Ultrastructural localization of calsequestrin in adult rat atrial and ventricular muscle cells

The distribution of calsequestrin in rat atrial and ventricular myocardial cells was determined by indirect immunocolloidal gold labeling of ultrathin frozen sections. The results presented show that calsequestrin is confined to the sarcoplasmic reticulum where it is localized in the lumen of the peripheral and the interior junctional sarcoplasmic reticulum as well as in the lumen of the corbular sarcoplasmic reticulum, but absent from the lumen of the network sarcoplasmic reticulum. Comparison of these results with our previous studies on the distribution of the Ca2+ + Mg2+-dependent ATPase of the cardiac sarcoplasmic reticulum show directly that the Ca2+ + Mg2+-dependent ATPase and calsequestrin are confined to distinct regions within the continuous sarcoplasmic reticulum membrane. Assuming that calsequestrin provides the major site of Ca2+ sequestration in the lumen of the sarcoplasmic reticulum, the results presented support the idea that both junctional (interior and peripheral) and specialized nonjunctional (corbular) regions of the sarcoplasmic reticulum are involved in Ca2+ storage and possibly release. Furthermore, the structural differences between the junctional and the corbular sarcoplasmic reticulum support the possibility that Ca2+ storage and/or release from the lumen of the junctional and the corbular sarcoplasmic reticulum are regulated by different physiological signals.     

Immunoelectron microscopic localization of sarcoplasmic reticulum proteins in cryofixed, freeze-dried, and low temperature-embedded tissue

Immunoelectron microscopic labeling of calsequestrin on ultra-thin sections of rat ventricular muscle prepared by quick-freezing, freeze-drying, and direct embedding in Lowicryl K4M was compared to that observed on ultra-thin sections prepared by chemical fixation, dehydration in ethanol, and embedding in Lowicryl K4M. Brightfield electron microscopic imaging of cryofixed, freeze-dried, osmicated, and Spurr-embedded rat ventricular tissue showed that the sarcoplasmic reticulum was very well preserved by cryofixation and freeze-drying. Therefore, the four structurally distinct regions of the sarcoplasmic reticulum (i.e., the network SR, the junctional SR, the corbular SR, and the cisternal SR) were easily identified even when myofibrils were less than optimally preserved. As previously shown by immunoelectron microscopic labeling of ultra-thin frozen sections of chemically fixed tissue, calsequestrin was confined to the lumen of the junctional SR and of a specialized non-junctional (corbular) SR, and was absent from the lumen of network SR in cryofixed, freeze-dried, Lowicryl-embedded myocardial tissue. In addition, a considerable amount of calsequestrin was also present in the lumen of a different specialized region of the non-junctional SR, called the cisternal sarcoplasmic reticulum. By contrast, relocation of calsequestrin to the lumen of the network SR was observed to a variable degree in chemically fixed, ethanol-dehydrated, and Lowicryl-embedded tissue. We conclude that tissue preparation by cryofixation, freeze-drying, and direct embedding in Lowicryl K4M for immunoelectron microscopic localization of diffusible proteins, such as calsequestrin, is far superior to that obtained by chemical fixation, ethanol dehydration, and embedding in Lowicryl K4M.     

