Adult cardiac muscle cells in long-term serum-free culture: myofibrillar organization and expression of myosin heavy chain isoforms.

A culture system for adult rat cardiac muscle cells has been established without exposure of cells to serum at any step of the procedure. The methodology has been standardized and optimized to obtain better quality and high yield of cells and culture. Subsequent to enzyme perfusion, the release of myocytes from enzyme-perfused tissues was carried out in enzyme-free Joklik's medium instead of exposing cells to proteolytic enzyme(s) as done previously. Approximately 5 million cylindrical muscle cells per ventricle were obtained. The culture medium contained Eagle's minimum essential medium with Earle's salts, basic fibroblast growth factor, epidermal growth factor, insulin, transferrin, selenium, norepinephrine, triiodothyronine (T3), bovine serum albumin, nonessential amino acids, and ascorbic acid. The plating efficiency of the experimental cultures was comparable to that of the control cultures grown in the presence of serum. The cells in the serum-free medium contained myofibrillar and myosin isoforms characteristics of the adult myocytes. The cells underwent cellular reorganization comparable to that of the controls. The initial phase of reorganization involved the breakdown of myofibrils and extrusion of mitochondria, degraded myofibrils, and other cellular organelles. The latter phase of reorganization included myofibrillogenesis and organellogenesis resulting in the development of myofibrillar apparatus with cellular organelles. Myocytes were contractile throughout the culture period. Cardiac myocytes grown in serum-free medium expressed the predominant myosin isoform V1 similar to their counterparts in vivo. T3 is essential for the expression of isomyosin V1.(ABSTRACT TRUNCATED AT 250 WORDS)

Z-band proteins in the flight muscle and leg muscle of the honeybee.

Monoclonal antibodies (mAb's) have been raised against proteins in preparations of Z-discs isolated from honeybee fibrillar flight muscle. These antibodies have identified four Z-disc antigens on immunoblots of honeybee fibrillar proteins. Antibody alpha binds to the 90-100 kD protein, alpha-actinin; mAb P interacts with the protein, projectin, an extremely large polypeptide (greater than 600kD) found in the connecting filaments which link thick filaments to the Z-band in insect asynchronous flight muscle. Two other mAb's recognize previously uncharacterized insect Z-band proteins. Monoclonal antibody Z(400) binds to a pair of proteins with molecular masses near 400 kD and 600 kD. Antibody Z(175) recognizes two components, 158 kD and 175 kD, that are not only immunologically similar but have nearly identical peptide maps. Indirect immunofluorescence microscopy studies show that the proteins recognized by mAb's alpha, Z(175) and Z(400) are located at the Z-band, while the mAb P antigen is found on either side of it. Three of the four antibodies we have obtained recognize leg muscle proteins. Monoclonal antibodies alpha and P comigrate on SDS gels with analogous components from flight muscle. Only the smaller of the two proteins identified in flight muscle by mAb Z(400) is found in leg muscle, however. Furthermore, no Z(175) antigens have been detected in the non-fibrillar tissue by either monoclonal or polyclonal antibodies. Immunofluorescence microscopy studies localize the alpha and Z(400) antigens at the Z-line in leg muscle fibrils. Surprisingly, however, mAb P binds within the A-bands of synchronous fibres, not between the A- and Z-bands as in asynchronous fibrillar muscle.

A myofibrillar protein of insect muscle related to vertebrate titin connects Z band and A band: purification and molecular characterization of invertebrate mini-titin.

We show that myofibrils of insect flight and leg muscle contain a doublet of polypeptides with apparent molecular weights of 700K (K = 10(3) Mr) (Hmp I) and 600K (Hmp II), respectively. In Locusta migratoria high ionic strength extraction solubilizes only Hmp II, which is readily purified in native form. It probably reflects a proteolytic derivative of the non-extractable Hmp I. On the basis of its viscosity radius and sedimentation coefficient, Hmp II has a molecular weight of 600K and seems to consist of a single polypeptide chain. The highly asymmetric structure of the molecule is confirmed by rotary shadowing. The flexible rods have a uniform diameter of 3-4 nm and an average length of 260 nm. Polyclonal antibodies show cross-reactivity between Hmp II and its putative precursor Hmp I. We discuss the similarities and differences between the larger titin I/titin II of vertebrate sarcomeric muscle and the smaller Hmp I/Hmp II of invertebrate muscle and conclude that the latter may reflect a mini-titin. In line with the smaller length, immunoelectron microscopy locates the insect mini-titin to the I band and a very short portion of the A band only, while vertebrate titin is known to connect the Z band to the M band. Mini-titin has also been purified from several other insects including Drosophila. Immunofluorescence microscopy on frozen sections shows that mini-titin is present in the sarcomeric muscles of various species from different invertebrate phyla. These include Annelida, Nematomorpha, Plathelmintha, Nemertea and Nematoda like Ascaris lumbricoides and Caenorhabditis elegans. This wide-spread occurrence of invertebrate mini-titin is confirmed by immunoblotting experiments.

