Monoclonal antibodies to the muscle isoform of alpha-actinin--a marker for the study of the differentiation of skeletal and cardiac muscles

A battery of monoclonals to the rabbit skeletal muscle alpha-actinin has been produced. The majority of monoclonals proved to be species-specific by indirect immunofluorescence on the isolated rabbit skeletal myofibrils and on the differentiating cultures of chicken and rat skeletal muscles. One monoclonal, EA-53, reacts with the skeletal muscle alpha-actinin of various species (rat, rabbit, chicken) in immunofluorescence and immunoblotting. The monoclonal EA-53 recognizes also heart muscle alpha-actinin in cultured cardiomyocytes of human, rat and mouse origin. EA-53 does not stain alpha-actinin in myoblasts, fibroblasts, and endothelial cells. The monoclonal antibody EA-53 discriminating muscle and nonmuscle alpha-actinin isoforms could be used as a tool to study the mechanisms of skeletal and cardiac myogenesis.     

Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.     

alpha-Actinin and tropomyosin interactions with a hybrid complex of erythrocyte-actin and muscle-myosin.

alpha-Actinin isolated from dog muscle was used to incite antibodies in rabbits, Antibodies, purified by affinity chromatography on CNBr-Sepharose coupled with alpha-actinin and then ferritin-labeled were found to localize on the Z disc of muscle sarcomeres. Molecules of alpha-actinin as an adsorbed monolayer on the surface of polystyrene Lytron particles could bind muscle-actin and tropomyosin from solution. Both the ATPase activity and superprecipitation of an erythrocyte-actin and muscle-myosin hybrid actomyosin complex were altered by alpha-actinin, while tropomyosin diminished these alpha-actinin effects. The binding properties of alpha-actinin are consistent with those of an anchoring protein for microfilaments in nonmuscle cells.     

Acute effects of desmin mutations on cytoskeletal and cellular integrity in cardiac myocytes

Mutations in desmin have been associated with a subset of human myopathies. Symptoms typically appear in the second to third decades of life, but in the most severe cases can manifest themselves earlier. How desmin mutations lead to aberrant muscle function, however, remains poorly defined. We created a series of four mutations in rat desmin and tested their in vitro filament assembly properties. RDM-G, a chimera between desmin and green fluorescent protein, formed protofilament-like structures in vitro. RDM-1 and RDM-2 blocked in vitro assembly at the unit-length filament stage, while RDM-3 had more subtle effects on assembly. When expressed in cultured rat neonatal cardiac myocytes via adenovirus infection, these mutant proteins disrupted the endogenous desmin filament to an extent that correlated with their defects in in vitro assembly properties. Disruption of the desmin network by RDM-1 was also associated with disruption of plectin, myosin, and alpha-actinin organization in a significant percentage of infected cells. In contrast, expression of RDM-2, which is similar to previously characterized human mutant desmins, took longer to disrupt desmin and plectin organization and had no significant effect on myosin or alpha-actinin organization over the 5-day time course of our studies. RDM-3 had the mildest effect on in vitro assembly and no discernable effect on either desmin, plectin, myosin, or alpha-actinin organization in vivo. These results indicate that mutations in desmin have both direct and indirect effects on the cytoarchitecture of cardiac myocytes.     

The effect of taurine on cardiac sarcoplasmic reticulum

A recent communication reported that the rate of calcium uptake by sarcoplasmic reticulum (SR) isolated from rat skeletal muscle could be increased by the isolation of the SR in 15 mM taurine, and that exposure of the SR to taurine throughout the isolation procedure resulted in an increased yield of SR. Because of these results in rat skeletal muscle SR studies were carried out on dog myocardial SR.Sarcoplasmic reticulum isolated from adult dog cardiac muscle was not affected by taurine in concentrations as high as 15 millimolar. The addition of taurine to isolation media did not affect calcium transport, ATPase, binding, or release. Sarcoplasmic reticulum fragments were stored and re-examined over a period of a week without appreciable difference in stability of activity between those isolated in the presence of taurine and the control group. This lack of effect suggests that the role of taurine in cardiac muscle metabolism is not likely to be found in regulation of the sarcoplasmic reticulum.     