The atrial myocardial cells of mouse heart: a structural and stereological study

Structural and stereological studies of mouse atrial myocardial cells, carried out in the same fashion as our previous investigations on mouse ventricle, demonstrate an extremely well-developed sarcoplasmic reticulum (SR) in atrial cells. The volume fraction (Vv) of the SR exceeds 12% in mouse atrial cells; perimyofibrillar network SR constitutes the major portion. We have confirmed the findings of Bossen et al. (1981, Tissue Cell 13, 71-77) of a difference between atria in terms of coupling density, the right atrium having a significantly lower incidence of interior junctional SR than the left. The SR of mouse atrium comprises a rich variety of specialized segments, including the IJSR, peripheral junctional SR, corbular SR, cisternal SR (including regions similar to fenestrated collars of striated skeletal muscle SR), as well as a peculiar form of extended junctional SR (EJSR). Although less frequent in occurrence than corbular SR, the EJSR seems closely related, since it occurs in multiple clusters at or near the Z-line regions, contains internal granular densities, and bears surface-connected structures resembling junctional processes. Seen in thin sections, mouse atrial EJSR elements are more complex than corbular SR, being larger in diameter and frequently circular in profile. Thick-section and serial-section analyses reveal that bodies of EJSR are in fact hollow spheroids. The transverse-axial tubular system of mouse atrium is rather poorly developed in comparison to its ventricular counterpart. The Golgi apparatus and associated specific atrial granules are prominent cell components. "Focal ellipsoidal deposits" (FEDs) previously described by Page and co-workers (1986, Amer. J. Physiol.) are consistently located adjacent to the Golgi region, but immunocytochemical staining for two different segments of atrial natriuretic peptide reveals no specific reaction in FEDs, whereas the SAGs are densely labeled for both antibodies.     

FREEZE FRACTURE OF SKELETAL MUSCLE FROM THE TARANTULA SPIDER : Structural Differentiations of Sarcoplasmic Reticulum and Transverse Tubular System Membranes

The structure of the membranes of sarcoplasmic reticulum (SR), tubular (T) system, and sarcolemma has been studied by freeze fracture in leg muscles of the Tarantula spider. Two regions of the sarcoplasmic reticulum can be differentiated by the distribution of particles on the fracture faces: a junctional SR, at the dyads, and a longitudinal SR, elsewhere. The dyads are asymmetric junctions, the disposition of particles in the apposed membranes of SR and T tubules being different from one another and from the regular arrangement of feet in the junctional gap. It is concluded that no channels can be visualized to directly connect SR- and T-system lumina.     

The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle

The subcellular distribution of the Ca(2+)-release channel/ryanodine receptor in adult rat papillary myofibers has been determined by immunofluorescence and immunoelectron microscopical studies using affinity purified antibodies against the ryanodine receptor. The receptor is confined to the sarcoplasmic reticulum (SR) where it is localized to interior and peripheral junctional SR and the corbular SR, but it is absent from the network SR where the SR-Ca(2+)-ATPase and phospholamban are densely distributed. Immunofluorescence labeling of sheep Purkinje fibers show that the ryanodine receptor is confined to discrete foci while the SR-Ca(2+)-ATPase is distributed in a continuous network-like structure present at the periphery as well as throughout interior regions of these myofibers. Because Purkinje fibers lack T- tubules, these results indicate that the ryanodine receptor is localized not only to the peripheral junctional SR but also to corbular SR densely distributed in interfibrillar spaces of the I-band regions. We have previously identified both corbular SR and junctional SR in cardiac muscle as potential Ca(2+)-storage/Ca(2+)-release sites by demonstrating that the Ca2+ binding protein calsequestrin and calcium are very densely distributed in these two specialized domains of cardiac SR in situ. The results presented here provide strong evidence in support of the hypothesis that corbular SR is indeed a site of Ca(2+)-induced Ca2+ release via the ryanodine receptor during excitation contraction coupling in cardiac muscle. Furthermore, these results indicate that the function of the cardiac Ca(2+)-release channel/ryanodine receptor is not confined to junctional complexes between SR and the sarcolemma.     

SPHEROIDAL BODIES IN THE JUNCTIONAL SARCOPLASMIC RETICULUM OF LIZARD MYOCARDIAL CELLS

The sarcoplasmic reticulum (SR) of lizard (Anolis carolinensis) myocardial cells has been examined, with particular attention being paid to the structural details of the peripheral couplings (junctional SR). Spheroidal bodies are present within the opaque core of junctional SR; these can be seen both in sections made en face and in sections cut to show the apposition of the junctional SR with the sarcolemma. Opaque junctional processes extend between the sarcolemma and the peripheral junctional SR. The myocardial cells in addition contain some SR cisternae deep within the cells which also possess opaque cores composed of spheroids. Although the significance of the junctional SR spheroidal bodies is unknown, it is thought that they could act as a matrix on which enzymes such as calcium-specific ATPase may be located.     

