Biochemical characterization and molecular cloning of cardiac triadin.

Triadin is an intrinsic membrane protein first identified in the skeletal muscle junctional sarcoplasmic reticulum and is considered to play an important role in excitation-contraction coupling. Using polyclonal antibodies to skeletal muscle triadin, we have identified and characterized three isoforms in rabbit cardiac muscle. The cDNAs encoding these three isoforms of triadin have been isolated by reverse transcription-polymerase chain reaction and cDNA library screening. The deduced amino acid sequences show that these proteins are identical in their N-terminal sequences, whereas the C-terminal sequences are distinct from each other and from that of skeletal muscle triadin. Based upon both the amino acid sequences and biochemical analysis, all three triadin isoforms share similar membrane topology with skeletal muscle triadin. Immunofluorescence staining of rabbit cardiac muscle with antibodies purified from the homologous region of triadin shows that cardiac triadin is primarily confined to the I-band region of cardiac myocytes, where the junctional and corbular sarcoplasmic reticulum is located. Furthermore, we demonstrate that the conserved region of the luminal domain of triadin is able to bind both the ryanodine receptor and calsequestrin in cardiac muscle. These results suggest that triadin colocalizes with and binds to the ryanodine receptor and calsequestrin and carries out a function in the lumen of the junctional sarcoplasmic reticulum that is important for both skeletal and cardiac muscle excitation-contraction coupling.

Purification, primary structure, and immunological characterization of the 26-kDa calsequestrin binding protein (junctin) from cardiac junctional sarcoplasmic reticulum.

Previously we identified a protein of apparent M(r) = 26,000 as the major calsequestrin binding protein in junctional sarcoplasmic reticulum vesicles isolated from cardiac and skeletal muscle (Mitchell, R. D., Simmerman, H. K. B., and Jones, L. R. (1988) J. Biol. Chem. 263, 1376-1381). Here we describe the purification and primary structure of the 26-kDa calsequestrin binding protein. The protein was purified 164-fold from cardiac microsomes and shown by immunoblotting to be highly enriched in junctional membrane subfractions. It ran as a closely spaced doublet on SDS-polyacrylamide gel electrophoresis and bound 125I-calsequestrin intensely. Cloning of the cDNA predicted a protein of 210 amino acids containing a single transmembrane domain. The protein has a short N-terminal region located in the cytoplasm, and the bulk of the molecule, which is highly charged and basic, projects into the sarcoplasmic reticulum lumen. Significant homologies were found with triadin and aspartyl beta-hydroxylase, suggesting that all three proteins are members of a family of single membrane-spanning endoplasmic reticulum proteins. Immunocytochemical labeling localized the 26-kDa protein to junctional sarcoplasmic reticulum in cardiac and skeletal muscle. The same gene product was expressed in these two tissues. The calsequestrin binding activity of the 26-kDa protein combined with its codistribution with calsequestrin and ryanodine receptors strongly suggests that the protein plays an important role in the organization and/or function of the Ca2+ release complex. Because the 26-kDa calsequestrin binding protein is an integral component of the junctional sarcoplasmic reticulum membrane in cardiac and skeletal muscle, we have named it Junctin.

Characterization and ultrastructural localization of a novel 90-kDa protein unique to skeletal muscle junctional sarcoplasmic reticulum.

Monoclonal antibodies were used to identify and characterize a novel 90 kDa protein that was specifically localized to the junctional sarcoplasmic reticulum of rabbit skeletal muscle. Biochemical experiments show that the 90 kDa protein is an integral membrane protein of the junctional face membrane and is a substrate for the intrinsic protein kinase in triads. Immunofluorescence staining of serial transverse sections of skeletal muscle with a monoclonal antibody to the 90 kDa protein showed preferential staining of type II "fast" fibers. Specific labeling was confined to the interphase between the A- and I-bands, where the triad structure is localized. Immunoelectron microscopical labeling further indicates that the 90 kDa protein, like the ryanodine receptor/Ca(2+)-release channel and triadin, is confined to the terminal cisternae of the sarcoplasmic reticulum. Western blot analysis with a combination of monoclonal antibodies against the 90 kDa protein shows that it is specifically expressed in skeletal muscle but not in cardiac muscle or brain. Similarly, specific immunofluorescence labeling to the 90 kDa protein was not detected in ventricular myocytes or vascular smooth muscle cells. The junctional localization and phosphorylation of this protein suggest that it may play an important regulatory or structural role in the skeletal muscle triad junction.

