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PubMed1995

Prenatal development of the rat sinuatrial node.

In order to study the origin and mode of differentiation of the cells which make up the sinuatrial node, samples of the sinuatrial junction of rat embryos of different ages were studied by transmission electron microscopy. From a seemingly morphologically homogeneous cell population at 11 days, an ultrastructural differentiation occurs from day 12. So, one could see: a) irregular-shaped cells with dark nucleus and medium-sized contractile apparatus which we have identified as ordinary ("working") atrial myocardiocytes and, b) pale cells with a clear spheroidal nucleus and cytoplasm containing few organelles and fine myofibrils which we have classified as nodal cells. Numerous undifferentiated cells of intermediate morphology appear intermingled with nodal and ordinary cells. Throughout development, nodal and ordinary cells progressively enhanced their mutual differences. Ordinary myocardiocytes become increasingly rich in myofibrils and mitochondria, and nodal cells contain scanty organelles and fine myofibrils, whereas undifferentiated cells are few at every stage. At the end of prenatal life, the sinuatrial node shows numerous unmyelinated axons of immature aspect but direct contacts between nodal cells and nerve fibers are not seen. The images we obtained suggest that the sinuatrial node must not be taken as an embryological remnant. Nodal cells are recognized from the 12th day of embryonic life as a particular form of differentiation of the cells which make up the sinuatrial region at the preceeding stages.

Animals↗
PubMed1994

Immunofluorescent confocal analysis of tropomyosin in developing hearts of normal and cardiac mutant axolotls, Ambystoma mexicanum.

Tropomyosin is a major component of the thin filaments in organized myofibrils of cardiac muscle cells. A purported role for this protein is to prevent myosin thick filaments from interacting with actin thin filaments until the cell contracts. Recently, tropomyosin has been implicated in actin filament formation/stabilization as well. In the cardiac mutant axolotl, Ambystoma mexicanum, heart development is arrested. Mutant embryos form hearts that fail to beat due to a lack of organized sarcomeric myofibrils. There is also a concomitant reduction in the amount of tropomyosin in mutant heart cells. Previous studies have suggested that the addition of purified tropomyosin to glycerinated homogenates of mutant hearts causes amorphous actin to polymerize (or stabilize) into thin filaments. The current study was undertaken to examine the three-dimensional distribution of tropomyosin during myofibrillogenesis in normal hearts over the course of development from the heart-beat stage (35) through the advanced embryonic stage (41) and to investigate whether myofibrils form and/or significant quantities of tropomyosin accumulate in developing mutant hearts of comparable ages.

Ambystoma mexicanum↗
PubMed1995

The mechanism of force generation in myosin: a disorder-to-order transition, coupled to internal structural changes.

We propose a molecular mechanism of force generation in muscle, based primarily on site-specific spectroscopic probe studies of myosin heads in contracting muscle fibers and myofibrils. Electron paramagnetic resonance (EPR) and time-resolved phosphorescence anisotropy (TPA) of probes attached to SH1 (Cys 707, in the catalytic domain of the head) have consistently shown that most myosin heads in contracting muscle are dynamically disordered, undergoing large-amplitude rotations in the microsecond time range. Some of these disordered heads are bound to actin, especially in the early (weak-binding, preforce) phase of the ATPase cycle. The small ordered population (10-20%) is rigidly oriented precisely as in rigor, with no other distinct angle observed in contraction or in the presence of intermediate states trapped by nucleotide analogs. These results are not consistent with the classical model in which the entire head undergoes a 45 degree transition between two distinct orientations. Therefore, it has been proposed that the catalytic domain of the myosin head has only one stereospecific (rigor-like) actin-binding angle, and that the head's internal structure changes during force generation, causing the distal light-chain-binding domain to rotate. To test this model, we have performed EPR and TPA studies of probes attached to regulatory light chains (RLCs) in rabbit and scallop myofibrils and fibers. The RLC results confirm the predominance of dynamic (microsecond) rotational disorder in both relaxation and contraction, and show that the different mechanisms of calcium regulation in the two muscles produce different rotational dynamics. In rabbit myofibrils, RLC probes are more dynamically disordered than SH1 probes, especially in rigor and contraction,indicating that the light-chain-binding domain undergoes rotational motions relative to the catalytic domain when myosin heads interact with actin. An SH1-bound spin label, which is sensitive to myosin's internal dynamics, resolves three distinct conformations during contraction, and time-resolved EPR shows that these transitions are coupled to specific steps in the ATPase cycle. We propose that force is generated during contraction by a disorder-to-order transition, in which myosin heads first attach weakly to actin in a nonstereospecific mode characterized by large-scale dynamic disorder, then undergo at least two conformational transitions involving large-scale structural (rotational) changes within the head, culminating in a highly ordered strong-binding state that bears force.