Comparison of ATPase activity of cardiac sarcoplasmic reticulum fraction from rat and dog

The purpose of the present study was to compare the ATPase activities of cardiac SR in two species in which the different intrinsic myocardial contractility can only partially be explained by the different properties of cardiac myosins. In cardiac SR isolated from rat heart, the total ATPase activity was 1512.5 +/- 23.3 nmol Pi/mg protein/min, nearly four times as high as in dog cardiac SR (408.8 +/- 28.9 nmol Pi/mg protein/min). The Ca2+-activated ATPase in rat cardiac SR represented only 23.8% of the total ATPase activity, while in dog cardiac SR it was approximately 50% of the total. Thus, the specific Ca2+-activated ATPase was nearly two times higher in the cardiac SR of the rat than in that of the dog. This higher rate of ATP hydrolysis in rat cardiac SR may be, at least in part, responsible for the increased intensity and shorter duration of the active state in the rat myocardium. Polyacrylamide gel electrophoresis of SR showed that the relative amount of Ca2+-pump protein was two times higher in dog heart, similar to the percentage of Ca2+-activated ATPase activity. At the same time, the specific Ca2+-activated ATPase activity and the relative amount of Ca2+ pump protein in both the rat and dog cardiac SR were inversely related.     

Electrical, contractile, and ultrastructural properties of adult rat and guinea-pig ventricular myocytes in long-term primary cultures

The electrical, contractile, and morphological characteristics of ventricular myocytes isolated from adult rat and guinea-pig hearts and maintained in cultures for 7-24 days are described. These cultured cells form different networks, depending on the species, when plated at certain density and maintained under specific conditions; the cells within the networks appear to be electrically coupled. Their resting and action potentials, their contractile activity (shortenings), and their pharmacological responses qualitatively resemble those of freshly isolated myocytes. Cultured cells from both species exhibit near-normal ultrastructural organization of sarcomeres, myofilaments, and mitochondria, as well as formation of intercellular contacts, or gap junctions. These data indicate that cultured adult rat and guinea-pig myocardial cells that make intercellular contacts possess electrical, contractile, and ultrastructural properties and responses to pharmacological agents similar to those of the respective adult myocardial tissues and the functionally intact freshly isolated cells from which these cultures are prepared. Thus, this study indicates that long-term cultures (7-24 days) of networked cardiac myocytes could be used as a valuable experimental model in various investigations of excitation-contraction coupling in cardiac muscle.     

Polymorphism of alpha-actinin. Electrophoretic and immunological studies of rabbit skeletal muscle alpha-actinins

Heterogeneity of alpha-actinins from rabbit skeletal muscles was studied. Polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate has made it possible to distinguish two closely related alpha-actinins from rabbit fast, white muscle. One isoprotein (designated type I alpha-actinin) appears to be preferentially located in the psoas muscle, while the other (designated type II alpha-actinin) appears to be preferentially located in the longissimus dorsi muscle. Electrophoretic analyses have further shown that the two isoproteins are present as mixtures in most rabbit white, fast-twitch muscles. A standard polyacrylamide gel--sodium dodecyl sulfate/polyacrylamide gel sequential electrophoretic procedure was developed to resolve the different alpha-actinin dimers and to determine their subunit compositions. By this technique, both type I and type II alpha-actinins appeared to be homodimers. No heterodimeric species of alpha-actinin were detected. alpha-Actinin of red, slow-twitch muscles was similar to type II alpha-actinin of fast, white muscle on one-dimensional and two dimensional gels. However, slow, red muscle alpha-actinin was significantly different from fast, white muscle alpha-actinins in terms of one-dimensional peptide mapping and immunological cross-reactivity.     

