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Location of Ryanodine and Dihydropyridine Receptors in Frog Myocardium

Pierre Tijskens^*,, Gerhard Meissner and Clara Franzini-Armstrong^*

^* Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6058; Department of Anatomy and Human Physiology, University of Padova, Italy; and Department of Biochemistry/Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260

Correspondence: Address reprint requests to Clara Franzini-Armstrong, B42 Anatomy-Chemistry Building, Dept. of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6058. Tel.: 215-898-3345; Fax: 215-572-2170; E-mail: armstroc{at}mail.med.upenn.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Frog myocardium depends almost entirely on calcium entry from extracellular spaces for its beat-to-beat activation. Atrial myocardium additionally shows internal calcium release under certain conditions, but internal release in the ventricle is absent or very low. We have examined the content and distribution of the sarcoplasmic reticulum (SR) calcium release channels (ryanodine receptors, RyRs) and the surface membrane calcium channels (dihydropyridine receptors, DHPRs) in myocardium from the two atria and the ventricle of the frog heart using binding of radioactive ryanodine, immunolabeling of RyR and DHPR, and thin section and freeze-fracture electron microscopy. In cells from both types of chambers, the SR forms peripheral couplings and in both chambers peripheral couplings colocalize with clusters of DHPRs. However, although a low level of high affinity binding of ryanodine is detectable and RyRs are present in peripheral couplings of the atrium, the ventricle shows essentially no ryanodine binding and RyRs are not detectable either by electron microscopy or immunolabeling. The results are consistent with the lack of internal calcium release in the ventricle, and raise questions regarding the significance of DHPR at peripheral couplings in the absence of RyR. Interestingly, the free SR membrane in both heart chambers shows a low but equal density of intramembrane particles representing the Ca²⁺ ATPase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The relative contributions of Ca²⁺ influx from extracellular spaces and Ca²⁺ release from internal stores (the sarcoplasmic reticulum, SR) to excitation–contraction (e–c) coupling of cardiac muscle cells vary in different regions of the heart and in hearts from different species (Bassingthwaighte and Reuter, 1974; Reuter, 1974; Lederer, et al., 1989; see Bers, 1991, for a review). In the ventricle of rat heart, the SR is quite abundant and block of the SR Ca²⁺ pump by thapsigargin, resulting in depletion of the SR Ca²⁺ stores, reduces twitch tension and Ca²⁺ transients by 80% (Inesi et al., 1998). At the other end of the spectrum, frog myocardium relies on extracellular Ca²⁺, SR Ca²⁺ release is of small magnitude and its role in e–c coupling has been debated (Kavaler, 1974; Anderson et al., 1977; Niedergerke et al., 1976; Niedergerke and Page, 1981a,b; Morad and Cleemann, 1987; Tunstall and Chapman, 1994). Indeed, it is thought that SR Ca²⁺ in frog myocardium is called into play only under conditions of adrenergic and/or P₂ purinergic stimulation (Niedergerke and Page, 1981b).

The influx of extracellular Ca²⁺ in cardiac muscle takes place either through the dihydropyridine receptors (DHPRs), L-type voltage sensitive Ca²⁺ channels, located in exterior membranes (plasmalemma and T-tubules; see Ashley et al., 1991; Stern and Lakatta 1992; Cannell et al., 1995) or through other mechanisms such as Na⁺/Ca²⁺ exchange. Ca²⁺ release from the SR membrane is either via the ryanodine receptors (RyRs; see Coronado et al., 1994; Meissner 1994; Franzini-Armstrong and Protasi, 1997; Sutko and Airey 1997, for reviews), or possibly via the IP3 receptor (Niedergerke and Page, 1981b).

In cardiac muscle, activation of RyR is dependent on Ca²⁺ entry through DHPRs, and this interaction takes place at junctions called peripheral couplings, and dyads, between specialized domains of the SR and either surface membrane or T-tubules, respectively. DHPRs and RyRs are clustered in close proximity with each other at dyads and peripheral couplings and these junctions are also called Ca²⁺ release units (CRUs) on the basis of their function. Activation of additional, extrajunctional RyR clusters located at some distance from DHPRs (Sommer et al., 1991) must occur via a less direct mechanism, probably mediated by the Ca²⁺ entering the cell and/or released at peripheral CRUs (Mackenzie et al., 2001; Trafford et al., 2002).

Caffeine and ryanodine, two pharmacological agents that specifically affect the open probability and permeability of RyRs (Pessah et al., 1987; Rousseau and Meissner, 1989; Coronado et al., 1994; Ogawa, 1994), have been used to probe for SR Ca²⁺ release in frog heart. Interestingly, the effects of caffeine and ryanodine are quite different in atrium and ventricle. In frog atrium, high concentrations of caffeine (10 mM) increase the strength of the contractures initiated by either high-potassium or sodium-free solutions, whereas low concentrations (10 mM) enhance the twitch response (Chapman and Miller, 1974; Chapman and Miller, 1972; Niedergerke and Page, 1981a). In ventricle, on the other hand, the effect of caffeine is either absent (Niedergerke and Page, 1981a) or very small (Chapman and Miller, 1974), and the latter may be attributable to a direct effect on the myofibrils at the high concentrations of caffeine used (S. Page, personal communication; see also Wendt and Stephenson, 1983). In frog atrium ryanodine leads to a transient increase in twitch tension if applied in the presence of caffeine, but seems to have no effect in its absence (Tunstall and Chapman, 1994). No effect of ryanodine was reported for the ventricle, even though a large range of concentrations was used (Ciofalo, 1973). However, inasmuch as no caffeine was used in these experiments, the results are not comparable with those in the atrium.