Adenosine Triphosphate↗
PubMed1994

The effect of L-carnitine on the AZT-induced destruction of human myotubes. Part II: Treatment with L-carnitine improves the AZT-induced changes and prevents further destruction.

BACKGROUND: Zidovudine (AZT) as used in the treatment of AIDS causes a mitochondrial myopathy characterized by enzymatic defects in the respiratory chain system, accumulation of lipid droplets, and carnitine deficiency. Human myotubes treated with AZT demonstrate abnormal mitochondria, accumulation of lipid, and increased lysosomes. Because L-carnitine plays a major role in the transport of long chain fatty acids across the inner mitochondrial membrane and facilitates the beta-oxidation of fatty acids, we examined whether L-carnitine can enhance the recovery of the affected myotubes after withdrawal of AZT and can improve the structural changes of the myotubes while AZT treatment continues. EXPERIMENTAL DESIGN: Myotubes, prepared from human muscle biopsies, were exposed to 250 microM of AZT for 3 to 6 weeks. After 3 weeks of AZT treatment, the cultures were treated with L-carnitine or medium for 3 weeks, while AZT treatment was either withdrawn or continued for 3 more weeks. The cultures were evaluated with: (a) light microscopy; (b) immunocytochemistry, to count the number of myotubes stained with antibodies to Leu-19; (c) oil red O stain, to assess the lipid droplet accumulation; and (d) electron microscopy, to count all the organelles within representative sections of the myotubes, at x24,000, and to calculate the volumetric density (Vvi) of each organelle per unit volume of tissue. RESULTS: In the post-AZT-treated cultures, L-carnitine increased the number of Leu-19-positive myotubes from 3.83 +/- 1.23 to 23 +/- 1.5 per field, normalized their mitochondria, decreased the lipid droplets, and increased the Vvi of the myofibrils. In the cultures treated with 3 weeks of L-carnitine while AZT treatment continued for 3 more weeks, the number of myotubes increased from 3.3 +/- 0.74 to 6.87 +/- 1.35; the absolute number of the mitochondria increased from 1.65 +/- 0.35 to 9.02 +/- 1.11 and their Vvi from 3.67 +/- 0.83 to 6.57 +/- 0.78 (p < 0.05); the Vvi of the myofibrils increased from 2.50 +/- 0.52 to 5.37 +/- 0.76 (p < 0.05); and the Vvi of the lipid droplets decreased from 5.06 +/- 1.44 to 2.72 +/- 0.72 (p < 0.05). In the AZT-treated cultures that did not receive L-carnitine, the mitochondria demonstrated extensive vacuolation, abnormal cristae, and paracrystalline inclusions; in contrast, in the L-carnitine-treated cultures, the mitochondria had substantially improved in spite of continuation of AZT. CONCLUSIONS: L-carnitine enhances the pace and degree of recovery of the AZT-associated destruction of human myotubes, restores and preserves the structure of mitochondria, mobilizes the endomyotubular fat, and allows the regeneration of myofibrils, even if AZT treatment continues. The findings may have potential clinical implications in improving the myotoxicity of AZT in patients with AIDS when the administration of AZT treatment must continue.