Isoforms of alpha-actinin from cardiac, smooth, and skeletal muscle form polar arrays of actin filaments

We have used a positively charged lipid monolayer to form two-dimensional bundles of F-actin cross-linked by alpha-actinin to investigate the relative orientation of the actin filaments within them. This method prevents growth of the bundles perpendicular to the monolayer plane, thereby facilitating interpretation of the electron micrographs. Using alpha-actinin isoforms isolated from the three types of vertebrate muscle, i.e., cardiac, skeletal, and smooth, we have observed almost exclusively cross-linking between polar arrays of filaments, i.e., actin filaments with their plus ends oriented in the same direction. One type of bundle can be classified as an Archimedian spiral consisting of a single actin filament that spirals inward as the filament grows and the bundle is formed. These spirals have a consistent hand and grow to a limiting internal diameter of 0.4-0.7 microm, where the filaments appear to break and spiral formation ceases. These results, using isoforms usually characterized as cross-linkers of bipolar actin filament bundles, suggest that alpha-actinin is capable of cross-linking actin filaments in any orientation. Formation of specifically bipolar or polar filament arrays cross-linked by alpha-actinin may require additional factors that either determine the filament orientation or restrict the cross-linking capabilities of alpha-actinin.     

Molecular properties and functions in vitro of chicken smooth-muscle alpha-actinin in comparison with those of striated-muscle alpha-actinins

alpha-Actinin purified from chicken gizzard smooth muscle was characterized in comparison with alpha-actinins from chicken striated muscles, or fast-skeletal muscle, slow-skeletal muscle, and cardiac muscle. The gizzard alpha-actinin molecule consisted of two apparently identical subunits with a molecular weight of 100,000 on SDS-polyacrylamide gel electrophoresis, as do striated-muscle alpha-actinins. Its isoelectric points in the presence of urea were similar to the striated-muscle counterparts. Despite these similarities, distinctive amino acid sequences between smooth-muscle alpha-actinin and striated-muscle alpha-actinins were revealed by peptide mapping using limited proteolysis in SDS. Gizzard alpha-actinin was immunologically distinguished from striated-muscle alpha-actinins. Gizzard alpha-actinin formed bundles of gizzard F-actin as well as of skeletal-muscle F-actin, but could not form any cross-bridges between adjacent actin filaments under conditions where skeletal-muscle alpha-actinin could. Temperature-dependent competition between gizzard alpha-actinin and tropomyosin on binding to gizzard thin filaments was demonstrated by electron microscopic observations. Gizzard alpha-actinin promoted Mg2+-ATPase activity of reconstituted skeletal actomyosin, gizzard acto-skeletal myosin, and gizzard actomyosin. This promoting effect was depressed by the addition of gizzard tropomyosin. These findings imply that, despite structural differences between gizzard and striated-muscle alpha-actinin molecules, they function similarly in vitro, and that gizzard alpha-actinin can interact not only with smooth-muscle actin (gamma- and beta-actin) but also with skeletal-muscle actin (alpha-actin).     

Subcellular origin of the oxalate- or inorganic phosphate-stimulated Ca2+ transport by smooth muscle microsomes: revisitation of the old problem by a new approach using saponin

Saponin, a cell-skinning reagent which perforates the cell membrane via its specific interaction with plasmalemmal cholesterol, was used to identify the subcellular origin of ATP-dependent Ca2+ accumulation in the presence and absence of inorganic phosphate and oxalate by microsomal fractions isolated from rat vas deferens and dog aorta. The purified plasma membranes from rat gastric fundus muscle, which elicit the stimulation of ATP-dependent Ca2+ accumulation by inorganic phosphate but not by oxalate, were used as a control reference. Saponin at concentrations effective for skinning smooth muscle fibres (10-50 micrograms/ml) inhibited Ca2+ binding in the absence of ATP to a similar extent in all fractions, but the inhibition of ATP-dependent Ca2+ accumulation was more pronounced in dog aorta microsomes and rat gastric fundus muscle plasma membranes than in rat vas deferens microsomes. The resistance of phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation to inhibition by saponin was much greater in rat vas deferens than in dog aorta microsomes. Our results suggest that phosphate- and oxalate-stimulated ATP-dependent Ca2+ accumulation also occurs in plasma membrane vesicles isolated from smooth muscle and is by no means an unique property of endoplasmic reticulum.     