Finally, adrenaline and ATP in conjunction with the action potential facilitate the release of Ca from the SR in frog heart, but the responses were more readily obtained in atrium than in ventricle. The concentration of ATP required to give a weak response in the ventricle is 10-fold larger than the concentration producing a stronger response in the atrium (Niedergerke and Page, 1981b).

All these results lead to the conclusion that, even though as most, if not all, the Ca²⁺ required for the activation of the twitch tension in frog heart cells is supplied by the inward Ca²⁺ current through the surface membrane (Tunstall and Chapman, 1994; Niedergerke et al., 1976), atrial and ventricular cells may also rely on Ca²⁺ release from the SR under special conditions and to a smaller extent than in mammalian and avian myocardium. In addition, the release may be due to different mechanisms in the two heart chambers (see above).

With this in mind, it is interesting to consider the structure of the SR in frog myocardium. Myocytes in both atrium and ventricle have small diameter and no T-tubules (Page, 1968). The cells basically contain few myofibrils and a network of relatively scarce, but well-organized SR. In both atrial and ventricular myocytes, flattened SR sacs have one surface closely associated with the surface membrane, forming peripheral couplings (Page and Niedergerke, 1972; Bossen and Sommer, 1984; Duvert and Verna, 1985), a type of CRU typical of cardiac muscle (Sommer and Johnson, 1980; Bossen and Sommer, 1984; Sommer et al., 1991). The rest of the SR is free and it is expected to contain the Ca²⁺ pump protein. The surface densities of SR are greater in atrium than in ventricle (0.441 ± 0.067 vs. 0.264 ± 0.034 µm²/µm³, respectively) whereas the surface density of junctional SR is the same in the two chambers (Bossen and Sommer, 1984).

None of the above structural data are sufficient to explain the functional differences in Ca²⁺ release in atrium and ventricle. We have examined the distribution of RyRs and DHPRs in the two chambers of the frog heart, using electron microscopy in combination with immunolabeling at the light microscope level and ryanodine binding. We find that RyRs are present in limited amounts in peripheral couplings of the atrium, but are virtually absent from the ventricle. Unexpectedly, however, the disposition of DHPRs in clusters related to the position of peripheral couplings is qualitatively similar in cells from the two chambers.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
[³H]ryanodine binding
The ryanodine receptor contents of atria and ventricle were determined by isolating the 3-[(3-cholamidopropyl) dimethyl-ammonio]-1-propanesulfonate (CHAPS)-solubilized, [³H]ryanodine-labeled 30S receptor complexes by sucrose density gradient centrifugation. The experiments were done in duplicate. Either combined atria from three hearts or a complete ventricle were homogenized in a glass/ground glass homogenizer in 1 ml 1 M NaCl, 20 mM Na Pipes, pH 7.4, 100 µM EGTA, 400 µM Ca²⁺, 1.5% CHAPS, 5 mg/ml 95% phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL), 5 mM AMP, 0.2 mM Pefabloc 20 µM leupeptin, and 1 mM dithiothreitol. Homogenates were labeled for 30 min at 24°C with 35 nM [³H]ryanodine (NEM Life Science Products, Boston, MA) in the absence and presence of 10 µM unlabeled ryanodine (Calbiochem, San Diego, CA). To localize RyRs on the gradients, 30S ryanodine receptor complexes were isolated by sucrose density gradient centrifugation (Anderson et al., 1989). Matched experiments were carried out with canine cardiac SR vesicles and the results were consistent with published data (Anderson et al., 1989). Radioactivity of the sucrose gradient fractions was counted by liquid scintillation to obtain [³H]ryanodine comigrating with 30S receptor complexes in gradient fractions.

Electron microscopy
Whole hearts from adult Rana temporaria were quickly removed, opened by a single longitudinal cut through atria and ventricle, pinned to Sylgard with the trabeculae in a slightly stretched position, rinsed in frog Ringer's solution (KCl 2.5 mM, NaCl 115 mM, CaCl₂ 1.8 mM, and NaPO₄ buffer 3 mM) for a few minutes, and fixed in 6% glutaraldehyde in 0.1-M cacodylate buffer, pH 7.4. For thin sectioning, trabeculae were gently teased out, postfixed in 2% OsO₄ in cacodylate buffer for 1 h, en bloc stained in saturated aqueous uranyl acetate over night, and embedded in Epon 812. Thin sections were stained in 4% uranyl acetate in 50% ethanol and with "Sato" lead (Sato, 1968).

For freeze-fracture, atrial and ventricular trabeculae were teased from the glutaraldehyde-fixed hearts, infiltrated with 30% glycerol, frozen in liquid-nitrogen-cooled propane, fractured, shadowed with platinum at 45°, and replicated with carbon in a model 400 Balzers freeze-fracture apparatus (Balzer, Fürstentum, Liechtenstein).