Carnitine↗
PubMed1994

The relation between latissimus dorsi skeletal muscle structure and contractile function after cardiomyoplasty.

Past reports suggest that structural changes within the latissimus dorsi muscle occur with chronic electrical stimulation during cardiomyoplasty. However, the specific changes in the structure of the latissimus dorsi muscle and the relation to muscle contractile function with cardiomyoplasty are unknown. Accordingly, this study examined regional changes in latissimus dorsi muscle structure and function after cardiomyoplasty. The left latissimus dorsi muscle was mobilized and wrapped around the heart in pigs with the use of standardized techniques and the latissimus dorsi muscle chronically paced at ambient heart rates (90 beats/min; 20 Hz, 5 V amplitude, n = 6). After 6 weeks, the paced latissimus dorsi muscle and the contralateral control muscle were removed and divided into proximal (0 to 3 cm), middle (3 to 6 cm), and distal (6 to 12 cm) regions. By computer-assisted morphometry, muscle cell myofibril volume, cross-sectional area, and collagen percent area were determined. In the paced latissimus dorsi muscle, myofibril volumes increased by more than 50% in the proximal and middle regions compared with those in the contralateral control muscle. However, myofibril volumes were significantly lower in the distal region of the paced latissimus dorsi muscle compared with those in control muscles (33% +/- 5% versus 20% +/- 3%, p < 0.05). In the paced latissimus dorsi muscle, cross-sectional area was significantly reduced from that of control muscles in all regions. A further reduction in cross-sectional area was noted in the distal region of the paced latissimus dorsi muscle compared with that in both the contralateral control muscle and the proximal and middle regions of the paced latissimus dorsi muscle. Collagen content significantly increased in the paced latissimus dorsi muscle compared with that in control muscle with a more fibrotic pattern observed in the distal region. Latissimus dorsi muscle strips (less than 2 mm2 cross-sectional area) were harvested, and peak and velocity of tension development were examined after field electrical stimulation at 0.2 to 1.2 Hz. At 0.2 Hz, the velocity of tension development was unchanged in the paced latissimus dorsi muscle compared with that in control muscle. However, peak tension development degraded by only 28% in the paced latissimus dorsi muscles but fell by 51% in control muscles with increased stimulation frequencies. In summary, the contractile function of the chronically stimulated latissimus dorsi muscle was associated with fatigue resistance and increased contractile protein content. However, more distal regions of the paced latissimus dorsi muscle demonstrated atrophy and fibrosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Formation of inter- and intramolecular disulfide bonds can activate cardiac troponin C.

Troponin C regulates contraction in striated muscle by alternating between the Ca(2+)-bound and apo conformations. We report here that spontaneous formation of an intramolecular disulfide bond between Cys-35 and Cys-84, or dimerization via an intermolecular disulfide bond between Cys-84 in cardiac troponin C, renders the protein Ca(2+)-independent when assayed in fast skeletal muscle myofibrils but to a much lesser extent in cardiac myofibrils. Formation of the intramolecular disulfide bond appears to expose hydrophobic surfaces, as indicated by an increase in fluorescence from hydrophobic fluorescent dyes, but does not alter the affinity of Ca(2+)-binding site II. These disulfide bonds constrain the protein into a conformation that either resembles or can substitute for the Ca(2+)-bound form of cardiac troponin C in fast skeletal muscle myofibrils.

Animals↗
PubMed1996

Susceptibility and reactivity of sheep to Trichinella spiralis infection.

