anisotropy↗

PubMed1995

Modulation of connexin43 expression: effects on cellular coupling.

INTRODUCTION: Gap junctions connect cardiac myocytes allowing propagation of action potentials. They contain intercellular channels formed by multiple different connexin proteins. The arrangement and type of gap junctions and the types, function, and interaction of connexin proteins determine intercellular resistance and can thereby influence conduction velocity and the potential for reentrant arrhythmias. Our goal was to develop genetically manipulable models to test the effects of altering expression of a major cardiac connexin (connexin43) on intercellular coupling and expression of other connexin proteins. METHODS AND RESULTS: BHK cells that are poorly coupled and BWEM cells that are well coupled were stably transfected with plasmids containing connexin43 cDNA in antisense and sense orientations. RNA blots confirmed expression of the transfected transcripts. Immunoblots showed that connexin43 protein was reduced in the BHK antisense transfectants and increased in the BHK sense transfectants compared to the parental cells. It was not detectably changed in the BWEM antisense transfectant line compared to the BWEM parental cells. Transfection of connexin43 cDNA did not affect production of connexin45 mRNA and protein nor did transfection induce expression of other previously unexpressed connexin mRNAs. Cell coupling was assessed by intercellular diffusion of microinjected Lucifer yellow in confluent cell populations. Lucifer yellow passed to a mean of 3 +/- 3 neighboring parental BHK cells, to 8 +/- 8 neighbors in the sense connexin43 transfected BHK cells, and to only 2 +/- 2 neighbors in the antisense connexin43 transfected BHK cells (P < 0.05). In contrast, dye transfer did not differ significantly between the parental BWEM cells (mean transfer = 19 +/- 14 cells) and the BWEM connexin43 antisense transfectants (mean transfer = 15 +/- 12 cells) (P = 0.20). CONCLUSIONS: These data demonstrate that stable transfection with connexin43 cDNA constructs can result in detectable changes in connexin43 expression and cellular coupling without inducing compensatory changes in the cell's connexin phenotype and, therefore, may provide a basis for future attempts at specifically modulating connexin expression and intercellular resistance in cardiac tissues.

Animals↗

PubMed1995

The biologic behavior of balloon hyperinflation-induced arterial lesions in hypercholesterolemic pigs depends on the presence of foam cells.

Lack of a large-animal model of accelerated atherosclerosis has limited study of the biologic behavior of atherosclerotic lesions. We hypothesized that mechanical vascular trauma combined with diet-induced hypercholesterolemia would result in rapid development of complex atherosclerosis-like lesions. Accordingly, we induced deep injury to a carotid artery by repetitive balloon hyperinflations in minipigs that were fed either an atherogenic diet (n = 30) or a standard diet (controls, n = 4) and examined the resultant lesions 1 month later. The neointimal lesions that evolved in 23 patent vessels from cholesterol-fed animals were complex, exhibiting infiltration of smooth muscle and foam cells and evidence of organized thrombus, recent thrombus, hemorrhage, and calcification. Lesions were separable histologically into two groups: foam-cell rich (n = 12), with 33 +/- 10 foam cells per high-power field, and foam-cell poor (n = 11), with 4 +/- 1 foam cells per high-power field. Minipigs with foam cell-rich lesions had higher serum cholesterol levels than those with foam cell-poor lesions (712 +/- 178 vs 468 +/- 240 mg/dL, P < .02). The incidence of intralesional thrombus was also significantly greater in foam cell-rich than in foam cell-poor lesions (50% vs 9%, P < .04). In addition, the degree of luminal stenosis was greater in the presence of lesions containing thrombus compared with those without thrombus (60 +/- 38% vs 30 +/- 29%, P = .05). Lesions in the control animals were fibrocellular and lacked foam cells and thrombus. Thus, hypercholesterolemia appeared to affect lesion composition and behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Expression of multiple connexins in cultured neonatal rat ventricular myocytes.