The sarcoplasmic reticulum of mouse heart: its divisions, configurations, and distribution

The sarcoplasmic reticulum (SR) is a prominent, highly ramified component of mouse myocardial cells. The use of ferrocyanide-reduced osmium tetroxide (OsFeCN) as a postfixative solution facilitates appreciation of both its extent and three-dimensional architecture. We have found that the individual volume fractions (Vv) of myofibrils, mitochondria, and SR are similar in cells of the right and left ventricular walls. Vv(total SR) is approximately 7%, a value considerably larger than previously reported. We attribute this disparity in large part to the recognition factor which comes into play with OsFeCN-treated tissue. Previous observations pertaining to the stereology of myocardial SR have likely substantially underestimated both volume fraction and surface density of this membrane system, since none to this point has utilized specific staining such as that conferred by the OsFeCN regimen. Our stereological measurements of different depths of the ventricular cell indicate that although considerable differences are found between SR configuration at peripheral and deep cell levels, no significant difference exists between the volume fractions of either the total SR or its individual constituents. Two different stereologic regimens gave close agreement on volume fractions of the various SR segments; the majority (approximately 92%) of the total SR is network SR, whereas the remainder is composed of the various categories of junctional SR (peripheral, apposed to the surface sarcolemma; interior, complexed with the transverse-axial tubular system; corbular, existing free of sarcolemmal contact). In the adult mouse, interior junctional SR greatly preponderates the other types of junctional SR; corbular SR is qualitively assessed to be a far more common component of atrial cells than of ventricular cardiomyocytes.     

Activation of the contractile system in crustacean muscle: Ultrastructural evidence for the role of the T system

Freeze-fracture and thin sections of lobster abdominal fast flexor muscle were used to study the morphology of the sarcoplasmic reticulum (SR) and T system of crustacean muscle. Tannic acid mordanting, which can result in a dense black deposit in the T system lumen, was used to distinguish T system from SR membranes. Ferritin was also used as an extracellular tracer to confirm the tannic acid method. The T system consists of an extensive network of flattened sacs which fills most of the space between the mycfibrils and is in close contact with them. The SR also appears as flattened sacs, sometimes with fenestrations. There is extensive junctional contact between the SR and T system. Quantitative estimates of the volume and surface area of the membranes show that the T system has about 50 % more surface area than the SR. The intramembrane particle (IMP) density of the PF face of the T system is about 1100/ μm² membrane, while the IMP density of the PF face of the SR is about 4800/ μm² membrane. In morphology, extent, and IMP density, the T system of lobster abdominal fast flexor muscle appears (AFF) adapted to provide at least part of the Ca²⁺ for muscle activation and the transport system for relaxation.     

Bridging structures spanning the junctioning gap at the triad of skeletal muscle

The membrane systems of skeletal muscle were examined after tannic acid fixation. A new structure consisting of bridges spanning the junctional gap is described, and a model is proposed in which the cytoplasmic but not the luminal membrane leaflets of the transverse tubule and of the junctional sarcoplasmic reticulum (SR) are continuous. The globular particles (presumably the Ca-binding proteins) within the terminal cisternae were arranged in longitudinal rows and appeared adherent to the junctional membrane. The junctional gap was present in negatively stained, frozen thin sections of fixed muscles. Negatively staining material occured within the junctional gap. The cytoplasmic leaflets of the longitudinal, intermediate, and terminal cisterna regions of the SR exhibited a thick coat of densely staining material compatible with the presence of the Ca-ATPase. Similar bridges were also observed at the surface membrane-SR close coupling sites of vascular smooth muscle.     

Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle

The subcellular distribution of phospholamban in adult canine ventricular myocardial cells was determined by the indirect immunogold-labeling technique. The results presented suggest that phospholamban, like the Ca2+-ATPase, is uniformly distributed in the network sarcoplasmic reticulum but absent from the junctional portion of the junctional sarcoplasmic reticulum. Unlike the Ca2+-ATPase, but like cardiac calsequestrin, phospholamban also appears to be present in the corbular sarcoplasmic reticulum. Comparison of the relative distribution of phospholamban immunolabeling in the sarcoplasmic reticulum with that of the sarcolemma showed that the density of phospholamban in the network sarcoplasmic reticulum was approximately 35-fold higher than that of the cytoplasmic side of the sarcolemma, which in turn was found to be three- to fourfold higher than the density of the background labeling. However, a majority of the specific phospholamban labeling within 30 nm of the cytoplasmic side of the sarcolemma was clustered and present over the sarcoplasmic reticulum in the subsarcolemmal region of the myocardial cells, suggesting that phospholamban is confined to the junctional regions between the sarcolemma and the sarcoplasmic reticulum, but absent from the nonjunctional portion of the sarcolemma. Although the resolution of the immunogold-labeling technique used (60 nm) does not permit one to determine whether the specific labeling within 30 nm of the cytoplasmic side of the sarcolemma is associated with the sarcolemma and/or the junctional sarcoplasmic reticulum, it is likely that the low amount of labeling in this region represents phospholamban associated with sarcoplasmic reticulum. These results suggest that phospholamban is absent from the sarcolemma and confined to the sarcoplasmic reticulum in cardiac muscle.     

Subcellular distribution of ryanodine receptors in the cardiac muscle of carp (Cyprinus carpio)

We examined the subcellular localization of ryanodine receptors (RyR) in the cardiac muscle of carp using biochemical, immunohistochemical, and electron microscopic methods and compared it with those of rats and guinea pigs. To achieve this goal, an anti-RyR antibody was newly raised against a synthetic peptide corresponding to an amino acid sequence that was conserved among all sequenced RyRs. Western blot analysis using this antibody detected a single RyR band following the SDS-PAGE of sarcoplasmic reticulum (SR) membranes from carp atrium and ventricle as well as from mammalian hearts and skeletal muscles. The carp heart band had slightly greater mobility than those of mammalian hearts. Although immunohistochemical staining showed evident striations corresponding to the Z lines in longitudinal sections of mammalian hearts, clusters of punctate staining, in contrast, were distributed ubiquitously throughout carp atrium and ventricle. Electron microscopic images of the carp myocardium showed that the SR was observed largely as the subsarcolemmal cisternae and the reticular SR, suggesting that the RyR is localized in the junctional and corbular SR.     

The three-dimensional organization of smooth endoplasmic reticulum in capillary endothelia: its possible role in regulation of free cytosolic calcium

A rise in cytosolic free Ca in capillary endothelia leads to increased permeability. It has been proposed that this Ca(2+)-regulated modulation of junctional permeability of vascular endothelia involves structural elements comparable to those involved in stimulus-contraction coupling in smooth muscle. To explore this analogy the three-dimensional organization of smooth-surfaced cisternae, vesicular membrane profiles, and tight junctions was examined in endothelia of diaphragm and heart capillaries of the rat. Three-dimensional reconstructions, based on consecutive sections of the capillaries, have demonstrated a population of small, irregular membrane profiles, occurring in individual thin sections of the endothelial cytoplasm. These profiles represent an elaborate system of smooth-surfaced cisternae, structurally similar to the sarcoplasmic reticulum (SR) of smooth muscle cells. Slender processes from the cisternae are often situated in parallel to the tight junctions at a distance of about 100 nm. The great majority of the characteristic circular membrane profiles represents caveolae and racemose invaginations of the endothelial plasma membrane, often in close relation to the cisternae. It is hypothesized that the endothelial cisternae and invaginations of the cell membrane are involved in regulation of free cytosolic calcium in the same way as the SR and caveolae in smooth muscle cells. The junction-related cisternal processes may play a role in the Ca(2+)-regulated modulation of junctional permeability.     