Susceptibility and reactive manifestation to Trichinella spiralis infection were studied in atypical hosts (sheep) for the period of 247 days. Sheep produced anti-Trichinella antibodies as early on Day 11 (low titer 1:200), with maximum reached at Day 35 (titer 1:800). From Day 42 the antibody level was declining with a negative result of examination on Day 70. Mice exhibited anti-Trichinella antibodies only on Day 32 (titer 1:200). This level was rising, reaching high titer (1:1600) on Day 56. This antibody level persisted until Day 156. In the following period, a rapid decrease in the titer was observed (Graph). On Day 32, T. spiralis larvae in sheep were present in all groups of the muscles examined. The highest larval counts during the entire experiment were detected in the masseter. The initially high counts in the diaphragm and tongue were reduced to only 1/4 or 1/10 at the end of the experiment. In mice, the larvae occurred evenly throughout the entire experiment (Tab. I). The first appearance of a capsule around the T. spiralis larva in muscles was observed on Day 32 p. i. No cell response was detected around the capsule (Fig. 1). Neither was any response observed around necrotizing larvae, even though the surrounding myofibrils were caused to die off (Fig. 2). Certain differences in the degree of myofibril degradation by larvae were evident as early as on Day 32. The least damaged myofibrils were those in the masseter, tongue and diaphragm. This finding correlates with the histological recovery of a different number of necrotized larvae from the individual muscle groups examined. Fresh blood extravassations around larvae were observed on Day 59 (Fig. 3). They could be caused by the migration of larvae to a parasitation site. Live uncapsulated larvae were also found on Day 115 p. i. (Fig. 4). An increased cellular presence around some larvae was observed on Day 84. The larvae surrounded by lymphocytes consequently died off, those without lymphocytic responses formed capsules and survived (Fig. 5). The necrotizing larvae were subject to a powerful phagocytic process, presented by histiocytes, forming multinuclear symplasms (Fig. 6). On Day 11 p. i., larvae inside a capsule were dying off as well. The initial stage of larval necrosis in a capsule is also accompanied by an increased lymphocytic response (Fig. 7). The condition of larvae in capsules and the cellular unresponsiveness as late as on Day 247 indicate the long-lasting viability of the larvae. The capsules surrounding T. spiralis larvae in mice were distinctly seen as early as on Day 32 p. i. Lymphocytic aggregations around the capsule were observed throughout the entire experiment (247 days)-Fig. 8.

Animals↗
PubMed1997

Studies on the function of rho A protein in cardiac myofibrillogenesis.

The aim of this study was to provide morphological evidence for the presence of rho A protein in developing cardiomyocytes and to investigate its possible role in myofibrillogenesis. Immunostaining with a monoclonal anti-rho antibody gave a diffuse pattern in the cytosol of cultured cardiomyocytes. Introduction of C3 exoenzyme into the cells by electroporation was used to inactivate rho A protein by ADP-ribosylation. An immunostaining with anti-vinculin, anti-talin, and anti-integrin antibodies showed the focal adhesions in electroporation control cardiomyocytes to be evenly distributed in the ventral sarcolemma; the costameric structure was also detected using these antibodies. In contrast, in C3 exoenzyme treated cells, focal adhesions were disassembled and costamere were absent; in addition, beta-actin-positive, non-striated fibrils were lost and assembly of M-protein, titin, and alpha-actinin into myofibrils was poor, as shown by diffuse and filamentous staining pattern. C3 exoenzyme treatment had a less marked effect on mature cardiomyocytes than on immature cells; in this case, cells became distorted and few myofibrils were seen. The intensity of anti-phosphotyrosine antibody staining of the focal adhesion was also decreased or diffuse in C3 exoenzyme-treated cardiomyocytes, suggesting dephosphorylation of focal adhesion components. We therefore conclude that small G protein rho A plays an important role in myofibril assembly in cardiomyocytes.

ADP Ribose Transferases↗
PubMed1998

N-cadherin is required for the differentiation and initial myofibrillogenesis of chick cardiomyocytes.

