Raphael Zahler

Expression of α isoforms of the Na,K-ATPase in human heart

We studied expression of isoforms of Na,K-ATPase in normal and diseased human hearts. Na,K-ATPase alpha-isoform mRNA in samples from normal human left ventricle (LV) was composed of 62.5%, alpha 1, 15% alpha 2 and 22.5% alpha 3 on average. There was an increase in expression of the alpha 3 isoform in samples from failing hearts, but expression of all three isoforms decreased in pressure-overloaded right ventricle (RV).

The α3 Isoform Protein of the Na+,K+-ATPase Is Associated With the Sites of Cardiac and Neuromuscular Impulse Transmission

The alpha (catalytic) subunit of the Na+ pump (Na+, K(+)-ATPase) has three isoforms; alpha1 is ubiquitous, skeletal muscle expresses predominantly alpha2, and alpha3 has been localized to specific types of neurons and, possibly, to axonal processes. The alpha3 isoform mRNA is also expressed in the rat cardiac conduction system. Thus, we studied rat heart and quadriceps muscles by immunohistochemistry using isoform-specific antibodies to the Na+ pump alpha subunit and labeled alpha-bungarotoxin as a probe for the neuromuscular junction (NMJ). We found that alpha3 pump protein is localized to three sites important for impulse transmission: the junctional complex between cardiac myocytes, the heart conduction system, and the NMJ. Specifically, all levels of the conduction system expressed alpha3 immunoreactive protein, as assessed by two isoform-specific antibodies and histological conduction system markers. Specific expression at the junctional complex was confirmed by immuno-EM. Double-labeling and denervation analysis indicated that alpha3-positive areas in skeletal muscle were presynaptic and adjacent to postsynaptic bungarotoxin-positive regions, which had the classic morphology of NMJs. Thus, specific Na+,K(+)-ATPase pump isoforms may be adapted to maintenance of membrane potential and/or intracellular ion concentrations required for impulse transmission in both heart and presynaptic motor terminals contacting skeletal muscle.

Na, K-ATPase isoform gene expression in normal and hypertrophied dog heart.

ObjectivesThe catalytic α subunit of the sodium-potassium ATPase, the target of digitalis glycosides, has three isoforms is tissuespecific and developmentally regulated. While the effect of pressure overload on Na, K-ATPase isoform expression has been studied in rodent heart, there are no systematic data on this question in hearts of larger animals, which differ from those of rodents both in isoform composition and in glycoside sensitivity. Thus, we investigated the expression of Na, K-ATPase isoforms in normal dog heart; we also examined the effect of experimental left ventricular hypertrophy on isoform expression.Methodshypertrophy was produced by aortic banding. Expression was assessed by quantitative Northern and Western blotting, immuno-fluorescence, and3H-ouabain binding.ResultsRNA blotting indicated that the α3 isoform represented 11% of Na, K-ATPase mRNA in normal dog LV. Normal dog LV expressed α1 and α3 protein, but no detectable α2; immunoreactive α1 and α3 protein were also present in Purkinje fibers. There was a statistically significant decrease in total expression of all α isoform mRNAs in hypertrophied dog LV, resulting in a greater proportion of α1. The expression level of the α3 isoform mRNA and protein was lower in hypertrophied hearts.ConclusionsThese results indicate a greater proportion of α1 isoform pumps in experimental canine hypertrophy. Thus, shifts in Na, K-ATPase isoforms occur in pressure-overloaded heart in large animals as well as rodents.

Kinetics of drug effect by distributed lags analysis: An application to cocaine

The same plasma drug concentration may induce different effects depending on whether the concentration is rising or falling. We describe a new method of modeling kinetics of drug effects that can handle such “dissociation” phenomena, that does not depend on compartmental theory, and that allows quantitative comparisons between experiments. As applied to cocaine, it is shown that the resulting models are independent of drug dose or route of administration and give good predictions of the time course of response.

The α3 isoform protein of the Na+/K+-ATPase is associated with the sites of neuromuscular and cardiac impulse transmission

In heart and skeletal muscle the sodium pump is crucial for maintenance of membrane potential and cellular integrity. Of the three α-subunit isoforms, α2 and α3 mRNA’s are concentrated in cardiac conduction system (CCS) of normal adult rat (6). We thus studied expression of pump isoform proteins in normal rat heart and skeletal muscle, and in vascular endothelial tissue, using immunofluorescence, immuno-EM, and Western blotting with isoform-specific antibodies.

Isolated right atrial tamponade by serous fluid simulating tricuspid stenosis

Echocardiography is a key diagnostic tool in the recognition of pericardial tamponade. A 56-year-old man in whom severe dyspnea developed 22 days after cardiac surgery is described. Echocardiography suggested tricuspid valve disease but showed no pericardial abnormalities. Catheterization revealed functional stenosis of a normal tricuspid valve caused by loculated serous pericardial fluid.

Transgenic Mice Expressing Human α3 Na,K‐ATPase Isoform in Heart

The Na,K-ATPase is an enzyme crucial for normal mechanical and electrical function of the heart, because it supports cell volume regulation, membrane potential, and ion gradients for transport of other solutes. The a3 pump isoform is associated with the sites of conduction of the cardiac impulse; expression of a3 in adult rat heart is confined to the cardiac conduction system and the junctional complex.’ Also, our laboratory and others have shown that the a3 isoform differs functionally from a l , having higher ouabain affinity, lower Na+ affinity, and greater maximum turnover Isoform differences could thus underlie the well known discrepancy between the inotropic and conduction-system effects of cardiac glycosides. Also, a major consequence of myocardial ischemia is cell swelling caused by Na influx. Because hypertrophied hearts have fewer Na+ pumps and a higher percentage of a1 pumps, their increased vulnerability to ischemia could be offset by restoring high-capacity a3 pumps. Thus, to investigate the consequences of manipulating pump isoforms on transport and contractility in heart cells, we constructed transgenic mice (TGM) with cardiac-specific overexpression of human a3 Na,K-ATPase. We verified that normal mouse heart does not express detectable amounts of a3 mRNA and protein via Northern and Western blotting with isofom-specific reagents. We then prepared a construct in which a 640-bp fragment of a-myosin heavy chain 5’-upstream region drove expression of the full-length human a3 cDNA, terminated by the SV40 small t antigen splice and polyadenylation signal. An Sph I-Pvu I fragment of this plasmid (containing all the aforementioned elements but excluding almost all plasmid sequence) was isolated, purified, and injected into mouse oocyte pronuclei. A line of mice was developed which was hemizygous for this transgene and whose hearts reproducibly expressed a 97-kD membrane protein immunoreactive with isoform-specific anti-a3 antibody by both Western blotting and immunocytochemistry. Such immunoreactivity was not found in normal mouse heart or in any organ of TGM except brain and (at low levels) lung. Expression of immunoreactive a1 protein was reduced in TGM heart compared to normal heart, suggesting that a compensatory decrease in a 1 could exaggerate the effect of the transgenic isoform. TGM hearts were morphologically normal, and heart weighthody weight did not differ from that of controls. However, quantitative competitive (3H)ouabain binding on microsomes from TGM heart showed that the high-affinity component in TGM