Muscle Z-band ultrastructure: titin Z-repeats and Z-band periodicities do not match

Vertebrate muscle Z-bands show zig-zag densities due to different sets of alpha-actinin cross-links between anti-parallel actin molecules. Their axial extent varies with muscle and fibre type: approximately 50 nm in fast and approximately 100 nm in cardiac and slow muscles, corresponding to the number of alpha-actinin cross-links present. Fish white (fast) muscle Z-bands have two sets of alpha-actinin links, mammalian slow muscle Z-bands have six. The modular structure of the approximately 3 MDa protein titin that spans from M-band to Z-band correlates with the axial structure of the sarcomere; it may form the template for myofibril assembly. The Z-band-located amino-terminal 80 kDa of titin includes 45 residue repeating modules (Z-repeats) that are expressed differentially; heart, slow and fast muscles have seven, four to six and two to four Z-repeats, respectively. Gautel et al. proposed a Z-band model in which each Z-repeat links to one level of alpha-actinin cross-links, requiring that the axial extent of a Z-repeat is the same as the axial separation of alpha-actinin layers, of which there are two in every actin crossover repeat. The span of a Z-repeat in vitro is estimated by Atkinson et al. to be 12 nm or less; much less than half the normal vertebrate muscle actin crossover length of 36 nm. Different actin-binding proteins can change this length; it is reduced markedly by cofilin binding, or can increase to 38.5 nm in the abnormally large nemaline myopathy Z-band. Here, we tested whether in normal vertebrate Z-bands there is a marked reduction in crossover repeat so that it matches twice the apparent Z-repeat length of 12 nm. We found that the measured periodicities in wide Z-bands in slow and cardiac muscles are all very similar, about 39 nm, just like the nemaline myopathy Z-bands. Hence, the 39 nm periodicity is an important conserved feature of Z-bands and either cannot be explained by titin Z-repeats as previously suggested or may correlate with two Z-repeats.     

New, rapid methods for purifying alpha-actinin from chicken gizzard and chicken pectoral muscle

We introduce two new, rapid procedures. One is specifically designed for isolating alpha-actinin from skeletal and the other for isolating alpha-actinin from smooth muscle. Approximately 20 mg of greater than 95% pure alpha-actinin can be obtained/100 g of ground chicken pectoral muscle in just 4 days. The smooth muscle protocol yields 2.7 mg of greater than 99% pure alpha-actinin/100 g of ground gizzard after just 5 days. Differences in protein contaminants and in the extractability of alpha-actinin necessitated the development of separate isolation procedures for the two muscle types. Antibody prepared against the purified gizzard alpha-actinin reacted with alpha-actinin from skeletal, cardiac, and smooth muscle in immunodiffusion. Anti-alpha-actinin reacted only with alpha-actinin from crude extracts of skeletal and smooth muscle on Staph A gels. Anti-alpha-actinin stained Z-bands from skeletal muscle in indirect immunofluorescence microscopy and stress fibers from baby hamster kidney fibroblasts and mouse mammary epithelial cells in the characteristic punctate pattern observed by other workers (Lazarides, E., and Burridge, K. (1975) Cell 6, 289-298). These two methods for purifying alpha-actinin from skeletal and smooth muscle represent a significant improvement over that published previously.     

Alpha-actinin from sea urchin eggs: biochemical properties, interaction with actin, and distribution in the cell during fertilization and cleavage

A protein similar to alpha-actinin has been isolated from unfertilized sea urchin eggs. This protein co-precipitated with actin from an egg extract as actin bundles. Its apparent molecular weight was estimated to be approximately 95,000 on an SDS gel: it co-migrated with skeletal-muscle alpha-actinin. This protein also co-eluted with skeletal muscle alpha-actinin from a gel filtration column giving a Stokes radius of 7.7 nm, and its amino acid composition was very similar to that of alpha-actinins. It reacted weakly but significantly with antibodies against chicken skeletal muscle alpha-actinin. We designated this protein as sea urchin egg alpha-actinin. The appearance of sea urchin egg alpha-actinin as revealed by electron microscopy using the low-angle rotary shadowing technique was also similar to that of skeletal muscle alpha-actinin. This protein was able to cross-link actin filaments side by side to form large bundles. The action of sea urchin egg alpha-actinin on the actin filaments was studied by viscometry at a low-shear rate. It gelled the F-actin solution at a molar ratio to actin of more than 1:20, at pH 6-7.5, and at Ca ion concentration less than 1 microM. The effect was abolished by the presence of tropomyosin. Distribution of this protein in the egg during fertilization and cleavage was investigated by means of microinjection of the rhodamine-labeled protein in the living eggs. This protein showed a uniform distribution in the cytoplasm in the unfertilized eggs. Upon fertilization, however, it was concentrated in the cell cortex, including the fertilization cone. At cleavage, it seemed to be concentrated in the cleavage furrow region.     