Biochemical characterization of ultrastructural localization of a major junctional sarcoplasmic reticulum glycoprotein (triadin).

Monoclonal antibodies were used to identify a 94-kDa protein that was greatly enriched in traids and junctional face membranes (9.3 +/- 0.2%) but not detected in the transverse tubular and nonjunctional sarcoplasmic reticulum membranes. The 94-kDa protein is a hydrophobic glycoprotein based on endoglycosidase H sensitivity, concanavalin A binding, and labelling with a hydrophobic probe. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of reducing agents suggests that this protein is present as a population of multimeric structures containing a variable number of the 94-kDa subunits. Immunofluorescent staining of serial transverse sections of skeletal muscle shows staining of all fiber types with preferential staining of type II fast fibers. Specific immunofluorescence staining in longitudinal sections of skeletal muscle is confined to the interface between the A- and I-bands where the triad structures are localized. Immunocolloidal gold labeling revealed the 94-kDa glycoprotein to be localized over a region of the junctional sarcoplasmic reticulum where the ryanodine receptor/Ca2+ release channel is localized. The distribution and high abundance of the 94-kDa glycoprotein in the junctional membrane suggest that it performs a structural or functional role in the storage or release of calcium from the junctional sarcoplasmic reticulum in skeletal muscle.

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.

Dystrophin-glycoprotein complex and laminin colocalize to the sarcolemma and transverse tubules of cardiac muscle.

The expression and subcellular distribution of the dystrophin-glycoprotein complex and laminin were examined in cardiac muscle by immunoblot and immunofluorescence analysis of rabbit and sheep papillary muscle. The five dystrophin-associated proteins (DAPs), 156-DAG, 59-DAP, 50-DAG, 43-DAG, and 35-DAG, were identified in rabbit ventricular muscle and found to codistribute with dystrophin in both papillary myofibers and Purkinje fibers. The DAPs and dystrophin codistributed not only in the free surface sarcolemma but also in interior regions of the myofibers where T tubules are present. Neither the DAPs nor dystrophin were detected in intercalated discs, a specialized region of cardiac sarcolemma where neighboring myocardial cells are physically joined by cell-cell junctions. Similarly, in bundles of Purkinje fibers, which lack T tubules, DAPs and dystrophin were also found to codistribute at the free surface sarcolemma but were not detected either in the region of surface sarcolemma closely apposed to a neighboring Purkinje fiber or in interior regions of these myofibers. Comparison between the distribution of the dystrophin-glycoprotein complex and laminin showed that laminin codistributes with the components of this complex in both papillary myofibers and Purkinje fibers. These results are consistent with previous findings demonstrating that the extracellularly exposed 156-DAG (dystroglycan) of the skeletal muscle dystrophin-glycoprotein complex binds laminin, a component of the basement membrane. Although we demonstrate that DAPs, dystrophin, and laminin colocalize to the sarcolemma in rabbit and sheep papillary myofibers as they do in skeletal myofibers, the most striking difference between the subcellular distribution of these proteins in cardiac and skeletal muscle is that the dystrophin-glycoprotein complex and laminin also localize to regions of the fibers where T tubules are distributed in cardiac but not in skeletal muscle. These results imply that the protein composition and thus possibly some functions of T tubules in cardiac muscle are distinct from those of skeletal muscle.

Frog cardiac calsequestrin. Identification, characterization, and subcellular distribution in two structurally distinct regions of peripheral sarcoplasmic reticulum in frog ventricular myocardium.

Calsequestrin is a calcium-binding protein known to sequester calcium accumulated in the sarcoplasmic reticulum (SR) of muscle cells during relaxation. In the present study, we used affinity-purified antibodies to chicken cardiac calsequestrin to identify a 60,000-Da calsequestrin in frog myocardium. Like previously identified cardiac calsequestrins, it is enriched in cardiac microsomes, it is enriched by biochemical procedures previously used to purify cardiac and skeletal calsequestrins, and it exhibits a pH-dependent shift in its apparent Mr on a two-dimensional gel system. Finally, the NH2-terminal amino acid sequence of this 60,000-Da immunoreactive protein purified by fast protein liquid chromatography was identical to that of rabbit skeletal and canine cardiac calsequestrin. Thus, we conclude that this protein corresponds to the calsequestrin isoform in frog ventricular muscle. Frog calsequestrin was localized in discrete foci present at the periphery but absent from the central regions of frog ventricular myocytes as determined by immunofluorescence labeling. Immunoelectron microscopic labeling demonstrated that calsequestrin was confined to the lumen of two structurally distinct regions of the SR, where it was localized in the subsarcolemmal region of the myofibers. One of these appeared to correspond to the terminal SR previously reported to be closely apposed to the sarcolemma of frog myofibers. The other region, although close to the sarcolemma, was not physically joined to it and appeared to correspond to corbular SR. It generally is believed that frog cardiac SR does not provide activator Ca2+ required for excitation-contraction coupling. However, the identification of a calsequestrin isoform very similar to mammalian cardiac calsequestrin that is confined to specialized regions of frog cardiac SR lends support to the idea that frog cardiac SR has the ability to store Ca2+ and thus function in some capacity in frog cardiac muscle contraction.

