High fludarabine exposure and relationship with treatment-related mortality after nonmyeloablative hematopoietic cell transplantation.

Despite its common use in nonmyeloablative preparative regimens, the pharmacokinetics of fludarabine are poorly characterized in hematopoietic cell transplantation (HCT) recipients and exposure-response relationships remain undefined. The objective of this study was to evaluate the association between plasma F-ara-A exposure, the systemically circulating moiety of fludarabine, and engraftment, acute GVHD, TRM and OS after HCT. The preparative regimen consisted of CY 50 mg/kg/day i.v. day -6; plus fludarabine 30-40 mg/m²/day i.v. on days -6 to -2 and TBI 200 cGy on day -1. F-ara-A pharmacokinetics were carried out with the first dose of fludarabine in 87 adult patients. Median (range) F-ara-A area-under-the-curve (AUC((0-∞))) was 5.0 µg h/mL (2.0-11.0), clearance 15.3 L/h (6.2-36.6), C(min) 55 ng/mL (17-166) and concentration on day(zero) 16.0 ng/mL (0.1-144.1). Despite dose reductions, patients with renal insufficiency had higher F-ara-A exposures. There was strong association between high plasma concentrations of F-ara-A and increased risk of TRM and reduced OS. Patients with an AUC((0-∞)) greater than 6.5 µg h/mL had 4.56 greater risk of TRM and significantly lower OS. These data suggest that clinical strategies are needed to optimize dosing of fludarabine to prevent overexposure and toxicity in HCT.

Reversible down-regulation of connexin43 expression in acute cardiac allograft rejection.

We tested the hypothesis that cardiac allograft dysfunction in acute cardiac rejection may be related, in part, to diminished expression of connexin43, a gap junction channel protein that facilitates intercellular communication and coordinates electrical and mechanical cardiac function. We measured connexin43 levels using quantitative confocal immunofluorescence microscopy of endocardial biopsies from heart transplant recipients with histologic evidence of either no rejection or acute cellular rejection. Expression of connexin43 diminished significantly during acute cellular rejection and returned to baseline levels following resolution of rejection. Reversible down-regulation of connexin43 may contribute to ventricular dysfunction in allograft rejection.

Effects of diminished expression of connexin43 on gap junction number and size in ventricular myocardium.

Gap junction number and size vary widely in cardiac tissues with disparate conduction properties. Little is known about how tissue-specific patterns of intercellular junctions are established and regulated. To elucidate the relationship between gap junction channel protein expression and the structure of gap junctions, we analyzed Cx43 +/- mice, which have a genetic deficiency in expression of the major ventricular gap junction protein, connexin43 (Cx43). Quantitative confocal immunofluorescence microscopy revealed that diminished Cx43 signal in Cx43 +/- mice was due almost entirely to a reduction in the number of individual gap junctions (226 +/- 52 vs. 150 +/- 32 individual gap junctions/field in Cx43 +/+ and +/- ventricles, respectively; P < 0.05). The mean size of an individual gap junction was the same in both groups. Immunofluorescence results were confirmed with electron microscopic morphometry. Thus when connexin expression is diminished, ventricular myocytes become interconnected by a reduced number of large, normally sized gap junctions, rather than a normal number of smaller junctions. Maintenance of large gap junctions may be an adaptive response supporting safe ventricular conduction.

Voltage-gated Na+ channel activity and connexin expression in Cx43-deficient cardiac myocytes.