To investigate initial stages of cardiac myofibrillogenesis, heart-forming mesoderm was excised from stage 6 chick embryos and explanted on fibronectin-coated coverglasses. The explants were fixed at various times and immunofluorescently stained with antibodies to N-cadherin, alpha-catenin, beta-catenin, sarcomeric myosin, pan and sarcomeric alpha-actinins, or rhodamine phalloidin. After 7 hours in culture the cells appeared epithelial. N-cadherin, alpha- and beta-catenin, pan alpha-actinin, and F-actin showed circumferential localization at cell borders. No cells in the explant were positive for sarcomeric alpha-actinin or sarcomeric myosin at this stage. Sarcomeric alpha-actinin and sarcomeric myosin were detected around 10 hours after plating. Sarcomeric alpha-actinin initially appeared as small beads along thin actin filaments. Mature Z-lines began to be organized at 20 hours, at the same time the cells started to contract. When the rat monoclonal antibody NCD-2, which inhibits N-cadherin function, was added to the culture at early time-points, cells lost cell-cell contacts, became spherical in shape, and contained tangled actin fibers. The expression of sarcomeric alpha-actinin and sarcomeric myosin was suppressed. These results indicate that 1) the precardiac mesoderm explant cells differentiate and form well-organized myofibrils in culture, 2) N-cadherin-mediated cell-cell interactions are necessary for early differentiation of cardiomyocytes and organization of myofibrils.

Animals↗
PubMed2000

Targeting of cardiac muscle titin fragments to the Z-bands and dense bodies of living muscle and non-muscle cells.

A 6.5-kb N-terminal region of embryonic chick cardiac titin, including the region previously reported as part of the protein zeugmatin, has been sequenced, further demonstrating that zeugmatin is part of the N-terminal region of titin, and not a separate Z-band protein. This Z-band region of cardiac titin, from both 7- and 19-day embryos as well as from adult animals, was found to contain six different small motifs, termed z-repeats [Gautel et al., 1996: J. Cell Sci. 109:2747-2754], of approximately 45 amino acids each sandwiched between flanking regions containing Ig domains. Fragments of Z-band titin, linked to GFP, were expressed in cultured cardiomyocytes to determine which regions were responsible for Z-band targeting. Transfections of primary cultures of embryonic chick cardiomyocytes demonstrated that the z-repeats play the major role in targeting titin fragments to the Z-band. Similar transfections of skeletal myotubes and non-muscle cells lead to the localization of these cardiac z-repeats in the Z-bands of the myofibrils and the dense bodies of the stress fibers. Over-expression of these z-repeat constructs in either muscle or non-muscle cells lead to the loss of the myofibrils or stress fibers, respectively. The transfection experiments also indicated that small domains of a protein, 40 to 50 amino acids, can be studied for their localization properties in living cells if a suitable linker is placed between these small domains and the much larger 28 kDa GFP protein.

Amino Acid Sequence↗
PubMed1979

The atrioventricular node and bundle in the ferret heart: a light and quantitative electron microscopic study.

The cells of the atrioventricular (AV) junction in the ferret heart were examined using light microscopy, a wax-model reconstruction and quantitative electron microscopy to determine their organization and characteristics. A series of subdivisions of the specialized tissues of the AV junction was apparent at both the light and electron microscopic levels. A transitional zone was observed interposed between the atrial muscle cells and the AV node. The AV node consisted of a coronary sinus portion, a superficial portion and a deep portion. The AV bundle had a segment above the anulus fibrosus, a segment which penetrated the right fibrous trigone, a non-branching segment below the anulus fibrosus and a branched segment. At the ultrastructural level the AV junctional conduction tissues had fewer irregularly oriented myofibrils than did working atrial myocardial cells. T-tubules, present in atrial muscle cells, were not observed in the modified muscle cells of the AV node and bundle. Conventional intercalated discs also were not observed between the cells of the AV node or the AV bundle. Atrial myocardial cells had the highest percentage of the plasma membrane occupied by desmosomes, fasciae adherentes and gap junctions. The AV bundle cells had the highest percentage of appositional surface membrane and a relatively large fraction of plasma membrane occupied by gap junctions. Cells of the superficial portion of the AV node had the smallest percentage of the plasma membrane composed of gap junctions, desmosomes or fasciae adherentes, as well as the smallest fraction of the cell membrane apposed to adjacent cells. The stereological data indicate that the most useful distinguishing characteristic between atrial muscle cells and conduction cells was that a smaller percentage of the conduction cell sarcoplasm was occupied by mitochondria and myofibrils. The most useful characteristics that could be used to differentiate between the regions of the AV junctional conduction tissues were the amounts and types of surface membrane specializations in the respective cell types.