Exchangeability of alpha-actinin in living cardiac fibroblasts and muscle cells

We have investigated the exchangeability of alpha-actinin in various structures of cultured chick cardiac fibroblasts and muscle cells using fluorescent analogue cytochemistry in combination with fluorescence recovery after photobleaching. Living cells were microinjected with tetramethylrhodamine-labeled alpha-actinin, which became localized in cellular structures. Small areas of labeled structures were then photobleached with a laser pulse, and the subsequent recovery of fluorescence was monitored with an image intensifier coupled to an image-processing system. In fibroblasts, fluorescence recovery was studied in stress fibers and in adhesion plaques. Bleached spots in adhesion plaques generally attained complete recovery within 20 min; whereas complete recovery in stress fibers occurred within 30 to 60 min. In muscle cells, alpha-actinin became localized in the Z-lines of sarcomeres, in punctate structures, and in apparently continuous bundle-like structures. Fluorescence recovery in Z-lines, punctate structures, and some bundle-like structures was extremely slow. Complete recovery did not occur within the 6- to 7-h observation period. However, some bundle-like structures recovered completely within 60 min, a rate similar to that of stress fibers in fibroblasts. These results indicate that fluorescently labeled alpha-actinin is more stably associated with structures in muscle cells than in fibroblasts. In addition, different structures within the same cell can display different alpha-actinin exchangeabilities which, in muscle cells, could be developmentally related.     

Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle.

Calsequestrin is a high-capacity Ca(2+)-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Two isoforms of calsequestrin, cardiac and skeletal muscle forms, have been described which are products of separate genes. Purified forms of the two prototypical calsequestrin isoforms, dog cardiac and rabbit fast-twitch skeletal muscle calsequestrins, serve as excellent substrates for casein kinase II and are phosphorylated on distinct sites (Cala, S.E. and Jones, L.R. (1991) J. Biol. Chem 266, 391-398). Dog cardiac calsequestrin is phosphorylated at a 50 to 100-fold greater rate than is rabbit skeletal muscle calsequestrin, and only the dog cardiac isoform contains endogenous Pi on casein kinase II phosphorylation sites. In this study, we identified and examined both calsequestrin isoforms in rat muscle cultures and homogenates to demonstrate that the cardiac isoform of calsequestrin in rat skeletal muscle was phosphorylated in vivo on sites which are phosphorylated by casein kinase II in vitro. Phosphorylation of rat skeletal muscle calsequestrin was not detected. In tissue homogenates, cardiac and skeletal muscle calsequestrin isoforms were both found to be prominent substrates for endogenous casein kinase II activity with cardiac calsequestrin the preferred substrate. In addition, these studies revealed that the cardiac isoform of calsequestrin was the predominant form expressed in skeletal muscle of fetal rats and cultured myotubes.     

Identification of myosin-like proteins included in chromatin

A myosin-like protein was purified 40-fold from rat liver chromatin (as determined by the K+-EDTA-ATPase activity equal to 0.013 and 0.442 M PPi/mg.min for chromatin and purified protein, respectively). Electrophoresis performed under non-denaturating conditions revealed that the overall ATPase activity of the sample is associated with one component whose migration is very similar to that of skeletal muscle myosin. The myosin components isolated from the nuclei and cytoplasm of rat cardiac muscle differ by their electrophoretic mobilities; those from nuclei of different tissues, i.e., liver and heart, have similar mobilities.     

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.