Biogenesis of transverse tubules and triads: immunolocalization of the 1,4-dihydropyridine receptor, TS28, and the ryanodine receptor in rabbit skeletal muscle developing in situ.

Our previous immunofluorescence studies support the conclusion that the temporal appearance and subcellular distribution of TS28 (a marker of transverse (T) tubules and caveolae in adult skeletal muscle [Jorgensen, A. O., W. Arnold, A. C.-Y. Shen. S. Yuan, M. Gover, and K. P. Campbell, 1990, J. Cell Biol. 110:1173-1185]), correspond very closely to those of T-tubules forming de novo in developing rabbit skeletal muscle (Yuan, S., W. Arnold, and A. O. Jorgensen, 1990, J. Cell Biol. 110:1187-1198). To extend our morphological studies of the biogenesis of T-tubules and triads, the temporal appearance and subcellular distribution of the alpha 1-subunit of the 1,4-dihydropyridine receptor (a marker of the T-tubules and caveolae) was compared to (a) that of TS28; and (b) that of the ryanodine receptor (a marker of the junctional sarcoplasmic reticulum) in rabbit skeletal muscle cells developing in situ (day 19 of gestation to 10 d newborn) by double immunofluorescence labeling. The results presented show that the temporal appearance and relative subcellular distribution of the alpha 1-subunit of the 1,4-dihydropyridine receptor (alpha 1-DHPR) are distinct from those of TS28 at the onset of the biogenesis of T-tubules. Thus, in a particular developing myotube the alpha 1-DHPR appeared before TS28 (secondary myotubes; day 19-24 of gestation). Furthermore, the alpha 1-DHPR was distributed in discrete foci at the outer zone of the cytosol, while TS28 was confined to foci and rod-like structures at the cell periphery. As development proceeded (primary myotubes; day 24 of gestation) approximately 50% of the foci were positively labeled for both TS28 and the alpha 1-DHPR, while approximately 20 and 30% of the foci were uniquely labeled for TS28 and the alpha 1-DHPR, respectively. The foci labeled for both TS28 and the alpha 1-DHPR and the foci uniquely labeled for TS28 were generally confined to the cell periphery, while the foci uniquely labeled for the alpha 1-DHPR were mostly confined to the outer zone of the cytosol. 1-2 d after birth, TS28 was distributed in a chickenwire-like network throughout the cytosol, while the alpha 1-DHPR was confined to cytosolic foci. In contrast, the temporal appearance and subcellular distribution of the alpha 1-DHPR and the ryanodine receptor were very similar, if not identical, throughout all the stages of the de novo biogenesis of T-tubules and triads examined.(ABSTRACT TRUNCATED AT 400 WORDS)

Biogenesis of transverse tubules: immunocytochemical localization of a transverse tubular protein (TS28) and a sarcolemmal protein (SL50) in rabbit skeletal muscle developing in situ.