Animals↗
PubMed2001

Sound-generating and -detecting motor system in catfish: design of swimbladder muscles in doradids and pimelodids.

Catfishes have evolved a diversity of swimbladder muscles serving in the generation of different sounds and probably other acoustic functions. In order to find out if anatomical and acoustical differences are parallelled by fine structural differences, I examined the sonic muscles of the doradid Platydoras and the pimelodid Pimelodus by gross dissections and ultrastructural methods. In Platydoras, the sound-generating (drumming) muscle (DM) inserts on a dorsal bony plate that vibrates the swimbladder. In pimelodids, the large DM attaches directly on the ventral surface of the swimbladder, whereas the small tensor tripodis muscle (TT) inserts on the rostral surface near the tripus, the most caudal Weberian ossicle. Fibers of all three muscles possess an extensive development of sarcoplasmatic reticulum (SR) in association with very thin myofibrils (MF) but differed widely in their arrangement. In Platydoras, ribbons of MFs are arranged radially around a central core. Mitochondria were found within the core and the peripheral sarcoplasm. Pimelodus does not have a differentiated core and the cross-sectional area of DM-MFs is about 15% larger as determined by stereological measurements. The TT possesses shorter sarcomeres and more mitochondria than DMs, which were primarily found between MFs. This suggests faster contraction properties and greater resistance to fatigue compared with sonic muscles. Data indicate that the higher amount of DM-myofibrils in pimelodids might result in stronger muscle contractions and, presumably, in higher sound intensities. The fine structure of the TT reveals that contractions most likely prevent transmission of swimbladder vibrations to the inner ear via the Weberian ossicles during vocalization.

Acoustic Stimulation↗
PubMed2003

Effects of platelet-derived growth factor-AA and -BB on embryonic cardiac development.

Several studies have shown that disruption of the normal expression patterns of platelet-derived growth factor (PDGF) ligands and receptors during development results in gross cardiac defects and embryonic or neonatal death. However, little is known about the specific role that PDGF plays in the differentiation of cardiac myocytes. In experiments complementing studies that utilized naturally-occurring Patch mice lacking the PDGFr alpha, or knockout animals lacking a PDGF ligand or receptor, we used rat and mouse whole-embryo culture (WEC) techniques to increase the exposure of embryos to the PDGF-AA or -BB ligands. Following a 48-hr culture period, we analyzed heart growth and cardiac myocyte differentiation. Exposure of rat embryos to 50 ng/ml of PDGF-AA resulted in a 42% increase in total protein levels in the heart, but did not result in a significant increase in heart growth, as determined by measurements of the atrioventricular length and the left ventricular length and width. Exposure of embryos to 50 ng/ml of PDGF-BB resulted in a 77% increase in total protein levels and a significant (P < 0.05) 8-15% increase in the measured heart parameters. Although a comparison of control and PDGF-AA-treated embryos showed no increase in the overall size of the heart, confocal microscopy showed an increase in the size and number of myofibrillar bundles in the developing myocardium. In addition, transmission electron microscopy (TEM) revealed an increase in the presence of sarcomeres, indicating that myofibrils were more highly differentiated in these areas of the treated embryos. In PDGF-BB-treated embryos, the compact zone of the myocardium was thicker and, as shown by confocal microscopy and TEM, f-actin and well-developed sarcomeres were more prevalent, indicating that the myofibrils were more differentiated in the treated embryos than in the control embryos. These studies indicate that increased exposure of embryonic hearts to PDGF-AA or -BB increases the rate of myocardial development.