Thin sections and replicas were examined at 60 kV in a Philips 410 electron microscope (Philips Electron Optics, Mahwah, NJ).

Immunolabeling
For cryosections, the heart was quickly removed and large portions of the heart, including most of either atrium or ventricle, were frozen in liquid-nitrogen-cooled propane. Cryostat sections, 5–10 µm, were cut at -20°C. The frozen sections were fixed in methanol at -20°C for 30 min and rehydrated in phosphate-buffered saline (PBS; NaCl 140 mM, KCl 2.7 mM, Na₂HPO₄ 15 mM, KH₂PO₄ 1.5 mM, pH 7.4). For whole mount immunolabeling, hearts were quickly removed, opened and pinned, rinsed in frog Ringer's solution for 20 min, fixed in 1% paraformaldehyde and 0.5% Triton in PBS for 20 min at room temperature, and washed in PBS for 15 min. Small groups of trabeculae were dissected free.

Sections and whole mounts were blocked with PBS containing 1% bovine serum albumin (BSA) and 10% goat serum at 4°C for 1 h, incubated with primary antibody at 4°C overnight, with Texas Red-labeled secondary antibody for 1 h at room temperature with three washes in PBS–BSA in between, and mounted in antibleaching solution (0.0025% para-phenylenediamine, 0.25% 1, 4 diazobicyclo 2, 2, 2 octane, and 5% n-propylgallate in glycerol). In three single-labeling experiments, atrial and ventricular tissues were processed in parallel. For double labeling, sections and/or whole mounts were simultaneously incubated with a mixture of two primary antibodies and then with a mixture of Texas Red- and fluorescein-labeled secondary antibodies. Specimens were examined with a confocal Zeiss LSM 510 microscope (Carl Zeiss, Switzerland). All antibodies were diluted into the PBS–BSA solution.

The primary antibodies used were: 34C, a mouse monoclonal antibody generated against avian RyR (Airey et al., 1990), which recognizes all types of RyRs across species (Developmental Studies Hybridoma Bank, The University of Iowa); CI2, a polyclonal antibody which recognizes the GST-tagged fusion protein containing residues 785–930 of the rabbit _1C subunit of the L-type Ca²⁺ channel (Gao et al., 2000; kindly provided by M.M. Hosey); and an anti-s--actinin rabbit polyclonal antibody (Ojima et al., 1999). The following IP3 antibodies were tried without success: Chemicon (Cat. #MAB3078), against type 1,2,3; a polyclonal antibody against the C-terminus of rat (Takei et al., 1992), kindly donated by P. DeCamilli; CT1, a polyclonal antibody raised against a C-terminus peptide of rat type 1 IP3R (Wojcikiewjcz et al., 1994); three monoclonals (T1NH, T2NH, and T3NH) against the amino terminal regions of rat types 1–3 IP3R (kindly donated by G.A. Mignery).

The secondary antibodies used were: Texas Red-conjugated goat anti-mouse IgG (from Molecular Probes, Cat. #T-862); Texas Red-conjugated goat anti-rabbit IgG (from Cappel, Cat. #55675 and from Jackson Immuno Research Laboratories, Cat. #111-075-144); fluorescein (FITC)-conjugated goat anti-rabbit IgG (H+L; from Jackson ImmunoResearch Laboratories, Cat. #111-095-045); and fluorescein (FITC)-conjugated goat anti-mouse IgG (H+L; from Jackson ImmunoResearch Laboratories, Cat. #111-095-146).

Measurements
All micrographs for the measurements were collected using a criterion of randomness. Specifically, either the first fiber or the first peripheral coupling to appear on the EM screen for each grid opening was photographed.

The width of the junctional gap between SR and surface membrane of peripheral couplings was determined by measuring the distance between the cytoplasmic edges of the two membranes in EM micrographs taken at a magnification of 24,400x and digitally scanned at high resolution (1200 dpi), using the "ruler" tool of Adobe Photoshop.

Sarcomere length (between the center of two Z-lines) and the center-to-center distances between the Z-lines and either peripheral couplings (in thin sections) or clusters of particles (in freeze-fracture) were measured with a ruler on micrographs printed at final magnifications of 20–34,000x.

The surface areas of particle clusters were measured using NIH Image, from micrographs of freeze-fracture replicas taken at the magnification of 33,900x.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Ryanodine binding assays
Sucrose gradient experiments and radioactive ryanodine binding assays indicate an average RyR content of 20–50 fmol for the duplicate experiments using three combined atria from three frogs and less than 3 fmol for duplicate data from a single ventricle. The latter value is below the detection limit. By comparison, the RyR content of mouse and rat hearts is 25 times higher than in frog atrium, when expressed on a protein basis (G. Meissner, unpublished data). Considering that the atria together have considerably smaller muscle mass than the ventricle (by a factor of 4, as indicated by a rough measurements of wet weights), this indicates that the content of RyR of atrium myocardium, even though much lower than in mammalian myocardium, greatly exceeds that in ventricle. Indeed, RyR appears to be essentially absent from ventricle. The RyR content of the atrium, however, is also very low compared with that in mammalian myocardium.

