The Cardiac Conduction System in the Rat Expresses the A2 and A3

Proc. Nati. Acad. Sci. USA Vol. 89, pp. 99-103, January 1992 Medical Sciences The cardiac conduction system in the rat expresses the a2 and a3 isoforms of the Na+,K+-ATPase (in situ hybridization/heart conduction system/atrioventricular node/Purkinje strand) RAPHAEL ZAHLER*t, MICHAEL BRINES*, MICHAEL KASHGARIANt, E. J. BENZ, JR.*, AND MAUREEN GILMORE-HEBERT§ Departments of *Internal Medicine, SPathology, and §Therapeutic Radiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510 Communicated by Vincent T. Marchesi, September 5, 1991 ABSTRACT The sodium pump is crucial for the function tially expressed in specialized cardiac conduction tissue. This of the heart and of the cardiac conduction system, which conjecture is reasonable because (i) the cardiac effects of initiates the heartbeat. The a (catalytic) subunit of this pump glycosides are especially prominent in the conduction system has three isoforms; the al isoform is ubiquitous, but the a2 and (although this action is also mediated indirectly via the a3 isoforms are localized to excitable tissue. Because rodent a2 nervous system), (ii) Purkinje fibers contain pumps that are and a3 isoforms are relatively sensitive to ouabain, which also more ouabain-sensitive than those of ventricular muscle, slows cardiac conduction, we studied heart-cell-specific expres- both by electrophysiologic and transport criteria (16-18), (iii) sion of pump isoform genes. Multiple conduction-system struc- electrophysiologic parameters have been found to differ tures, including sinoatrial node, bundle branches, and Pur- between Purkinje myocytes and working ventricular fibers kinje strands, had prominent, specific hybridization signal for (19-22). We thus used the technique of in situ hybridization a2 and a3 isoforms compared with adjacent working myo- to address this issue. cytes. This gene-expression approach may be useful for labeling conduction tissue and also for localizing specific membrane AND channels and receptors in this system. MATERIALS METHODS We used hearts from adult male rats to prepare the histologic The sodium pump (Na+,K+-ATPase), a ubiquitous enzyme sections. Sprague-Dawley rats (Charles River Breeding Lab- that extrudes sodium from the cell in exchange for potassium, oratories) were anesthetized with enflurane, and hearts were has many important physiologic functions in the heart, such removed through a midline thoracotomy. Hearts were im- as regulation of cell volume, generation of ion gradients for mersed in 4% (wt/vol) paraformaldehyde/phosphate- solute transport, and maintenance of voltage gradients in buffered saline for 2 hr and 15% sucrose/phosphate-buffered excitable tissue. Digitalis glycosides, an important class of saline for 3 hr (both at 4°C) and then bisected through the long cardiac drugs, are highly specific inhibitors of this enzyme. axis. These tissues were then embedded in OCT, frozen in Recently, Na+,K+-ATPase activity has been found to arise isopentane, and stored at -70°C until use. Frozen sections from a complex pattern ofgene expression involving, at least, (10 ,um) were cut at -20°C, thaw-mounted, and then frozen six loci. The functional enzyme is made up of distinct a and at -700C. ,l protein chains and possibly a third y subunit; the a subunit Isoform-specific antisense oligonucleotides (30-mers) for has three isoforms, and the P subunit has, at least, two the a isoforms were synthesized as described (8, 9) in such a isoforms. All three a isoforms are expressed in mammalian way that each oligonucleotide has 100o homology with the heart in developmentally complex ways (1-4). Thus, recently corresponding Na+,K+-ATPase isoform, but cross-homol- discovered changes (both regional, developmental, and ogy between a isoform-specific oligonucleotides is <23%. pathophysiologic) in Na+,K+-ATPase isoform distribution The specificity of these probes has been previously validated (5, 6) are likely to be important in cardiac physiology. by Northern (RNA) analysis (8). Because we had difficulty A complex cell-type-specific distribution of isoforms is achieving low background with the al oligonucleotide probe, already known to occur in several tissues, including brain and labeled RNA probe was prepared from a rat al isoform transport epithelia (7-9) at both the mRNA and protein levels fragment (23). This fragment, cloned into the pGEM4 vector, (10). In the heart, as in most tissues, al is the predominant was linearized and shortened by digestion with Nco I and a isoform; yet the pattern of developmental change and BamHI. Labeled RNA probe was synthesized as described response to stimuli are complex for the other isoforms. (24). Oligonucleotides were 3'-end-labeled with poly(adeno- Although not all previous workers found a3 isoform in rat sine [3 S-thio]triphosphate) using terminal transferase (New heart, recent data indicate that high-affinity isoform protein England Nuclear). Probes were diluted so that