To study the biogenesis of transverse tubules, the temporal appearance and distribution of TS28 (a specific marker of transverse tubules absent from the sarcolemma in adult skeletal muscle; 28,000 Mr) and SL50 (specifically associated with the sarcolemma and absent from the region of the transverse tubules in adult rabbit skeletal muscle) (Jorgensen, A.O., W. Arnold, A. C.-Y. Shen, S. Yuan, M. Gaver, and K.P. Campbell. 1990. J. Cell Biol. 110:1173-1185) were determined in rabbit skeletal muscle developing in situ (day 17 of gestation to day 15 newborn) by indirect immunofluorescence labeling. The results presented show that the temporal appearance and subcellular distribution of TS28 is distinct from that of SL50 at the developmental stages examined. TS28 was first detected in some, but not all, multinucleated myotubes on day 17 of gestation. At this stage of development, SL50 and the Ca2(+)-ATPase of the sarcoplasmic reticulum were already present in all myotubes. TS28 first appeared in discrete foci mostly confined to the cell periphery of the myotubes. At subsequent stages of development (days 19-24 of gestation), TS28 was also found in shoft finger-like structures extending obliquely and transversely from the cell periphery towards the center of the myotubes. 1-2 d after birth, TS28 was observed in an anastomosing network composed of transversely oriented chickenwire-like networks extending throughout the cytoplasm and interconnected by longitudinally oriented fiber-like structures. As development proceeded, the transversely oriented network became increasingly dominant. By day 10 of postnatal development, the longitudinally oriented component of the tubular network was not regularly observed. At none of the developmental stages examined was TS28 observed to be uniformly distributed at the cell periphery. SL50, like TS28, first appeared in discrete foci at the cell periphery. However, shortly after its first appearance it appeared to be distributed along the entire cell periphery. Although the intensity of SL50 labeling increased with development, it remained confined to the sarcolemma and was absent from the interior regions of the myofibers, where transverse tubules were present at all subsequent developmental stages examined. Immunoblotting of cell extracts from skeletal muscle tissue at various stages of development showed that SL50 was first detected on day 24 of gestation, while TS28 was not detected until days 1-2 after birth. Comparison of these results with previous ultrastructural studies of the formation of transverse tubules supports the idea that the temporal appearance and subcellular distribution of TS28 correspond very closely to that of the distribution of forming transverse tubules in rabbit skeletal muscle developing in situ.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of novel proteins unique to either transverse tubules (TS28) or the sarcolemma (SL50) in rabbit skeletal muscle.

Novel proteins unique to either transverse tubules (TS28) or the sarcolemma (SL50) have been identified and characterized, and their in situ distribution in rabbit skeletal muscle has been determined using monoclonal antibodies. TS28, defined by mAb IXE112, was shown to have an apparent relative molecular mass of 28,000 D. Biochemical studies showed that TS28 is a minor membrane protein in isolated transverse tubular vesicles. Immunofluorescence and immunoelectron microscopical studies showed that TS28 is localized to the transverse tubules and in some subsarcolemmal vesicles possibly corresponding to the subgroup of caveolae connecting the transverse tubules with the sarcolemma. In contrast, TS28 is absent from the lateral portion of the sarcolemma. Immunofluorescence studies also showed that TS28 is more densely distributed in type II (fast) than in type I (slow) myofibers. Although TS28 and the 1,4-dihydropyridine receptor are both localized to transverse tubules and subsarcolemmal vesicles, TS28 is not a wheat germ agglutinin (WGA)-binding glycoprotein and does not appear to copurify with the 1,4-dihydropyridine receptor after detergent solubilization of transverse tubular membranes. SL50, defined by mAb IVD31, was shown to have an apparent relative molecular mass of 50,000 D. Biochemical studies showed that SL50 is not related to the 52,000-D (beta subunit) of the dihydropyridine receptor but does bind to WGA-Sepharose. Immunofluorescence labeling imaged by standard and confocal microscopy showed that SL50 is associated with the sarcolemma but apparently absent from the transverse tubules. Immunofluorescence labeling also showed that the density of SL50 in type II (fast) myofibers is indistinguishable from that of type I (slow) myofibers. The functions of TS28 and SL50 are presently unknown. However, the distinct distribution of TS28 to the transverse tubules and subsarcolemmal vesicles as determined by immunocytochemical labeling suggests that TS28 may be directly involved in excitation-contraction coupling. Our results demonstrate that, although transverse tubules are continuous with the sarcolemma, each of these membranes contain one or more unique proteins, thus supporting the idea that they each have a distinct protein composition.

Subcellular distribution of the 1,4-dihydropyridine receptor in rabbit skeletal muscle in situ: an immunofluorescence and immunocolloidal gold-labeling study.