Immunohistochemistry
Fig. 1 shows longitudinally oriented optical sections from confocal images of atrial and ventricular trabeculae labeled with antibodies for RyR, -actinin, and DHPR, singly or in combination. In both chambers, myocytes are very narrow and elongated. In a thin (1-µm thick) optical section, the cell surface is seen either as parallel lines separated by distances of 5–10 µm where the optical section cuts the cells in the middle, or as a wide strip where the section is tangent to the cell surface. Images from whole mounts and frozen sections did not differ in any significant detail.

View larger version (132K): [in this window] [in a new window]   FIGURE 1  Whole mounts (A,B) and frozen sections (C–G) of atrial and ventricular myocardium labeled with antibodies against RyR, DHPR, and -actinin, a Z-line protein. Longitudinal axes of fibers are approximately horizontal. In the atrium, RyR-positive foci (A–C) are aligned along parallel lines that delimit the borders of the narrow cells (from left to right in the image). The spacing between adjacent RyR spots in the longitudinal direction is equal to the sarcomere length (A, arrows). Either a single or a double set of RyR foci are located at each sarcomere (arrows and double arrows, A and B). Where the plane of the confocal image happens to be parallel to one of the wider sides of a cell, the RyR foci are aligned in an approximately transverse direction (arrowheads). Double labeling for RyR and -actinin (C) shows that RyR foci are located at or close to the position of the Z-lines. DHPRs are located in foci with a disposition similar to that of RyR foci (D), and double labeling for the two proteins indicates a frequent but not obligatory overlap of the foci position (E and F, yellow spots). In the ventricle (G), DHPR antibody labeling gives a Z-line-related position similar to that in the atrium, but RyR antibody (See Fig. 2) gives no labeling.

View larger version (63K): [in this window] [in a new window]   FIGURE 2  Comparison of labeling for RyR using 34C antibody in atrium (A) and ventricle (B). The image for the ventricle was recorded at a high gain in the confocal microscope, thus enhancing the finely granular nonspecific background which is also seen in the atrium between the positively labeled foci. The ventricle shows no periodically disposed brightly fluorescent foci. If taken with the same gain as Fig. 2 A, Fig. 2 B would be totally dark.

CI2, a primary antibody against the 1-cardiac subunit of the DHPR, reveals foci of DHPRs at the periphery of atrial cells (Fig. 1 D). The labeling pattern is similar to that shown by RyR, although the images show more nonspecific background staining. The DHPR foci are located in longitudinal rows at the intersection of the cell surface with the optical section, and they show a sarcomere-related periodicity (Fig. 1 D, arrowheads). Where the section grazes the cell surface, DHPR positive spots are aligned in small transversely oriented rows located at 2-µm intervals.

Double immunolabeling of frozen sections from atrial trabeculae with antibodies 34C and CI2 reveals that foci of the two proteins occupy closely related positions and that there is frequent but not complete colocalization (yellow) of RyR (red) and DHPR (green) foci (Fig. 1, E and F). The colocalization is not perfect, as either the green (RyR) or the red (DHPR) labels extend in many instances beyond the area of overlap between the two, and also some foci are labeled exclusively by either antibody. This indicates that groups of RyRs and DHPRs are located in close proximity to each other, but that both proteins are not always simultaneously present at the same site and over an equivalent area of membrane.

In ventricle, no RyR foci were detected even using the highest intensity gain in the confocal microscope (Fig. 2). The presence of RyR-antibody clusters in atrium and the virtual absence in ventricle were confirmed in three separate experiments using parallel immunolabeling of both whole mounts and frozen sections from the two chambers. Given the fact that the antibody used recognizes different RyR in a large variety of vertebrate muscles (Airey et al., 1990; Olivares et al., 1991), and given the positive labeling of the atrium RyR, it is unlikely that lack of labeling in the ventricle could be assigned to a failure of the antibody to bind to RyR rather than to an absence of the protein. Ryanodine binding experiments (see above) and thin sections electron microscopy (see below) fully confirm this assumption. Labeling of ventricular trabeculae with anti-DHPR antibodies, on the other hand, revealed clusters of DHPR in foci arranged along transverse lines at sarcomeric spacings (Fig. 1 G) which are similar to those in trabeculae from atrium.

Electron microscopy
Thin sections
In both atrium and ventricle of frog myocardium, the SR forms a network of elongated sacs and flat cisternae. The latter lie mostly in the space between the myofibrils and the surface membrane (Fig. 3), as previously described (Page and Niedergerke, 1972; Bossen and Sommer, 1984). Here we focus on the flat cisternae that are closely apposed to the surface membrane. Most of the flat cisternae are in close apposition to the surface membrane from which they are separated by a small distance (Fig. 4, A–E). We consider all such cisternae as components of peripheral couplings, although close examination reveals that they are not all equal (see below). Cisternae that are not apposed to the surface and SR elements with smaller sectional profiles (not shown, but see Page and Niedergerke, 1972) are considered to be part of the free SR.

View larger version (152K): [in this window] [in a new window]   FIGURE 3  Cross section of two adjacent cells in the atrium. The narrow extracellular space runs obliquely in the image. The cell at the left is sectioned at the edges of the A-band and shows the transition between A- and I-bands (A, I). The cell on the right is sectioned approximately at the level of the Z-line and shows Z-line and I-band (Z, I). Three flat SR cisternae are closely apposed to the surface membrane (between arrows) and are part of peripheral couplings. A fourth SR vesicle (between arrowheads) is probably not a component of a peripheral coupling, but rather is part of the free SR, inasmuch as its lumen is empty (that is, it contains no calsequestrin).