Shape, size, and distribution of Ca(2+) release units and couplons in skeletal and cardiac muscles.

Excitation contraction (e-c) coupling in skeletal and cardiac muscles involves an interaction between specialized junctional domains of the sarcoplasmic reticulum (SR) and of exterior membranes (either surface membrane or transverse (T) tubules). This interaction occurs at special structures named calcium release units (CRUs). CRUs contain two proteins essential to e-c coupling: dihydropyridine receptors (DHPRs), L-type Ca(2+) channels of exterior membranes; and ryanodine receptors (RyRs), the Ca(2+) release channels of the SR. Special CRUs in cardiac muscle are constituted by SR domains bearing RyRs that are not associated with exterior membranes (the corbular and extended junctional SR or EjSR). Functional groupings of RyRs and DHPRs within calcium release units have been named couplons, and the term is also loosely applied to the EjSR of cardiac muscle. Knowledge of the structure, geometry, and disposition of couplons is essential to understand the mechanism of Ca(2+) release during muscle activation. This paper presents a compilation of quantitative data on couplons in a variety of skeletal and cardiac muscles, which is useful in modeling calcium release events, both macroscopic and microscopic ("sparks").     

Structures located at the levels of the Z bands in mouse ventricular myocardial cells

Within ventricular myocardial cells of the mouse, the myoplasmic regions located immediately adjacent to the Z lines of the sarcomeres contain a variety of structures. These include: (1) transversely oriented 10 nm (‘intermediate’) filaments that apparently contribute to the cytoskeleton of the myocardial cell; (2) the majority of the transverse elements of the T-axial tubular system; (3) specialized segments of the sarcoplasmic reticulum (SR) that are closely apposed to the sarcolemma or T-axial tubules (junctional SR); (4) ‘extended junctional SR’ (‘corbular SR’) that exists free of association with the cell membrane; (5) ‘Z tubules’ of SR that are intimately apposed to the Z line substance; and (6) leptofibrils. In addition, fasciae adherentes supplant Z lines where myofibrils insert into the transverse borders (intercalated discs) of the cells. The concentration of these myocardial components at the level of the Z lines suggests that a particular specialization of structural and physiological activities exists in the Z-level regions of the myoplasm. In particular, it appears that the combination of intermediate filaments, T tubules, and Z-level SR elements forms a series of parallel planar bodies that extend across each myocardial cell to impart transverse rigidity. The movement and compartmentation of calcium ion (Ca²⁺) would seem especially active near the Z lines of the myofibrils, in view of the preferential location there of Ca²⁺-sequestering myocardial structures such as T tubules, junctional SR, extended junctional SR and Z tubules.     

Identification of the cardiac ryanodine receptor channel in membrane blebs of sarcoplasmic reticulum

Blebs of the sarcoplasmic reticulum (SR) membrane of heart muscle cells were generated after saponin perforation of the plasma membrane followed by complete hypercontraction of the cell. Although characteristic proteins of the plasma membrane, namely the beta1-adrenoreceptor and Galphai, were stained by monoclonal antibodies in the hypercontracted cells, these proteins could not be detected in the adjacent blebs. Monoclonal antibodies to the cardiac ryanodine receptor (RyR2), calsequestrin and SERCA2 bound at different amounts to surface components of the blebs and to components of the hypercontracted cells. From the immunofluorescence signals we conclude that the blebs are mainly constituted of corbular and junctional SR membrane, and only to a lesser extent of network SR membrane. Deconvolution microscopy revealed that the membrane location of RyR2, calsequestrin and SERCA2 in the bleb is comparable to native SR membrane. At the bleb membrane giga-ohm seals could be obtained and patches could be excised in a way that single-channel currents could be measured, although these are not completely identified.     

Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle

We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.     