Animals↗
PubMed1989

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.

Actins↗
PubMed1993

Localization of CapZ during myofibrillogenesis in cultured chicken muscle.

Actin filaments undergo dramatic changes in their organization during myofibrillogenesis. In mature skeletal muscle, both CapZ and the barbed end of the actin filaments are located at Z-discs. In vitro, CapZ binds the barbed end of actin filaments and prevents actin subunit addition and loss; CapZ also nucleates actin polymerization in vitro. Taken together, these properties suggest that CapZ may function to organize actin filaments during myofibrillogenesis. We report here that the amount of CapZ in myofibrils from adult chicken pectoral muscle is sufficient to "cap" each actin filament of the sacromere. Double immunofluorescence microscopy of skeletal muscle cells in culture was used to determine the spatial and temporal distributions of CapZ relative to actin, alpha-actinin, titin, and myosin during myofibrillogenesis. Of particular interest was the assembly of CapZ at nascent Z-discs in relation to the organization of actin filaments in nascent myofibrils. In myoblasts and young myotubes, CapZ was diffusely distributed in the cytoplasm. As myotubes matured, CapZ was initially observed in a uniform distribution along non-striated actin filaments called stress fiber-like structures (SFLS). CapZ was observed in a periodic pattern characteristic of mature Z-discs along the SFLS prior to the appearance of a striated staining pattern for actin. In older myotubes, when actin was observed in a pattern characteristic of I-bands, CapZ was distributed in a periodic pattern characteristic of mature Z-discs. The finding that CapZ was assembled at nascent Z-discs before actin was observed in a striated pattern is consistent with the hypothesis that CapZ directs the location and polarity of actin filaments during I-band formation in skeletal muscle cells. The assembly of CapZ at nascent Z-disc structures also was observed relative to the assembly of sarcomeric alpha-actinin, titin, and thick filaments. Titin and myosin were observed in structures having the organization of mature sarcomeres prior to the appearance of CapZ at nascent Z-discs. The distribution of CapZ and sarcomeric alpha-actinin in young myotubes was not coincident; in older myotubes, both CapZ and alpha-actinin were co-localized at Z-discs. In cardiac myocytes, CapZ was detected at Z-discs and was distributed in a punctate pattern throughout the cytoplasm. CapZ also was co-localized with A-CAM and vinculin at cell-cell junctions formed by the myocytes.

Actinin↗
PubMed1994

Sequential appearance of muscle-specific proteins in myoblasts as a function of time after cell division: evidence for a conserved myoblast differentiation program in skeletal muscle.