Three gap junction proteins have been identified in mammalian cardiac myocytes: connexin43 (Cx43), connexin45 (Cx45), and connexin40 (Cx40). These proteins form channels with different electrophysiological properties and have different distributions in cardiac tissues with disparate conduction properties. We characterized the expression, phosphorylation, turnover, and subcellular distribution of these connexins in primary cultures of neonatal rat ventricular myocytes. Cx43, Cx45, and Cx40 mRNA were specifically detected in RNA blots. Immunofluorescent staining with antibodies specific for Cx43 and Cx45 revealed punctate labeling at appositional membranes, but no immunoreactive Cx40 was detected. Double-label immunofluorescence confocal microscopy of cultured myocytes revealed colocalization of Cx43 and Cx45. Cx43 and Cx45 were both identified by immunoprecipitation from [35S]methionine-labeled cultures, but anti-Cx40 antibodies did not precipitate any radiolabeled protein. Phosphorylated forms of both Cx45 and Cx43 were immunoprecipitated from cultures metabolically labeled with [32P]orthophosphate. Phosphoamino acid analysis demonstrated that Cx45 was modified on serine residues, and Cx43 was phosphorylated on serine and threonine residues. Pulse-chase labeling experiments demonstrated that the half-lives of Cx43 and Cx45 were 1.9 and 2.9 hours, respectively. Thus, both Cx43 and Cx45 turn over relatively rapidly, suggesting that myocardial gap junctions have the potential for dynamic remodeling. The results implicate multiple mechanisms of gap junction regulation that may differ for different connexins.

Animals↗

PubMed1995

Relative densities of muscarinic cholinergic and beta-adrenergic receptors in the canine sinoatrial node and their relation to sites of pacemaker activity.

The site of earliest extracellular electrical activation in the sinoatrial node (SAN) is known to shift in response to autonomic stimuli, but the mechanisms underlying this phenomenon and the determinants of the location of dominant pacemaker activity have not been elucidated. The present study was designed to characterize the spatial distribution of muscarinic cholinergic and beta-adrenergic receptors in the canine SAN and to determine whether a consistent relationship exists between autonomic receptor densities and the site of dominant pacemaker activity. We used quantitative light-microscopic autoradiography of radioligand binding sites to characterize the spatial distribution of muscarinic cholinergic and beta-adrenergic receptor subtypes in tissue sections containing the SAN and adjacent right atrial muscle from 18 canine hearts. Muscarinic receptor density was 5.4 times greater in SAN cells than in atrial myocytes (P < .01). Total beta-adrenergic receptor density was more than 3 times greater in SAN cells than in atrial myocytes (P < .0001), due entirely to the significantly greater number of beta 1-adrenergic receptors in the SAN. The region of dominant pacemaker activity, localized in 4 hearts with in vitro mapping, consistently exhibited greater densities of muscarinic and beta 1-adrenergic receptors than other SAN regions. Muscarinic receptor density in the dominant pacemaker region was 18 +/- 2% and 29 +/- 7% higher than in adjacent superior and inferior regions, respectively. beta 1-Receptor density in the dominant site was 53 +/- 5% and 26 +/- 4% higher than in adjacent superior and inferior SAN regions, respectively. Thus, the SAN is richly endowed with both muscarinic cholinergic and beta 1-adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

The carboxy terminus of tissue factor pathway inhibitor is required for interacting with hepatoma cells in vitro and in vivo.

Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that directly inhibits coagulation Factor Xa and also inhibits tissue factor-initiated coagulation. Normal human plasma TFPI exists both as the full-length molecule and as variably carboxy-terminal truncated forms. We reported recently that the low density lipoprotein receptor-related protein mediates the cellular degradation of TFPI after TFPI binding to the hepatoma cell surface. To examine whether the carboxy terminus of TFPI was required for interacting with hepatoma cells, a mutant of TFPI lacking the third Kunitz-type domain and basic carboxy terminus was generated. We found that this mutant, TFPI-160, did not compete with full-length 125I-TFPI-160 for binding to hepatoma cells. We were also unable to demonstrate specific binding of 125I-TFPI-160 to hepatoma cells at 4 degrees C. At 37 degrees C, significantly less 125I-TFPI-160 was internalized and degraded via low density lipoprotein receptor-related protein than full-length 125I-TFPI. Full-length 125I-TFPI binding to hepatoma cells could be inhibited > 90% by heparin and other highly charged molecules. Since TFPI, but not TFPI-160, was capable of effectively binding to cultured hepatoma cells, the fates of TFPI and TFPI-160 in vivo were examined. Both 125I-TFPI and 125I-TFPI-160 disappeared rapidly from the circulation after their intravenous administration into rats. The initial plasma half-life of 125I-TFPI was approximately 30 s whereas the half-life of 125I-TFPI-160 was approximately 4 min. 125I-TFPI was cleared predominantly by the liver. In contrast, 125I-TFPI-160 accumulated in the outer cortex of the kidney. Using microscopic autoradiography, we demonstrate that 125I-TFPI clearance is largely hepatocellular, whereas 125I-TFPI-160 accumulates mainly in the cells of the kidney proximal tubules. Together our findings demonstrate that the carboxy-terminal region(s) distal to amino acid 160 of TFPI mediates TFPI binding to hepatoma cells both in vitro and in vivo.

Animals↗

PubMed1995

Targeted mutagenesis of the transcription factor GATA-4 gene in mouse embryonic stem cells disrupts visceral endoderm differentiation in vitro.

Transcription factor GATA-4 belongs to a family of zinc finger proteins involved in lineage determination. GATA-4 is first expressed in yolk sac endoderm of the developing mouse and later in cardiac tissue, gut epithelium and gonads. To delineate the role of this transcription factor in differentiation and early development, we studied embryoid bodies derived from mouse embryonic stem (ES) cells in which both copies of the Gata-4 gene were disrupted. Light and electron microscopy demonstrated that embryoid bodies formed from wild-type and heterozygous deficient ES cells were covered with a layer of visceral yolk sac endoderm, whereas no yolk sac endoderm was evident on the surface of the homozygous deficient embryoid bodies. Independently selected homozygous deficient cell lines displayed this distinctive phenotype, suggesting that it was not an artifact of clonal variation. Biochemical markers of visceral endoderm formation, such as alpha-feto-protein, hepatocyte nuclear factor-4 and binding sites for Dolichos biflorus agglutinin, were absent from the homozygous deficient embryoid bodies. Examination of other differentiation markers in the mutant embryoid bodies, studies of ES cell-derived teratocarcinomas and chimeric mouse analysis demonstrated that GATA-4-deficient ES cells have the capacity to differentiate along other lineages. We conclude that, under in vitro conditions, disruption of the Gata-4 gene results in a specific block in visceral endoderm formation. These homozygous deficient cells should yield insights into the regulation of yolk sac endoderm development and the factors expressed by visceral endoderm that influence differentiation of adjoining ectoderm/mesoderm.

Animals↗

PubMed1995

Cardiac intercellular communication: consequences of connexin distribution and diversity.

Gap junctions contain channels which allow the exchange of ions and small molecules between adjacent cells. In the heart, these channels are crucial for normal intercellular current flow and the propagation of action potentials throughout the myocardium. Molecular cloning studies have demonstrated that these channels are formed by members of a family of related proteins called connexins each containing conserved and unique regions. There are several consequences of this multiplicity of connexins. Multiple connexins are expressed in differing, but sometimes overlapping, distributions within cardiovascular and other tissues. Connexin40, connexin43, and connexin45 are all found in cardiac myocytes, but their abundance differs in specialized cardiac regions with disparate conductive properties. Individual connexins form channels with differing voltage-dependence, conductance, and permeability properties, as demonstrated by functional expression of the cloned sequences. Connexins differ in their modification by phosphorylation, which may contribute to physiological regulation of intercellular communication. Expression of multiple connexins may lead to the formation of multiple channel types in a single tissue or cell and potentially allows mixing to form heterotypic and/or heteromeric channels. Thus, multiple connexins may contribute to the differences in intercellular resistance in cardiac regions with differing conductive properties and possibly may allow differences in the signalling molecules that pass between cells.

Animals↗