Axial Tubules of Rat Ventricular Myocytes Form Multiple Junctions with the Sarcoplasmic Reticulum

Ryanodine receptors (RyRs) are located primarily on the junctional sarcoplasmic reticulum (SR), adjacent to the transverse tubules and on the cell surface near the Z-lines, but some RyRs are on junctional SR adjacent to axial tubules. Neither the size of the axial junctions nor the numbers of RyRs that they contain have been determined. RyRs may also be located on the corbular SR and on the free or network SR. Because determining and quantifying the distribution of RyRs is critical for both understanding and modeling calcium dynamics, we investigated the distribution of RyRs in healthy adult rat ventricular myocytes, using electron microscopy, electron tomography, and immunofluorescence. We found RyRs in only three regions: in couplons on the surface and on transverse tubules, both of which are near the Z-line, and in junctions on most of the axial tubules—axial junctions. The axial junctions averaged 510 nm in length, but they occasionally spanned an entire sarcomere. Numerical analysis showed that they contain as much as 19% of a cell's RyRs. Tomographic analysis confirmed the axial junction's architecture, which is indistinguishable from junctions on transverse tubules or on the surface, and revealed a complexly structured tubule whose lumen was only 26 nm at its narrowest point. RyRs on axial junctions colocalize with Ca_v1.2, suggesting that they play a role in excitation-contraction coupling.     

The structure of calsequestrin in triads of vertebrate skeletal muscle: a deep-etch study

We have examined the structure of calsequestrin in three-dimensional images from deep-etched rotary-replicated freeze fractures of skeletal muscle fibers. We selected a fast-acting muscle because the sarcoplasmic reticulum has an orderly disposition and is rich in internal membranes. Calsequestrin forms a network in the center of the terminal cisternae and is anchored to the sarcoplasmic reticulum membrane, with preference for the junctional portion. The anchorage is responsible for maintaining calsequestrin in the region of the sarcoplasmic reticulum close to the calcium-release channels, and it corroborates the finding that calsequestrin and the spanning protein of the junctional feet may interact with each other in the junctional membrane. Anchoring filaments may be composed of a protein other than calsequestrin.     

Immunolocalization of myotonic dystrophy protein kinase in corbular and junctional sarcoplasmic reticulum of human cardiac muscle

The subcellular localization of myotonic dystrophy protein kinase has been examined in human cardiac muscles with confocal laser-scanning microscopy and electron microscopy. A polyclonal antibody was produced against the synthesized peptide from a human kinase cDNA clone. We checked the antibody specificity for cardiac myotonic dystrophy protein kinase using an immunoblotting technique. Immunoblotting of extract from human cardiac muscles showed mainly 70 kDa and 55 kDa molecular weight bands. Confocal images of the protein kinase immunostaining showed striated banding patterns similar to those of skeletal muscles. In addition, the kinase was strongly detected around the intercalated disc. Immunoelectron microscopy showed that the kinase was mainly expressed in both corbular and junctional sarcoplasmic reticulum, but not in network sarcoplasmic reticulum. These results suggest that myotonic dystrophy protein kinase may be involved in the modulation of Ca2+ homeostasis in cardiac myofibres. © 1998 Chapman & Hall     

The unraveling architecture of the junctional sarcoplasmic reticulum

The sarcoplasmic reticulum (SR) of skeletal muscle controls the contraction-relaxation cycle by raising and lowering the myoplasmic free-Ca²⁺ concentration. The coupling between excitation, i.e., depolarization of sarcolemma and transvers tubule (TT) and Ca²⁺ release from the terminal cisternae (TC) of SR takes place at the triad. The triad junction is formed by a specialized region of the TC, the junctional SR, and the TT. The molecular architecture and protein composition of the junctional SR are under active investigation. Since the junctional SR plays a central role in excitation-contraction coupling and Ca²⁺ release, some of its protein constituents are directly involved in these processes. The biochemical evidence supporting this contention is reviewed in this article.