The subcellular distribution of the 1,4-dihydropyridine receptor was determined in rabbit skeletal muscle in situ by immunofluorescence and immunoelectron microscopy. Longitudinal and transverse cryosections (5-8 microns) of rabbit gracilis muscle were labeled with monoclonal antibodies specific against either the alpha 1-subunit (170,000-D polypeptide) or the beta-subunit (52,000-D polypeptide) of the 1,4-dihydropyridine receptor by immunofluorescence labeling. In longitudinal sections, specific labeling was present only near the interface between the A- and I-band regions of the sarcomeres. In transverse sections, specific labeling showed a hexagonal staining pattern within each myofiber however, the relative staining intensity of the type II (fast) fibers was judged to be three- to fourfold higher than that of the type I (slow) fibers. Specific immunofluorescence labeling of the sarcolemma was not observed in either longitudinal or transverse sections. These results are consistent with the idea that the alpha 1-subunit and the beta-subunit of the purified 1,4-dihydropyridine receptor are densely distributed in the transverse tubular membrane. Immunoelectron microscopical localization with a monoclonal antibody to the alpha 1-subunit of the 1,4-dihydropyridine receptor showed that the 1,4-dihydropyridine receptor is densely distributed in the transverse tubular membrane. Approximately half of these were distributed in close proximity to the junctional region between the transverse tubules and the terminal cisternae. Specific labeling was also present in discrete foci in the subsarcolemmal region of the myofibers. The size and the nonrandom distribution of these foci in the subsarcolemmal region support the possibility that they correspond to invaginations from the sarcolemma called caveolae. In conclusion, our results demonstrate that the 1,4-dihydropyridine receptor in skeletal muscle is localized to the transverse tubular membrane and discrete foci in the subsarcolemmal region, possibly caveolae but absent from the lateral portion of the sarcolemma.

Two structurally distinct calcium storage sites in rat cardiac sarcoplasmic reticulum: an electron microprobe analysis study.

The elemental composition of subcellular organelles in resting rat papillary muscle was measured by electron probe x-ray microanalysis of cryosections of flash-frozen tissue. Nonmitochondrial electron-dense structures (50-100 nm in diameter) with a phosphorous concentration larger than 375 mmol/kg dry wt were identified in the interfibrillar spaces of the I band region. They were not visible in the proximity of transverse tubules. The sodium, magnesium, phosphorus, sulfur, chlorine, and potassium content of the electron dense structures showed a normal distribution, consistent with a uniform composition of a specific subcellular organelle. However, the distribution of the calcium concentrations in these electron-dense structures was bimodal, suggesting that they are composed of at least two subpopulations. One subpopulation had relatively high calcium (up to 53 mmol/kg dry wt) content with a mean value of 12.5 +/- 1.1 mmol/kg dry wt, while the other one had a relatively low calcium content with a mean value of 2.8 +/- 0.3 mmol/kg dry wt. The mean calcium concentration in the junctional sarcoplasmic reticulum (j-SR) in rat papillary muscle with calcium concentrations larger than 6 mmol/kg dry wt was 14.6 +/- 2.0 mmol/kg dry wt. We propose that the electron-dense structures described above correspond to nonjunctional sarcoplasmic reticulum and that the population containing relatively high calcium concentrations is calsequestrin-containing corbular sarcoplasmic reticulum (c-SR) confined to the I band region, while the population containing relatively low calcium concentrations corresponds to anastomosing regions of the network sarcoplasmic reticulum that lack calsequestrin.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody to the Ca2+-ATPase of cardiac sarcoplasmic reticulum cross-reacts with slow type I but not with fast type II canine skeletal muscle fibers: an immunocytochemical and immunochemical study.

Ca2+-ATPase of the sarcoplasmic reticulum was localized in cryostat sections from three different adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with monoclonal antibodies to the Ca2+-ATPase. Type I (slow) myofibers were strongly labeled for the Ca2+-ATPase with a monoclonal antibody (II D8) to the Ca2+-ATPase of canine cardiac sarcoplasmic reticulum; the type II (fast) myofibers were labeled at the level of the background with monoclonal antibody II D8. By contrast, type II (fast) myofibers were strongly labeled for Ca2+-ATPase of rabbit skeletal sarcoplasmic reticulum. The subcellular distribution of the immunolabeling in type I (slow) myofibers with monoclonal antibody II D8 corresponded to that of the sarcoplasmic reticulum as previously determined by electron microscopy. The structural similarity between the canine cardiac Ca2+-ATPase present in the sarcoplasmic reticulum of the canine slow skeletal muscle fibers was demonstrated by immunoblotting. Monoclonal antibody (II D8) to the cardiac Ca2+-ATPase binds to only one protein band present in the extract from either cardiac or type I (slow) skeletal muscle tissue. By contrast, monoclonal antibody (II H11) to the skeletal type II (fast) Ca2+-ATPase binds only one protein band in the extract from type II (fast) skeletal muscle tissue. These immunopositive proteins coelectrophoresed with the Ca2+-ATPase of the canine cardiac sarcoplasmic reticulum and showed an apparent Mr of 115,000. It is concluded that the Ca2+-ATPase of cardiac and type I (slow) skeletal sarcoplasmic reticulum have at least one epitope in common, which is not present on the Ca2+-ATPase of sarcoplasmic reticulum in type II (fast) skeletal myofibers. It is possible that this site is related to the assumed necessity of the Ca2+-ATPase of the sarcoplasmic reticulum in cardiac and type I (slow) skeletal myofibers to interact with phosphorylated phospholamban and thereby enhance the accumulation of Ca2+ in the lumen of the sarcoplasmic reticulum following beta-adrenergic stimulation.