INTRODUCTION: Dynamic interplay between active and passive electrical properties of cardiac myocytes is based on interrelationships between various channels responsible for depolarizing and repolarizing ionic currents and intercellular conductances. Mice with targeted disruption of the connexin43 (Cx43) gene have hearts completely devoid of Cx43, the principal gap junctional protein expressed in mammalian hearts. METHODS AND RESULTS: To determine whether cardiac myocytes that develop in an abnormal environment of reduced intercellular coupling have altered active membrane properties, we studied whole cell action potentials, Na+ channel currents, and Na+ channel expression and distribution via immunoblotting and confocal immunofluorescence in neonatal ventricular myocytes isolated from Cx43 wild-type, heterozygous, and homozygous null hearts. Action potential morphology, peak Na+ current, activation and inactivation kinetics, and Na+ channel protein expression and distribution were not different among myocytes isolated from wild-type, heterozygous, or null hearts. Active membrane properties and Na+ channel activity were completely normal in Cx43-deficient myocytes isolated from hearts that have been shown to exhibit markedly reduced Cx43 expression, gap junction number, and epicardial conduction delay. CONCLUSION: Despite a genetic inability to produce Cx43 and a developmental history that culminates in marked gross cardiac morphologic abnormalities, premature death, and myocardial inexcitability ex vivo, cardiac Na+ channel distribution and function appear to be normal in Cx43 null hearts. Although intimate structural and functional interrelationships have been described between ion channels and gap junction channels, expression and function of Na+ channels is not affected by the absence of Cx43.

Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes.

OBJECTIVES: To elucidate signal transduction pathways regulating expression of myocardial gap junction channel proteins (connexins) and to determine whether mediators of cardiac hypertrophy might promote remodeling of gap junctions, we characterized the effects of angiotensin II on expression of the major cardiac gap junction protein connexin43 (Cx43) in cultured neonatal rat ventricular myocytes. BACKGROUND: Remodeling of the distribution of myocardial gap junctions appears to be an important feature of anatomic substrates of ventricular arrhythmias in patients with heart disease. Remodeling of intercellular connections may be initiated by changes in connexin expression caused by chemical mediators of the hypertrophic response. METHODS: Cultures were exposed to 0.1 micromol/liter angiotensin II for 6 or 24 h, and Cx43 expression was characterized by immunoblotting, confocal microscopy and electron microscopy. RESULTS: Immunoblot analysis revealed a twofold increase in Cx43 content in cells treated for 24 h with angiotensin II (n=4, p < 0.05). This response was inhibited by the presence of 1.0 micromol/liter losartan, an AT1-receptor blocker. Confocal and electron microscopy demonstrated enhanced Cx43 immunoreactivity and increases in the number and size of gap junction profiles in cells exposed to angiotensin II for 24 h. These effects were also blocked by losartan. Immunoprecipitation of Cx43 from cells metabolically labeled with [35S]methionine demonstrated 2.4- and 2.9-fold increases in Cx43 radioactivity after 6 and 24 h exposure to angiotensin II, respectively (p < 0.03 at each time point). CONCLUSIONS: Angiotensin II up-regulates gap junctions in cultured neonatal rat ventricular myocytes by increasing Cx43 synthesis. Signal transduction pathways activated by angiotensin II under pathophysiologic conditions could initiate remodeling of conduction pathways, leading to the development of anatomic substrates of arrhythmias.

Differential expression of gap junction proteins in the canine sinus node.

Electrical coupling of pacemaker cells at gap junctions appears to play an important role in sinus node function. Although the major cardiac gap junction protein, connexin43 (Cx43), is expressed abundantly in atrial and ventricular muscle, its expression in the sinus node has been a subject of controversy. The objectives of the present study were to determine whether Cx43 is expressed by sinus node myocytes, to characterize the spectrum of connexin expression phenotypes in sinus node pacemaker cells, and to define the spatial distribution of different connexin phenotypes in the intact sinus node. To fulfill these objectives, we performed high-resolution immunohistochemical analysis of disaggregated adult canine sinus node preparations. Using enhanced tissue preservation and antigen retrieval techniques, we also performed immunohistochemical studies on sections of intact canine sinus node tissue. Analysis of disaggregated sinus node preparations revealed three populations of pacemaker cells distinguished on the basis of connexin immunohistochemical phenotype: approximately 55% of cells expressed only connexin40 (Cx40); 30% to 35% of cells expressed Cx43, connexin45 (Cx45), and Cx40; and the remaining cells had no detectable connexin expression. In immunostained sections of intact sinus node, Cx43- and Cx45-positive cells were limited in their distribution and were observed in discrete bundles that appeared to abut atrial myocytes. In contrast, Cx40 immunoreactive signal was widely distributed in the sinus node region. These results indicate that subsets of pacemaker cells express distinct connexin phenotypes. Differential expression of connexins could create regions within the sinus node with different conduction properties, thereby contributing to the nonuniform conduction properties seen in this tissue.