each section is present in this tissue despite the overall low glycoside received 3 x 105 dpm. sensitivity, suggesting that the isoenzymes may differ in their In all experiments, pairs of slides with serial sections were contribution to cardiac physiology (11). We and others have processed at the same time in the same solutions, with one shown regional variations in isoform gene expression in both slide of each pair incubated with antisense probe and the normal and pressure-overloaded heart (12-15), but the sig- other slide incubated with control probe. Paired slides were nificance of these changes is unknown. also exposed and developed together. Following the proce- The cardiac conduction system is responsible for the dure of Wilcox et al. (25), slides were thawed, fixed in 4% initiation and propagation of the heartbeat. Because in adult paraformaldehyde/phosphate-buffered saline at 4°C, and animals a3 isoform is found almost exclusively in the central washed in 0.5 X standard saline citrate buffer before and after nervous system, we suspected that this isoform is preferen- deproteination with 0.2 M HCl. For prehybridization, each section was covered with 50 ,ul of hybridization buffer [RNA M mM The publication costs of this article were defrayed in part by page charge probes: 50% (vol/vol) formamide/0.3 NaCl/20 Tris/5 payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 99 Downloaded by guest on September 26, 2021 100 Medical Sciences: Zahler et al. Proc. Natl. Acad. Sci. USA 89 (1992) Ilford K4 emulsion (Polysciences), and exposed for 7-21 Svc -- days. Slides were then developed in Kodak D-19 solution, counterstained with hematoxylin/eosin, and photographed SAN -- both by conventional light microscopy and with a confocal -- LBB epipolarization microscope system (Bio-Rad). AVN p RESULTS The main structures of the cardiac conduction system are shown in Fig. 1: the impulse is initiated in the sinoatrial node, travels through the atrioventricular node, and spreads through the right and left bundles to the Purkinje fibers, p which form strands branching out from the endocardium adjacent to the papillary muscles of both ventricles. At the FIG. 1. Sketch ofcardiac conduction system. SVC, superior vena level of the Purkinje strands (Figs. 2 and 3), we observed cava; SAN, sinoatrial node; AVN, atrioventricular node; RBB/ hybridization signal for the a2 and a3 probes in the special- LBB, right/left bundle branches; P, Purkinje strand; LV, left ven- ized conducting cells, which are round and pale-staining but tricle; RV, right ventricle; LA, left atrium; RA, right atrium. not in the more elongated, striated working muscle cells. Fig. 2B also shows that cells in the Purkinje strand and immediate mM EDTA/1 x Denhardt's solution/10% dextran sulfate/10 subendocardial area have much more intense a2 signal than mM dithiothreitol; oligonucleotide probes: 30% (vol/vol) those deeper in the septal myocardium, again suggestive of formamide/2x standard saline citrate/i x Denhardt's solu- conduction-system localization. tion/10% dextran sulfate/salmon sperm DNA at 100 ,g/ml, The sinoatrial node, a discrete structure at the junction of yeast at mM in a tRNA 100 ,g/ml/10 dithiothreitol], placed superior vena cava and right atrium (Fig. 4 A and B, straight humidified box, and incubated for 1-3 hr. Then, 10 ,l of arrows) also has intense signal for the a2 isoform when labeled probe was added directly to the buffer overlying each compared with neighboring vascular and atrial tissue (curved section, and the slides were incubated overnight at 55°C arrows). In the region of the right and left bundle branches (RNA probes) or 37°C (oligonucleotide probes)-5°C lower (Fig. 5A), the conduction tissue is recognizable as suben- than the calculated melting temperature Tm. After hybridiza- docardial bands oflighter-staining cells (arrows). High-power tion, slides were treated with RNase A (RNA probes only) for views of these bands probed for a3 isoform (Fig. 5 B and C) background reduction and were washed in 0.2x standard show deposition of silver grains in a linear pattern (arrows), saline citrate/10 mM mercaptoethanol/1 mM EDTA for 2 hr consistent with c3 mRNA in the right bundle (a linear pattern at 65°C (RNA probes) or 1.5 hr at 60°C (oligonucleotide of increased O3 signal was also seen in the left bundle, and probes). The slides were dehydrated, dried, coated with both bundles were also positive for a2 isoform; data not ..S~~~~~z.;@.~~~Y,Ai... *. ..........A FIG. 2. Purkinje strand, branching from septum into left ventricular (LV) cavity, probed for a2 isoform. (A) Trans- mission photograph, showing silver grains as black dots. Straight arrows, specialized conducting cells; curved ar- row, striated muscle cell. (x2400.) (B) Lower-power dark-field confocal image (montage ofthree frames), showing silver grains as white dots. (x1550.) Downloaded by guest on September 26, 2021 Medical Sciences: Zahler et al.

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