Structural change of troponin C molecule and its domains upon Ca2+ binding in the presence of Mg2+ ions measured by a solution X-ray scattering technique.

A small-angle X-ray scattering study on troponin C showed that two domains of the molecule move closer to each other and the molecule shrinks along its long axis upon Ca2+ binding in the absence of Mg2+ ions (Fujisawa, T., Ueki, T., & Iida S. (1988) J. Biochem. 105, 377-383). When Mg2+ ions bind to troponin-C, the radius of gyration changes from 27.8 to 24.3 A and the average radius of gyration of the two domains is estimated to be 15.1 A. These radii indicate that the distance between the centers of the two domains is 38.1 A. Such a change is analogous to the previous result for troponin C with two Ca2+ ions bound at the high-affinity sites. Thus, the structural behavior of troponin C molecule is essentially the same when Ca2+/Mg2+ ions bind to its high-affinity sites. On the other hand, the effect of Ca2+ binding to the low-affinity sites in the presence of Mg2+ ions is quite different from the previous result. The binding of Ca2+ ions causes a dimerization of troponin C molecules with an apparent constant of 511 M-1. Such a characteristic behavior, implying the occurrence of a surface property change, may be related to the physiological role of troponin C molecule in the muscle. The scattering experiments on the tryptic fragments of troponin C also had interesting and important results: the C-domain shrinks, with the radius of gyration changing from 17.0 to 14.9 A while the N-domain swells from 13.9 to 15.0 A upon Ca2+ binding. Such an opposite change is consistent with the results of circular dichroism and spectroscopic studies of the domains.

Fast and slow isoforms of troponin I and troponin C. Distribution in normal rabbit muscles and effects of chronic stimulation.

Polyclonal antibodies were raised against troponin I (TnI) and troponin C (TnC) purified from fast-twitch and slow-twitch rabbit muscles. These antibodies were used to elucidate the distribution of fast and slow isoforms of TnI and TnC in normal and chronically stimulated rabbit hind limb muscles by immunoblots of one-dimensional and two-dimensional electrophoreses. In contrast to the multiplicity of fast and slow troponin T (TnT) isoforms, TnI and TnC were present as unique fast and slow isoforms. Whereas no charge variants were detected for slow TnI, fast TnI was present in at least three charge variants. As judged from the results of alkaline phosphatase digestion, these charge variants represent differently phosphorylated forms. Fast and slow TnC both exist as two charge variants which, however, were unaffected by alkaline phosphatase treatment. Chronic low-frequency stimulation of fast-twitch muscles induced progressive increases in the slow isoforms of TnC and TnI at the expense of their fast isoforms. The extent of the fast-to-slow transition was more pronounced in the case of TnC than in that of TnI. Long-term stimulated muscles with a complete fast-to-slow transition, at the level of the TnT isoforms, still contained fast and slow isoforms of both TnI and TnC. The coexistence of fast and slow isoforms of the three troponin subunits in the transforming muscle was interpreted as indicating the presence of hybrid troponin molecules composed of fast and slow isoforms. Studies at the mRNA level showed changes similar to those at the protein level. However, in long-term stimulated muscles, the fast-to-slow transition of TnI was more pronounced at the mRNA level than at the protein level.

Tropomyosin-troponin complex stabilizes the pointed ends of actin filaments against polymerization and depolymerization.