View larger version (228K): [in this window] [in a new window]   FIGURE 4  Details of peripheral couplings in the atrium (A–C, F) and the ventricle (D, E). In atrial cells, some peripheral couplings show a row of fairly evenly spaced feet in the junctional gap between the SR vesicle and the sarcolemma (A, B, F between asterisks). Other peripheral couplings have feet that do not completely fill the gap (arrows in B and F). Finally a fairly high proportion of the junctions show no feet (C). In ventricular cells none of the vesicles associated with the surface membrane show rows of feet (D), but a very small percentage show few single feet (arrows, E). F is a higher magnification of B.

Peripheral couplings in ventricles lack periodically disposed feet (Fig. 4 D). A few (14% from a total of 175 junctions in four frogs) have occasional densities that may represent feet; however, these are not clearly delineated and are not periodically disposed (Fig. 4 E, arrows).

The size of the junctional gap separating SR and surface membranes is fairly constant in the peripheral couplings from the atrium that contain periodically disposed feet. In contrast, peripheral couplings that lack feet in atrium and ventricle have variable distances between SR and surface membrane, and sometimes the gap is large (Fig. 4 C).

The width of the junctional gap between the apposed SR and surface membranes was measured in peripheral couplings of atrial and ventricular myocytes from three frogs. For atrium, junctions were divided into three categories: those that contain a periodically disposed row of feet; those that have few, not clearly aligned feet; and those that lack feet. In all cases, the gap width was measured only along the portions of cisternal surfaces that are approximately parallel to the surface membrane. Segments of vesicle profiles that are clearly detached were not considered. The values for the gap width in the three types of junctions defined above are, respectively: (8.3 ± 1.5 nm, n = 19; 7.4 ± 1.2 nm, n = 18; 7.2 nm ± 1.6, n = 86; mean ± 1 SD, n = number of junctions). In ventricle junctions where few feet may be present, the gap is wider (9.0 ± 2.0 nm, n = 19) than in dyspedic junctions (7.4 ± 2.0, n = 117). The differences between junctions containing and lacking feet in atrium and ventricle are significant at P-levels of 0.0072 and 0.0015, respectively (Student's t-test).

Calsequestrin has a recognizable structural signature in the electron microscope. It appears as an electron-dense, fine-grained content in the lumen of junctional SR (Sommer, 1995; Jorgensen et al., 1985; Jewett et al., 1971; Jones et al., 1998). Calsequestrin is present in frog myocardium (MacLeod et al., 1991) and it is visible as granular density in the SR of atrial peripheral couplings with feet (Fig. 4, A and B). However, calsequestrin is less visible and/or absent in peripheral couplings that have no or few feet in both atrium and ventricle. It is also absent from the rest of the SR.

Freeze-fracture
In freeze-fracture replicas of both atrium and ventricle the cytoplasmic leaflet of the surface membrane shows clearly visible specialized membrane domains, containing clusters of large, tall particles (Fig. 5). Particles of variable and smaller sizes are mostly excluded from the special membrane domains. Exclusion of smaller particles is less complete in ventricle than in atrium leaflets. The large particles within the cluster are of a fairly uniform height and diameter, indicating a homogenous protein composition. The average particle diameters measured perpendicular to the direction of shadowing are 10.0 ± 1.6 nm for atrium and 9.4 ± 1.6 nm for the ventricle (mean ± 1 SD, from 100 particles each). The minor difference between the two chambers is attributable to slight variations in the thickness of the platinum shadow.

View larger version (230K): [in this window] [in a new window]   FIGURE 5  Images from freeze-fracture replicas of the plasmalemma in atrial (A, B) and ventricular (C, D) myocardial cells. Clusters of large particles are visible in the cytoplasmic leaflet. The size of the domains occupied by large particles and the total number of particles in each cluster vary, but in general both parameters are larger in atrium than in ventricle. Two characteristics of these clusters of particles resemble those of DHPR-containing membrane domains in avian myocardium: the particles are unusually tall and large in diameter, and smaller particles are mostly excluded, although the exclusion is not as stringent in the ventricle as it is in the atrium. The outlines in (A) and (C) indicate the approximate boundaries that were used to delimit the areas to be measured (see text for discussion of measurement accuracy).

View larger version (89K): [in this window] [in a new window]   FIGURE 6  Freeze-fracture of atrial (A and B) and ventricular (C) myocytes showing SR membranes. (A) Cytoplasmic leaflets of two elongated, free SR vesicles. The relatively scarce small particles are fractures through the SR ATPase. The SR approaches the surface membrane at the arrowhead and presumably continues right under the membrane. The adjacent surface membrane contains a patch of large intramembranous particles (between arrows). (B) The luminal leaflet of the SR between arrows shows small pits surrounded by shallow bumps characteristic of the fractured intramembrane region of RyRs in the SR membrane (Block et al., 1988; Felder and Franzini-Armstrong, 2002). The transition from generic to large particle patch is seen in the adjacent surface membrane, where some large particles are present (arrowheads) and smaller particles are mostly absent. (C) Luminal (arrow) and cytoplasmic (arrowhead) leaflets of free SR in the ventricle. The cytoplasmic leaflet has a small number of intramembranous particles.