Structural determinants of slow conduction in the canine sinus node.

INTRODUCTION: To elucidate the role of tissue structure as a determinant of the unique conduction properties of the sinus node, we compared the spatial distribution of intercellular connections at gap junctions in the sinus node to the more rapidly conducting crista terminalis and left ventricle, which have been studied previously. METHODS AND RESULTS: Samples of four canine sinus nodes were prepared for electron microscopy. The total number and spatial orientation of neighboring myocytes connected by ultrastructurally identified intercalated disks and gap junctions to nine randomly selected index cells were determined by sequentially examining subserial sections. Sinus node cells were sparsely interconnected compared to the extent of interconnections observed previously in other tissues. A typical sinus node cell was connected to only 4.8 +/- 0.7 neighbors compared with 11.3 +/- 2.2 cells in the left ventricle and 6.4 +/- 1.7 cells in the crista terminalis. Sinus node interconnections occurred at small intercalated disks that usually connected cells in partial side-to-side and end-to-end juxtaposition. In contrast, left ventricular myocytes are interconnected at large intercalated disks that adjoin many cells in pure side-to-side and end-to-end orientations. Crista terminalis myocytes are connected primarily in end-to-end fashion. The aggregate gap junction profile length per unit myocyte area was 26.5 times greater in the left ventricle and 5.0 times greater in the crista terminalis than in the sinus node. CONCLUSION: Sinus node myocytes exhibit small, sparsely distributed gap junctions that interconnect cells in complex patterns of lateral and terminal apposition. These structural features are consistent with the unique conduction properties of the sinus node.

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)

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.

Multiple connexins colocalize in canine ventricular myocyte gap junctions.

We have recently shown that adult canine ventricular myocytes express three distinct gap junction channel proteins, connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45). These proteins have unique cytoplasmic domains that likely confer connexin-specific physiological properties. To determine whether the three distinct channel proteins are distributed in identical or different populations of gap junctions, we performed double-label immunofluorescence on disaggregated canine ventricular myocytes incubated simultaneously with a mouse monoclonal anti-Cx43 and affinity-purified polyclonal rabbit antibodies against Cx40 or Cx45. Analysis of double-labeled cardiac myocytes using laser scanning confocal microscopy revealed virtually identical patterns of immunoreactivity for both the Cx43/Cx40 and Cx43/Cx45 pairs. Double-label immunoelectron microscopy confirmed that ultrastructurally identified cardiac myocyte gap junctions contain multiple channel proteins. Thus, three channel proteins colocalize in canine cardiac myocyte gap junctions. The presence of multiple functionally distinct connexins suggests complex possibilities regarding the composition of individual channels and the regulation of intercellular coupling.

Blood pressure, cigarette smoking and heart attack in the WHO co-operative trial of clofibrate.

In the WHO sponsored trial of clofibrate and its follow-up, about 15,000 men were observed for a mean period of 13.2 years. As expected, incidence of heart attacks (HA) was directly related to serum cholesterol, blood pressure (BP) and cigarette smoking. The previously reported lower incidence of HA in men receiving clofibrate compared with controls was most noticeable in hypertensive heavy smokers (P less than 0.01). BP was slightly lower in smokers than non-smokers (P less than 0.01). The difference in BP was greater in the trial visit before HA. Smokers also had higher plasma fibrinogen levels (P less than 0.05). The combination of reduced diastolic BP, and therefore myocardial perfusion pressure, with an increased thrombogenic tendency, might explain the high incidence of HA in smokers. Clofibrate apparently reduced fibrinogen levels, which might account for its specially good effect in preventing HA in smokers. However, the ill effects of smoking are still evident at a lower level in the men taking clofibrate and the drug is no substitute for giving up the habit. Clofibrate is not recommended for widespread use, except in subjects with marked lipid and/or blood fibrinogen abnormalities after dietary measures have been tried.