A O Jorgensen

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.

Immunohistochemical and electron microscopic assessment of childhood rhabdomyosarcoma. Increased frequency of diagnosis over routine histologic methods.

Histologic examination was carried out in 65 cases of childhood rhabdomyosarcoma (RMS), 53 embryonal, and 12 alveolar. Cross‐striations were seen on light microscopy in 12 (23%) embryonal and 4 (33%) alveolar tumors. The capacity of immunohistochemical staining (PAP technique) to increase diagnostic accuracy was assessed, using antibodies against myoglobin, the MM isoenzyme of creatine kinase, desmin, calcium magnesium‐dependent ATPase of sarcoplasmic reticulum and calsequestrin. Myoglobin was detected in 16 (30%) embryonal and eight (67%) alveolar RMS, higher numbers than obtained by viewing cross‐striations on light microscopy. The creatine kinase antibody was slightly better than the antibody to myoglobin and 15 of 25 (60%) embryonal RMS were positive when both specificities were used. The remaining three antibodies were less useful. Of 13 (two alveolar and 11 embryonal) RMS studied by electron microscopy, four showed cross‐striations, contained late myoblasts, and were positive for myoglobin. Three additional cases showed only late myoblasts and one of these was positive for myoglobin. Thus, 16 of 25 (64%) of the embryonal and seven of nine (78%) of the alveolar RMS showed either positive immunostaining or ultrastructural features of RMS. This study indicates that a combination of immunohistochemical staining, using antimyoglobin and anticreatine kinase (MM isoenzyme) antibodies, and electron microscopy are useful markers in the diagnosis of childhood RMS.

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.

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 organdie. However, the distribution of the calcium concentrations hi 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 sarcopiasmic 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 sarcopiasmic reticulum and that the population containing relatively high calcium concentrations is calsequestrin-containing corbular sarcopiasmic reticulum (c-SR) confined to the I band region, while the population containing relatively low calcium concentrations corresponds to anastomosing regions of the network sarcopiasmic reticulum that hick calsequestrin. These results are consistent with the idea that both the corbular sarcopiasmic reticulum and the junctional sarcopiasmic reticulum are potential sources of Ca2+ released into the myofibrillar space during excitation-contraction coupling in cardiac muscle.

Chapter 8 Assembly of the Sarcoplasmic Reticulum during Muscle Development

Publisher Summary This chapter focuses on the assembly of the sarcoplasmic reticulum during muscle development. The sarcoplasmic reticulum membrane from mammalian or avian skeletal muscle, like a number of other membrane systems whose synthesis has been successfully probed, has certain unique features that recommend its study. The membrane has relatively few major proteins, all of which have been well characterized with respect to their location and orientation within the membrane. Advances in recombinant DNA technology may open the potential for a genetic approach to the assembly of the sarcoplasmic reticulum. The sarcoplasmic reticulum is an organelle system wholly contained within muscle cells, where it functions in the control of intracellular free Ca 2+ concentrations. It is composed of about one-third phospholipid and neutral lipids and two-thirds protein and carries out a differentiated function in muscle cells. The precursors to multinucleated myotubes—the myoblasts or satellite cells—do not have an extensive organellar network for the control of Ca 2+ , yet such a network develops in multinucleated myotubes. The combined biochemical and morphological studies described so far have shown that there is a gradual increase of the Ca 2+ transport system in microsomal vesicles during development. Extracellular Ca 2+ is an important determinant of the fusion of myoblasts in culture to form myotubes. If medium Ca 2+ is kept below a concentration of a few hundred micromolar, fusion is inhibited. This observation has led to a variety of experiments in which manipulation of fusion has been studied as a determinant of differentiation.

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.

Evidence for the presence of calsequestrin in both peripheral and interior regions of sheep Purkinje fibers.

Localization of calsequestrin in sheep Purkinje fibers was determined by indirect immunofluorescence labeling of cryostat sections of sheep myocardium from the intraventricular wall. The results presented show that calsequestrin is present in discrete foci at the peripheral, as well as the interior regions of the cytoplasm. Since Purkinje fibers lack transverse tubules, the presence of calsequestrin at specific foci in the interior regions of the cytoplasm in these cells suggests that calsequestrin is localized in the lumen of peripheral junctional sarcoplasmic reticu-lum, as well as in the lumen of corbular sarcoplasmic reticulum present in the I band region of the myofibrils. Assuming that the function of calsequestrin is to sequester calcium into the lumen of the sarcoplasmic reticulum, these results imply that two structurally different regions of the sarcoplasmic reticulum function as calcium storage sites in mammalian Purkinje fibers and raises the possibility that calcium storage and/or release from these two sites might be regulated differently.