View larger version (166K): [in this window] [in a new window]   FIGURE 7  Montage of images from longitudinal sections of atrial and ventricular trabeculae. Each image shows narrow strips at the fibers' edges. The sites of peripheral couplings are not clearly visible at this magnification, but are indicated by semicircles. Most of peripheral couplings are located at or near the Z-lines (Z) of the nearest myofibrils. A few are located opposite the central region of the A-band.

View larger version (48K): [in this window] [in a new window]   FIGURE 8  Montage of low magnification images from a freeze-fracture replica of the plasmalemma in an atrial cell. Arrows point to slight furrows that indicate the Z-line position of the underlying myofibrils. The positions of particle clusters (visible at higher magnification) are indicated.

View larger version (29K): [in this window] [in a new window]   FIGURE 9  Frequency distribution of the ratios between the Z-line-to-peripheral coupling distance and the sarcomere length measured from thin sections (dotted bars), and of the ratios between the Z-line-to-particle cluster distance and the sarcomere length measured from freeze-fracture replicas (solid bars). A ratio of 0.5 indicates a position opposite the center of the A-band. The majority of peripheral couplings in both atrium (A) and ventricle (B) are located at or in proximity to the Z-line; fewer are opposite the A-band of the neighboring myofibrils.

Overall density of large clustered particles
To determine the total density of DHPRs per cell surface area, we determined the mean density of DHPR clusters per surface area, and counted the number of particles per cluster. The surface density of DHPR clusters relative to the cell surface area is approximately three times higher in atrial than in ventricular cells. The clusters were counted on montages of electron micrographs covering extended areas of cell surface (see Fig. 8) and the results indicate densities of 0.7 ± 0.2 clusters/µm² of cell surface n = 5 cells and 0.2 ± 0.1 clusters/µm², n = 8 respectively for atrium and ventricle. Atrial clusters contain 37 ± 23 particles (mean ± 1 SD, n = 67 clusters), vs. 20 ± 14 (n = 47) or ventricular clusters. The accuracy of particle counts is affected by the fact that the boundaries of the cluster are not always very clear, and thus it is sometimes difficult to decide whether some of the more peripheral particles belong to the cluster or not. This affects the data for ventricle more strongly than those for atrium, because in the latter the clusters are larger. An idea of the error involved can be derived by considering that if five particles at the periphery of each patch have uncertain assignment, the maximum error is 13% in atrium and 23% in the ventricle. The average error will be no more than one-half of those values.

The density of DHPRs was estimated by measuring the surface area of each DHPR cluster and obtaining the ratio of particle number/surface area independently for each cluster. The surface area measurements are affected by an uncertainty due to the precise position of the boundary line used to measure the area. Repeated measurements on the patch shown in Fig. 5 A, made by drawing boundary lines that followed the outline of the patch either very closely or more peripherally gave a variations of 8% in the areas measured. The total area is also affected by the uncertainty in assigning some peripheral particles to the patch (see above), but the particle density is not affected by this source of variation because if particles are excluded from the measurements, so is the area in which they are located. The surface area of the clusters is larger in the atrium than in the ventricle (25.7 ± 17.7 x 10^-3 µm², n = 67 clusters; 11.6 ± 10.5 x 10^-3 µm², n = 47 respectively), but the density of particles within the cluster is not very different between the two chambers: 1,555 ± 392 particles/µm² for the atrium, and 1,876 ± 426 particles/µm² for the ventricle.

From the above data we calculate that the average density of clustered large particles, in relation to the total cell surface area, is 28/µm² for atrium and 4/µm² for ventricle.

Correlations between EM and immunolabeling data
Comparison of images from single- and double-immunolabeled optical sections and from the two EM techniques allows a better understanding of the distribution of RyR and DHPRs. First, the overall distribution of RyR- and DHPR-positive foci in the optical images is quite similar, particularly in showing foci that are aligned along parallel longitudinal lines. In the case of DHPRs, which are surface membrane channels, this must occur where the optical section bisects the cell surface. In the case of the RyR foci, the longitudinal lines might indicate a location of RyR positive spots either at the cell surface or between the two to three myofibrils that are present in each cell. The latter situation, however, is excluded by the observation that feet-bearing SR cisternae are observed only at the cell surface and that the positions of RyR- and DHPR-positive foci coincide.

When comparing immunolabeling images (Fig. 1) and thin section electron micrographs (Fig. 7) of atrial cells it might seem at first sight that the frequency of RyR-positive foci seen at the light microscope level far exceeds that of feet-bearing SR cisternae, particularly considering that only 30% of peripheral couplings appear to have feet. The effect, however, is simply due to differences in section thickness, which is at least 10-fold higher in the optical images (1–2 µm), than in the thin sections (100 nm). The effect of section thickness is graphically illustrated in Fig. 10. The figure was obtained using three micrographs in a random series from thin sections of atrial cells similar to those illustrated in Fig. 7, as follows. The position of each peripheral coupling was marked by a large dot and the locations of Z-lines were noted. Twelve profiles of the fiber edges were copied and superimposed, using the Z-lines for alignment, to mimic the amount of material that would be included in a 1.2-µm thick section. Sarcomere lengths were adjusted to superimpose the Z-lines. The actual images were then eliminated, leaving only the positions of the Z-lines and of peripheral couplings, which are illustrated in Fig. 10. Note that at the level of each Z-line there are several couplings, so that even if only 30% of the couplings present have feet, each Z-line would have at least one RyR-positive spot in its proximity, as seen in the confocal images. Also, some spots are centered on the Z-lines, whereas others lie on either side of them, as also seen in the confocal images.