In striated muscle the pointed ends of polar actin filaments are directed toward the center of the sarcomere. Formed filaments keep a constant length of about 1 micron. As polymerization and depolymerization at free pointed ends are not sufficiently slow to account for the constant length of the filaments, we searched for proteins which occur in sarcomeres and can stabilize the pointed ends of actin filaments. We observed that tropomyosin-troponin complex reduces the rate of association and dissociation of actin molecules at the pointed ends more than 30-fold. On the average, every 600 s one association or dissociation reaction has been found to occur at the pointed ends near the critical actin monomer concentration.

Inhibition reactivation myofibrillar ATPase technique for demonstration of three fiber types in a single cryostat muscle section.

An inhibition reactivation technique was used for histochemical staining of human skeletal muscle sections. Myofibrillar ATPase activity was inhibited by sodium hydroxymercuribenzoate (2.5 mM in 0.1 M Tris-HCl buffer, pH 7.2-7.5, 30 min) and successively reactivated by cysteine which was added to incubation solution (10 mM cysteine-HCl, 2.5 mM ATP-disodium salt, 50 mM potassium chloride and 27 mM calcium chloride in barbital buffer, pH 9.4, 35 min at 37 C). This technique allows the distinction of three fiber categories with different staining intensities in single cross-section. Dark, intermediate and light fibers correspond to IIB, I, and IIA types, respectively. Storage of air dried sections in the freezer at -20 C for one month had no influence on staining characteristics.

Characterization of components of Z-bands in the fibrillar flight muscle of Drosophila melanogaster.

Twelve monoclonal antibodies have been raised against proteins in preparations of Z-disks isolated from Drosophila melanogaster flight muscle. The monoclonal antibodies that recognized Z-band components were identified by immunofluorescence microscopy of flight muscle myofibrils. These antibodies have identified three Z-disk antigens on immunoblots of myofibrillar proteins. Monoclonal antibodies alpha:1-4 recognize a 90-100-kD protein which we identify as alpha-actinin on the basis of cross-reactivity with antibodies raised against honeybee and vertebrate alpha-actinins. Monoclonal antibodies P:1-4 bind to the high molecular mass protein, projectin, a component of connecting filaments that link the ends of thick filaments to the Z-band in insect asynchronous flight muscles. The anti-projectin antibodies also stain synchronous muscle, but, surprisingly, the epitopes here are within the A-bands, not between the A- and Z-bands, as in flight muscle. Monoclonal antibodies Z(210):1-4 recognize a 210-kD protein that has not been previously shown to be a Z-band structural component. A fourth antigen, resolved as a doublet (approximately 400/600 kD) on immunoblots of Drosophila fibrillar proteins, is detected by a cross reacting antibody, Z(400):2, raised against a protein in isolated honeybee Z-disks. On Lowicryl sections of asynchronous flight muscle, indirect immunogold staining has localized alpha-actinin and the 210-kD protein throughout the matrix of the Z-band, projectin between the Z- and A-bands, and the 400/600-kD components at the I-band/Z-band junction. Drosophila alpha-actinin, projectin, and the 400/600-kD components share some antigenic determinants with corresponding honeybee proteins, but no honeybee protein interacts with any of the Z(210) antibodies.

Structure and function of contractile proteins in human dilated cardiomyopathy.

The pathogenesis of reduced systolic left ventricular function in dilated cardiomyopathy is yet unclear. To analyze a possible involvement of contractile protein, function and structure of left ventricular myofibrils were examined in hearts of patients with advanced cardiomyopathy undergoing heart transplantation and in normal control hearts (from renal transplant donors). Myosin and actin content of the left ventricular myocardium was slightly reduced in cardiomyopathic hearts. Myofibrillar polypeptide composition was determined using two-dimensional electrophoresis and immunoblotting. No differences in constituting polypeptides were apparent, including Z-line proteins and proteins of the endosarcomeric lattice. M-line-bound creatine kinase was identical in both groups. Further, basal and maximal myofibrillar adenosine triphosphatase (ATPase) activities were unaltered in dilated cardiomyopathy. The structure of purified myosin was identical in both groups by the following criteria: electrophoretic mobility of native myosin, identical pattern of light chains after isoelectric focusing, identical cleavage peptides of myosin's heavy chain, and identical patterns after immunoblotting of heavy chain cleavage peptides using polyclonal antibodies generated against myosin from normal and cardiomyopathic ventricles. Ca2+-activated, K+-EDTA-activated and actin-activated myosin ATPase activities were identical in control and cardiomyopathic hearts. A structural alteration or functional defect of myofibrils does not seem to be primarily involved in the pathogenesis of reduced myocardial contractility in dilated cardiomyopathy.