Based on the assumption that a conserved differentiation program governs the assembly of sarcomeres in skeletal muscle in a manner analogous to programs for viral capsid assembly, we have defined the temporal and spatial distribution of 10 muscle-specific proteins in mononucleated myoblasts as a function of the time after terminal cell division. Single cells in mitosis were identified in monolayer cultures of embryonic chicken pectoralis, followed for selected time points (0-24 h postmitosis) by video time-lapse microscopy, and then fixed for immunofluorescence staining. For convenience, the myoblasts were termed x-h-old to define their age relative to their mitotic "birthdate." All 6 h myoblasts that emerged in a mitogen-rich medium were desmin+ but only 50% were positive for a alpha-actin, troponin-I, alpha-actinin, MyHC, zeugmatin, titin, or nebulin. By 15 h postmitosis, approximately 80% were positive for all of the above proteins. The up-regulation of these 7 myofibrillar proteins appears to be stochastic, in that many myoblasts were alpha-actinin+ or zeugmatin+ but MyHC- or titin- whereas others were troponin-I+ or MyHC+ but alpha-actinin- or alpha-actin-. In 15-h-old myoblasts, these contractile proteins were organized into nonstriated myofibrils (NSMFs). In contrast to striated myofibrils (SMFs), the NSMFs exhibited variable stoichiometries of the sarcomeric proteins and these were not organized into any consistent pattern. In this phase of maturation, two other changes occurred: (1) the microtubule network was reorganized into parallel bundles, driving the myoblasts into polarized, needle-shaped cells; and (2) the sarcolemma became fusion-competent. A transition from NSMFs to SMFs took place between 15 and 24 h (or later) postmitosis and was correlated with the late appearance of myomesin, and particularly, MyBP-C (C protein). The emergence of one, or a string of approximately 2 mu long sarcomeres, was invariably characterized by the localization of myomesin and MyBP-C to their mature positions in the developing A-bands. The latter group of A-band proteins may be rate-limiting in the assembly program. The great majority of myoblasts stained positively for desmin and myofibrillar proteins prior to, rather than after, fusing to form myotubes. This sequential appearance of muscle-specific proteins in vitro fully recapitulates myofibrillar assembly steps in myoblasts of the myotome and limb bud in vivo, as well as in nonmuscle cells converted to myoblasts by MyoD. We suggest that this cell-autonomous myoblast differentiation program may be blocked at different control points in immortalized myogenic cell lines.

Animals↗
PubMed1994

Switching of the dominant calcium sequestering protein during skeletal muscle differentiation.

A major Ca(2+)-storing protein in endoplasmic reticulum (ER) of non-muscle cells is calreticulin (CR), which is considered to be functionally homologous to calsequestrin. Calsequestrin is a Ca(2+)-binding protein in sarcoplasmic reticulum (SR) of striated muscle, which stores Ca2+ during muscle relaxation. In order to investigate the expression and distribution of calsequestrin and calreticulin during skeletal muscle differentiation, cultured chick embryonic skeletal muscles were observed by immunofluorescence using anti-calsequestrin, anti-calreticulin, anti-desmin, and anti-sarcomeric myosin antibodies and rhodamine-phalloidin. Within 6 hours in culture, myoblasts started to express desmin. Desmin-positive cells demonstrated the reticular staining of calreticulin, as did desmin-negative cells. Around fusion, calsequestrin and sarcomeric myosin started to appear in desmin-positive cells. The expression of calsequestrin slightly preceded that of sarcomeric myosin. As the myotubes matured, the fluorescent dots of calsequestrin increased and spread to the cell periphery along the myofibrils, while the reticular pattern of calreticulin gradually disappeared. Double labeling showed that calsequestrin colocalized with calreticulin. In mature myotubes, anti-calsequestrin staining demonstrated many dots along myofibrils, whereas calreticulin was barely seen except at the perinuclear region. These results suggest that the expression of calsequestrin and calreticulin are switched during skeletal muscle differentiation.

Animals↗
PubMed1978

The developmental morphology of Torpedo marmorata: electric organ--myogenic phase.

The early development of the electric organ of Torpedo marmorata has been examined by light and electron microscopy to the 40-mm stage of embryo growth. The myogenic nature of this tissue is confirmed ultrastructurally by the presence of myoblasts and myotubes both containing myofibrils cross striated with Z,A and I bands. Fusion between these cells is also found taking place. A scheme is presented to explain the development of the overall structural plan of the organ and specifically the formation of the future electrocyte columns. AT 40 mm, a series of morphological transformations signals the onset of a divergent developmental pattern ultimately leading to the establishment of mature electrocyte columns. These features include rounding up of myotubes, dissolution of myofibrils and the appearance of intermediate size filaments (11 nm) and perhaps a non-muscular actin (5.5 nm). This early myogenic phase of development occurs in the absence of any specific nervous contact even though electromotor nerves are always in close proximity.

Animals↗