View larger version (5K): [in this window] [in a new window]   FIGURE 10  Predicted position and number of peripheral couplings visible in a optical section of 1.2-µm thickness at the periphery of an atrial cell. The figure was constructed from thin section electron microscopy images as described in the text. The positions of Z-lines are indicated by bars. Each dot represents one peripheral coupling.

The values from the above analysis indicate that there should be a predominance of DHPR- versus RyR-containing membrane regions in the atrium. The double immunolabeling image shown in Fig. 1 was selected from an area exhibiting a maximum amount of overlap between RyR and DHPR foci, demonstrating colocalization. Other areas of the confocal images from the double labeling, however, show a predominance of labeling for DHPRs over that for RyR.

SR membrane particles
The membrane of the free SR is occupied by randomly disposed particles of variable but mostly small size on the cytoplasmic leaflet (Fig. 6, A and C). The particles resemble in size and disposition those representing the calcium pump ATPase (Franzini-Armstrong and Ferguson, 1985). The density of particles was counted in fractured SR profiles from atrium and ventricle, giving average values of 1256 ± 662/µm² (mean ± SD, n = 39 vesicles) in atrium and 1200 ± 429/µm² (n = 32) in ventricle.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Our observations provide a structural foundation for the unusual properties of e–c coupling in frog myocardium. The basis for this work is our ability to detect and quantitate the content of ryanodine and dihydropyridine receptors. RyRs were identified by their characteristic structure in electron micrographs, with the confirmation by immunolabeling and ryanodine binding studies. Three sets of data are internally consistent. As in the case of skeletal and other cardiac muscles (Jorgensen et al., 1993; Junker et al., 1994; Takekura et al., 1994; Sun et al., 1995; Protasi et al., 1996), we find that the presence and location of feet in thin sections of atrium coincides with the presence and location of RyR foci detected by confocal microscopy using immunolabeling. The relatively low level of ryanodine binding to the atrium, as compared to the 25 times higher binding levels to mouse and rat hearts, corresponds well with the scarcity of feet seen in thin sections. In ventricle, the absence of RyR foci and the undetectable level of ryanodine binding fit well with the EM observations indicating lack of feet.

DHPRs were identified by comparing electron microscopy and immunolabeling data as previously published (See Franzini-Armstrong and Protasi, 1997, for a review). Freeze-fracture indicates the presence and location of large intramembrane particles within well delimited clusters that closely correspond to the location of DHPR foci detected by immunolabeling in both atrium and ventricle. The particles have the same size (10.0 nm for atrium and 9.4 nm for the ventricle) as those identified with DHPRs in cells of skeletal muscle origin (9.9 nm, Protasi et al., 1998) and in chick cardiac muscle (8.5 nm, Sun et al., 1995), allowing us to extend the identification to the frog myocardium. From the correspondence in the positions of DHPR clusters and of peripheral couplings, we deduce that DHPRs occupy domains of the surface membrane that are associated with underlying SR cisternae.

The most striking result from these studies is the observation that ryanodine receptors are virtually absent in the ventricle of the frog myocardium. The finding is not unexpected in view of the physiological evidence for a lack of a calcium-releasing action of caffeine on the ventricle (see Introduction), but it does put the frog ventricle in a very special category. To our knowledge, all muscle cells and a large variety of nonmuscle cells contain ryanodine receptors, and release from intracellular stores via these channels contributes significantly to calcium transients. Frog ventricle seems to rely exclusively on calcium entry through the surface membrane. This is not inappropriate in cells that have very small diameters. However, a variety of muscle fibers that have very large inward Ca²⁺ currents, numerous T-tubules and/or small cell diameters, such as scorpion muscle (Gilly and Scheuer, 1984), body muscle of amphioxus (Benterbusch and Melzer, 1992; Benterbusch et al., 1992), fast adductor muscles of the scallop (Nunzi and Franzini-Armstrong, 1981), smooth muscles of vertebrates (Devine et al., 1972), atrial myocardium in many species, and all avian myocardium (Junker et al., 1994; Carl et al., 1995), do have the machinery for internal calcium release via RyRs. The frog ventricular myocytes remains for the time being the only known muscle to lack this internal calcium release mechanism.

The frog atrium seems to conform more closely than the ventricle to the standard calcium release unit composition of mammalian and avian cardiac myocytes, inasmuch as at least a portion of peripheral couplings in frog atrium show the usual close spatial association of DHPR and RyRs. The sparse presence of couplings is in accord with observations that release of Ca²⁺ from the SR in the atrium plays a minor role in steady-state e–c coupling and may come into play only in the case of -adrenergic and/or P₂-purinergic stimulation (Niedergerke and Page, 1981b). The underlying structural cause for this reluctance to activate the internal release may lie in the relative scarcity of RyRs in the peripheral couplings of this muscle. Thus extra stimulatory mechanisms may be necessary to obtain a detectable release of Ca²⁺ from the SR in frog atrium.