Equilibrium distribution of ions in a muscle fiber.

We have developed a mathematical description of the equilibrium (Donnan) distribution of mobile ions between two phases containing fixed charges. This differs from the classical Donnan derivation by including mobile polyvalent ions such as those present in intact muscle fibers and in solutions used with skinned muscle fibers. Given the average concentrations of ionic species present in intact frog muscle, we calculate that the myofibrillar fixed charge density (-42 meq/liter cytoplasmic fluid) is in close agreement with estimates from amino acid analysis of myofibrillar proteins. As expected, with negative fixed charges in the myofibril, anions are excluded from the myofibrillar space while cations are concentrated in this space; the ratio between the average intra- and extramyofibrillar concentrations for an ion of valence n is (1.11)n. This model allowed us to design a bathing solution for skinned muscle fibers in which the intramyofibrillar ion concentrations closely approximate those found in intact frog muscle cells. Our model, applied to the A- and I-bands of the sarcomere, suggests that likely differences in fixed charge densities in these regions accounts for only a small fraction of the extreme concentration of phosphocreatine observed in the I-bands of intact frog muscle.

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins.

Electrophoresis of the high-molecular-mass proteins (greater than 500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoretic dye front. Inclusion of 10 mM 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mM 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.

Short-term stability and muscle adaptation after mandibular advancement surgery with and without suprahyoid myotomy in juvenile Macaca mulatta.

The purpose of this study was to examine the short-term adaptations that occur within the mandible and anterior digastric muscle complex after mandibular advancement with and without suprahyoid myotomy in 20 juvenile rhesus monkeys. The results showed that the animals that did not undergo myotomy experienced relapse equivalent to 13% of the surgical advancement. Those animals that underwent a myotomy of the digastric muscle complex showed complete stability of the surgical lengthening of the mandible. Both groups of animals grew normally after the fixation period when compared to age-matched control animals. Analysis of adaptations within the digastric muscle complex was performed with the use of radiopaque muscle and tendon markers. The results showed an immediate lengthening of the entire digastric muscle complex with mandibular advancement surgery in the group that underwent advancement without myotomy. Further analysis showed that most lengthening in these animals occurred at the connective tissue interfaces of the complex--at the muscle-bone and muscle-tendon interfaces. No significant changes in sarcomere or fiber length were found in the group that did not undergo myotomy, although there was a significant shortening of muscle fibres resulting from loss of serial sarcomeres in the myotomy group. Comparison of histochemical characteristics of the anterior digastric muscle before and after surgery revealed the following findings: (1) there were no significant differences in percentage of composition between control and experimental muscles; (2) despite fixation of the jaws and myotomy, there was no evidence of atrophy of the anterior digastric muscle at any experimental interval; and (3) the type I fibers of the anterior digastric muscle underwent significant stretch-induced hypertrophy after lengthening. The results of this study support the hypothesis that tension produced by stretching of the connective tissues associated with the digastric muscle complex can contribute to postsurgical relapse of the surgically advanced mandible. However, no adverse effect on future growth of the mandible was observed from stretching the digastric muscle complex by mandibular advancement surgery in juvenile subjects.

Differential response of myofibrillar and cytoskeletal proteins in cells treated with phorbol myristate acetate.