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.

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.

Localization of phospholamban in slow but not fast canine skeletal muscle fibers. An immunocytochemical and biochemical study.

Phospholamban, originally described as a cardiac sarcoplasmic reticulum protein, was localized in cryostat sections of three adult canine skeletal muscles (gracilis, extensor carpi radialis, and superficial digitalis flexor) by immunofluorescence labeling with highly specific phospholamban antibodies. Only some myofibers were strongly labeled with phospholamban antibodies. The labeling of myofibers with phospholamban antibodies was compared to the distribution of Type I (slow) and Type II (fast) myofibers as determined by staining adjacent sections cytochemically for the alkali-stable myosin ATPase, a specific marker for Type II myofibers. All the skeletal myofibers labeled for phospholamban above background levels corresponded to Type I (slow) myofibers. The presence of phospholamban in microsomal fractions isolated from canine superficial digitalis flexor (89 +/- 3% Type I) and extensor carpi radialis skeletal muscle (14 +/- 6% Type I) was confirmed by immunoblotting. Antiserum to cardiac phospholamban bound to proteins of apparent Mr values of 25,000 (oligomeric phospholamban) and 5,000-6,000 (monomeric phospholamban) in sarcoplasmic reticulum vesicles from both muscles. Quantification of phospholamban in sarcoplasmic reticulum vesicles from cardic, slow, and fast skeletal muscle tissues following phosphorylation with [gamma-32P] ATP suggested that superficial digitalis flexor and extensor carpi radialis skeletal muscle contained about 16 and 3%, respectively, as much phospholamban as cardiac muscle per unit of sarcoplasmic reticulum. The presence of phospholamban in both Type I (slow) and cardiac muscle fibers supports the possibility that the Ca2+ fluxes across the sarcoplasmic reticulum in both fiber types are similarly regulated, and is consistent with the idea that the relaxant effect of catecholamines on slow skeletal muscle is mediated in part by phosphorylation of phospholamban.

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.

Monoclonal antibodies to the Ca2+ + Mg2+-dependent ATPase of sarcoplasmic reticulum identify polymorphic forms of the enzyme and indicate the presence in the enzyme of a classical high-affinity Ca2+ binding site.

In order to determine whether polymorphic forms of the Ca2+ + Mg2+-dependent ATPase exist, we have examined the cross-reactivity of five monoclonal antibodies prepared against the rabbit skeletal muscle sarcoplasmic reticulum enzyme with proteins from microsomal fractions isolated from a variety of muscle and nonmuscle tissues. All of the monoclonal antibodies cross-reacted in immunoblots against rat skeletal muscle Ca2+ + Mg2+-dependent ATPase but they cross-reacted differentially with the enzyme from chicken skeletal muscle. No cross-reactivity was observed with the Ca2+ + Mg2+-dependent ATPase of lobster skeletal muscle. The pattern of antibody cross-reactivity with a 100,000 dalton protein from sarcoplasmic reticulum and microsomes isolated from various muscle and nonmuscle tissues of rabbit demonstrated the presence of common epitopes in multiple polymorphic forms of the Ca2+ + Mg2+-dependent ATPase. One of the monoclonal antibodies prepared against the purified Ca2+ + Mg2+-dependent ATPase of rabbit skeletal muscle sarcoplasmic reticulum was found to cross-react with calsequestrin and with a series of other Ca2+-binding proteins and their proteolytic fragments. Its cross-reactivity was enhanced in the presence of EGTA and diminished in the presence of Ca2+. Its lack of cross-reactivity with proteins that do not bind Ca2+ suggests that it has specificity for antigenic determinants that make up the Ca2+-binding sites in several Ca2+-binding proteins including the Ca2+ + Mg2+-dependent ATPase.