Beside RyR, another Ca²⁺ release channel, the IP3 receptor, may contribute to internal Ca²⁺ release in frog heart, being activated as a result of the effect of external ATP (Niedergerke and Page, 1981b). IP3 receptors (Berridge, 1993) are known to be present in some cardiac muscles (Kijima and Fleischer, 1992; Kijima et al., 1993; Lipp et al., 2000). However, we were not able to detect IP3 receptors in the frog myocardium despite using a variety of available antibodies, perhaps because these antibodies did not cross-react with the frog isoform. If IP3 receptors were located in the cisternae of peripheral couplings, their presence, perhaps together with RyRs, would explain why the average junctional gap of frog peripheral couplings (between 7 and 9 nm) is always smaller than the gap in peripheral couplings of other cardiac and skeletal muscles that contain well-defined arrays of feet (12 nm in mouse skeletal muscle, Takekura et al., 1995; 11 nm in chick cardiac muscle, Protasi et al., 1996).

Despite the difference in content of ryanodine receptors, SR of frog atrial and ventricular myocytes have essentially the same density of Ca²⁺ ATPase in the SR membrane, as shown by the similar density of intramembranous particles in the free SR. The free SR is the site where the Ca²⁺ ATPase is located and the density of membrane particles in this membrane is directly related to the density of Ca²⁺ ATPase (Franzini-Armstrong and Ferguson, 1985). The maximum ATPase/volume density in frog myocardium can be calculated using the data of Bossen and Sommer (1984) for the SR surface/volume ratio and assuming that all intramembrane particles in the freeze-fractured free SR membrane represent clusters of two to four (average, three) ATPase molecules (see Franzini-Armstrong and Ferguson, 1985). The ATPase density is at the most 1659/µm³ in atrial and 945/µm³ in the ventricular cells. These values are 10 times lower than the content of SR ATPase in slow- and fast-twitch skeletal muscle fibers (Ferguson and Franzini-Armstrong, 1988) and 200-fold lower than the ATPase content in a super-fast skeletal muscle (Appelt et al., 1991). Thus the frog cardiac SR has a limited role in relaxation, consistent with its limited role during excitation–contraction coupling. The calcium ions that are taken up by the ventricular SR may be necessary for maintaining the housekeeping roles of the membrane system (chaperoning proteins, etc.) and may exit the SR either via a very limited number of RyRs, or via IP3 receptors.

DHPRs in frog myocardium are clustered at sites of peripheral couplings, even though the latter contain no feet in the ventricle and often lack feet in the atrium. This is not entirely surprising, in view of the fact that DHPRs are appropriately targeted to calcium release units in "dyspedic" skeletal muscles resulting from null mutations of RyR1. Thus targeting of DHPRs to junctional sites is independent of the presence of RyR (Protasi et al., 1998; Takekura and Franzini-Armstrong, 1999; Felder et al., 2002). In skeletal muscle the signal to target DHPRs in the triad is located in the C-terminus of the DHPR _1s subunit (Flucher et al., 2000). Perhaps the same targeting signal is used by the DHPR _1c subunit of frog cardiac muscle, leading to DHPR clusters. Location of clusters near Z-lines is fascinating because it is at the same location where DHPRs are found in cardiac muscle having RyRs. During differentiation of avian cardiac muscle and mammalian skeletal muscle, docking of SR to surface membrane/T-tubules precedes delivery and/or detention of DHPRs and RyRs at CRUs, indicating that SR docking guides the location of the junctional sites (Protasi et al., 1996; Takekura et al., 2001). However, this leaves the functional meaning of DHPR clustering at junctional domains open to question. Why should DHPRs be clustered at sites of peripheral coupling if, particularly in the case of the ventricle, they do not interact either functionally or structurally with RyRs?

The density of large particles present in the junctional domains of the plasmalemma, when referred to the total cell surface area, is larger in atrial than in ventricular cells (28 vs. 5/µm²). This is in strong contrast to the measured values of L-type Ca²⁺ current densities, which are higher in ventricle than in atrium, 3.4 ± 2.5 µA/cm² vs. 1.6 ± 2.5 µA/cm² (Hartzell and Simmons, 1987). There are two possible explanations for this apparent discrepancy. One is simply that atrium and ventricle differ in the extent of DHPR clustering, so that in ventricular cells the majority of Ca²⁺ channels are dispersed over the cell surface and a small proportion are in the junctional clusters, whereas in the atrium the majority of DHPRs are clustered. Our approach detects only the clustered channels and cannot identify the channels that are dispersed. A second, more interesting possibility is that the open probability of calcium channels under basal conditions is not the same in atrium and ventricle. Bean et al. (1984) showed that ß-adrenergic stimulation increases the average number of functional Ca channels per cell in frog ventricle. Perhaps the atrium has an excess of channels which are less active.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
We thank Dr. Sally Page for extensive reviewing of the manuscript and numerous stimulating suggestions, and Dr. Donald Bers for a discussion of DHPR content in cardiac muscle.

This research was supported by grant RO1 HL 48093 from the National Institutes of Health.

Submitted on June 17, 2002; accepted for publication October 17, 2002.

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