Muscle-specific and nonmuscle contractile protein isoforms responded in opposite ways to 12-o-tetradecanoyl phorbol-13-acetate (TPA). Loss of Z band density was observed in day-4-5 cultured chick myotubes after 2 h in the phorbol ester, TPA. By 5-10 h, most I-Z-I complexes were selectively deleted from the myofibril, although the A bands remained intact and longitudinally aligned. The deletion of I-Z-I complexes was inversely related to the appearance of numerous cortical, alpha-actinin containing bodies (CABs), transitory structures approximately 3.0 microns in diameter. Each CAB consisted of a filamentous core that costained with antibodies to alpha-actin and sarcomeric alpha-actinin. In turn each CAB was encaged by a discontinuous rim that costained with antibodies to vinculin and talin. Vimentin and desmin intermediate filaments and most cell organelles were excluded from the membrane-free CABs. These curious bodies disappeared over the next 10 h so that in 30-h myosacs all alpha-actin and sarcomeric alpha-actinin structures had been eliminated. On the other hand vinculin and talin adhesion plaques remained prominent even in 72-h myosacs. Disruption of the A bands was first initiated after 15-20 h in TPA (e.g., 15-20-h myosacs). Thick filaments of apparently normal length and structure were progressively released from A segments, and by 40 h all A bands had been broken down into enormous numbers of randomly dispersed, but still intact single thick filaments. This breakdown correlated with the formation of amorphous cytoplasmic aggregates which invariably colocalized antibodies to myosin heavy chain, MLC 1-3, myomesin, and C protein. Complete elimination of all immunoreactive thick filament proteins required 60-72 h of TPA exposure. The elimination of the thick filament-associated proteins did not involve the participation of vinculin or talin. In contrast to its effects on myofibrils, TPA did not induce the disassembly of the contractile proteins in stress fibers and microfilaments either in myosacs or in fibroblastic cells. Similarly, TPA, which rapidly induces the translocation of vinculin and talin to ectopic sites in many types of immortalized cells, had no gross effect on the adhesion plaques of myosacs, primary fibroblastic cells, or presumptive myoblasts. Clearly, the response to TPA of contractile protein and some cytoskeletal isoforms not only varies among phenotypes, but even within the domains of a given myotube the myofibrils respond one way, the stress fibers/microfilaments another.

Detection of a specific isoform of alpha-actinin with antisera directed against dystrophin.

We have characterized a protein immunologically related to dystrophin, the protein product of the Duchenne muscular dystrophy gene. We identify this related protein as a fast-twitch glycolytic isoform (mouse extensor digitorum longus-specific) of myofibrillar alpha-actinin. This specific isoform of alpha-actinin exhibits a more restricted pattern of expression in skeletal muscle than fast-twitch-specific isoforms of both myosin and Ca2+-ATPase. Our results provide evidence that dystrophin and myofibrillar alpha-actinin are related proteins, reinforcing the previous data concerning the sequence homologies noted between nonmuscle cytoskeletal alpha-actinin and dystrophin. In addition, we describe the first antisera directed against a specific myofibrillar skeletal muscle isoform of alpha-actinin.

A silver-reducing component in rat striated muscle. I. Selective localization at the level of the terminal cistern/transverse tubule system. Light and electron microscope studies with a new histochemical procedure.

This study reports the presence of a silver-reducing constituent in rat striated muscle fiber located selectively at the level of the terminal cistern/transverse tubule system. It is related to the T tubule network at or near sites that participate in junctions with terminal cisternae, i.e., at both sides of the T tubule in skeletal muscle (triad) and, predominantly, at one side in the ventricle (dyad). Little reactivity is present in the auricle due to the scarcity of those membrane systems. The longitudinal sarcoplasmic reticulum, the sarcolemma, mitochondria and myofibrils are not outlined by the reaction product. Extraction of low molecular weight substances, nucleic acids and lipids did not suppress the chemical reaction. A new argentaffin (Hg--Ag) technique is described. Ethanol or aldehyde fixed muscles were passed to water, postfixed 6-24 h with mercuric acetate (5% w/v in 1% acetic acid), washed with 1% acetic acid and distilled water, stained 12-24 h at 43 degrees C with ammoniacal silver nitrate (60% w/v) and washed in 10% sodium sulfite (three changes) and water. All steps were carried out in darkness. Postfixation with mercuric acetate proved to be essential for immobilizing the argentaffin component without interfering with its strong argentaffinity. The procedure also provides a simple method for tracing the pathway of transversally oriented membrane systems in skeletal and cardiac muscle cells.

Myofibril assembly is linked with vinculin, alpha-actinin, and cell-substrate contacts in embryonic cardiac myocytes in vitro.

The relationship of nascent myofibrils with the accumulation of adhesion plaque proteins and the formation of focal cell contacts was studied in embryonic chick cardiac myocytes in vitro. The cultures were double-stained with various combinations of the specific antiactin drug phalloidin and antibodies against vinculin, alpha-actinin, connectin (titin), myosin heavy chain, fibronectin, and desmin and examined under fluorescence and interference reflection microscopy. In the areas of myofibril assembly, vinculin and alpha-actinin plaques were formed at the ventral sarcolemmae. These areas overlapped with the sites of cell-to-substrate focal contacts and extracellular fibronectin. Because the myofibrils always ran in a straight line between these sites, polarized lines appeared to be generated within the cells in response to their physical (e.g., stress) and/or biochemical environment (e.g., adhesion plaque proteins). The possible presence of other factors cannot be ruled out for the proper alignment of myofibrils. As soon as myofibrils came to span between these adhesion sites, they exhibited typically mature cross-striated characteristics. Thus, the formation of these inferred lines has some relation to, or is in fact necessary for, the maturation of myofibrils, in addition to the directional arrangement of sarcomeric proteins. Additionally, synthesis and distribution of myosin and connectin were tightly linked during early developmental (premyofibril and myofibril) stages. The spatial deployment of desmin was not coupled with vinculin. Thus, connectin and desmin do not appear to form the initial scaffold of sarcomeres.

Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. III. Generation of fasciae adherentes and costameres.

To study whether the first myofibrils are separate from or firmly bound to the myocytic cell membranes, whole mount preparations of 6-12-somite-stage chick embryonic hearts were examined by fluorescence microscopy after double labeling with antibodies to vinculin (fluorescein-conjugated) and rhodamine-phalloidin, or with antibodies to titin (rhodamine-conjugated) and nitrobenz-oxadiazole-phallacidin. When a small number of myofibrils appeared for the first time at the nine somite stage, most of them were already bound to the cell membranes through zonulae adherentes, fasciae adherentes, or costameres. In the outer of the two myocardial cell layers, in which the myocytes were closely in contact with each other along polygonal boundaries, fasciae adherentes and costameres developed at the boundaries, apparently by conversion of preexisting zonulae adherentes. On the other hand, in the inner cell layer, in which myocytes were more loosely associated with each other, both costameres and fasciae adherentes appeared to develop de novo, the former in association with the inner surface of the myocardial wall and the latter at the intercellular boundaries. The myofibrillar tracks in the inner layer followed long and smooth courses and were as a whole aligned in the circumferential direction of the tubular heart wall from the earliest stage of myofibril formation. Those in the outer layer were arranged in a pattern of two- or three-dimensional networks in the 9-10 somite stage, although many myofibrils were also circumferentially directed. The fact that the majority of the first myofibrils were already bound to the cell membranes in a directed manner suggests that myocytes at the earliest stage of myofibril formation are endowed with spatial information that directs the organization of nascent myofibrils. It is proposed that the myocyte cell membranes perform an essential role in cardiac myofibrillogenesis.

Immunocytochemical studies of spectrin in hamster cardiac tissue.

The spectrins are a family of cytoskeletal-membrane proteins that have a wide tissue distribution. In the present study, we employed polyclonal antibodies made against mammalian and avian erythroid spectrins as well as mammalian brain spectrin to assess their presence and distributions in the mammalian heart. Western blot analyses revealed that all three antibodies were specific for a 240,000 molecular weight alpha-spectrin subunit found in hamster erythrocyte ghost homogenates, whole hamster heart, and isolated hamster cardiac myofibril homogenates. Spectrin staining was absent from the Triton X-100-extracted supernatant fraction of myofibril preparations, suggesting that the protein is linked to the myofibril precipitate after exposure to the detergent. Frozen, unfixed, 2-microns-thick; sections of adult. Syrian golden hamster cardiac tissue exhibited strong immunofluorescent staining of intercalated discs and Z-bands using all three antibodies. In addition, the mammalian erythroid spectrin antibodies showed staining of the sarcolemma, and in cross section, revealed a delicate internal network of staining that appears to surround individual myofibrils. This may be T-tubule-associated staining. Myofibrils isolated from cardiac myocytes using Triton X-100 show positive Z-band staining using all three antibodies. Double staining with Texas Red-labeled monoclonal desmin and FITC-labeled polyclonal spectrin antibodies revealed that both stained the myofibrillar Z-line regions. These results demonstrate that spectrin is closely associated with the membranes, myofibrils, and intermediate filaments in the mammalian heart.