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PubMed1988

Differential effects of pharmacological modulators of cardiac myofibrillar ATPase activity in normal and myopathic (BIO 14.6) hamsters.

The effects of four Ca2+ binding protein modulators (bepridil, perhexiline, calmidazolium and trifluoperazine) on cardiac myofibrillar ATPase activity in Triton-purified myofibrils prepared from normal dogs, normal hamsters and age/sex (male, 16-20 weeks)-matched myopathic hamsters (BIO 14.6) have been quantitated. When compared with normal hamsters, myopathic hamster myofibrils have a markedly depressed maximum MgATPase activity (178 +/- 2 vs. 119 +/- 3 nmol/mg per min, respectively) and a slightly increased requirement of Ca2+ for half-maximal activation (ED50; 0.66 vs. 0.75 microM free Ca2+, respectively). Calmidazolium, trifluoperazine and bepridil lower the ED50 for Ca2+ in myopathic myofibrils. Moreover, bepridil and trifluoperazine increase maximum myofibrillar MgATPase activity in myopathic hamster myofibrils. In contrast, calmidazolium depresses maximum and stimulates basal MgATPase activities in myopathic and normal hamster myofibrils. Qualitatively, different effects are apparent when these agents are examined in canine myofibrils. Thus, the pharmacological effects of Ca2+ binding protein modulators on cardiac myofibrillar ATPase activity are dependent upon species and/or pathological state. It is possible to directly enhance a pathological consequence of cardiac myopathy, depression in maximum myofibrillar MgATPase activity, with pharmacological agents.

Adenosine Triphosphatases↗
PubMed1992

Desmin at myotendinous junctions.

Myofibrils are linked to the cell membrane at myotendinous junctions located at the ends of muscle fibers, and at costameres, sites positioned periodically along lateral surfaces of muscle cells. Both of these sites are enriched in proteins that link active components of myofibrils to the cell membrane. Costameres are also enriched in desmin intermediate filaments that link passive components of myofibrils to the lateral surfaces of muscle cells. In this study, the possibility that desmin is also found between the terminal Z-disk of myofibrils and the myotendinous junction membrane is examined by immunocytochemistry and by KI-extraction procedures. Data presented show that desmin is located in the filamentous core of cellular processes at myotendinous junctions at sites 30 nm or more from the membrane. This core lies deep to subsarcolemmal material previously shown to contain talin, vinculin, and dystrophin. The distance from desmin to the membrane suggests desmin does not interact directly with membrane proteins at the junction. Immunoblots and indirect immunofluorescence of junctional regions of muscle compared to nonjunctional regions show no apparent enrichment of desmin at junctional sites, although vinculin, another costameric and junctional component, is significantly enriched at junctional regions. These findings show that passive elements of myofibrils may be continuous from myotendinous junctions of muscle origin to insertion via desmin filaments located between terminal Z-disks and the junctional membrane. This can provide a system in parallel to that involving thin filaments, vinculin, and talin for linking myofibrils to the cell membrane at myotendinous junctions.

Animals↗
PubMed1995

Myofibrillogenesis in rodent skeletal muscle in vitro: two pathways involving thick filament aggregates.

Thick filament aggregates play an important role in myofibrillogenesis in rodent skeletal muscle in vitro. This ultrastructural study describes these aggregates, shows their involvement in the process of myofibril formation, and correlates their appearance and function with current models of myofibrillogenesis. Initially, following myoblast fusion in normal mouse skeletal muscle in vitro, abundant stress fiber-like structures (SFLS) are found near the periphery of early myotubes. These undergo internal rearrangements, forming subcortical sarcomeres and early myofibrils. However, additional thick filaments are synthesized, and some join appositionally to the nascent myofibrils, increasing their diameter. More interiorly, this thick filament synthesis accelerates, with filaments aligning into aggregates resembling discrete A-bands, usually with M-lines and M-regions. The ends of these 'A-band' aggregates are infiltrated with ribosomes and capped by flocculent material. Ultimately, aggregates are incorporated into preexisting myofibrils or associate end-to-end to form new, parallel myofibrils, the flocculent material forming putative I-bands with diminished Z-lines and few thin filaments. As differentiation continues, Z-lines and thin filaments appear, forming true myofibrils. Dysgenic mouse skeletal muscle develops similarly, but when this non-contractile cell matures (i.e., generates action potentials), filaments and their organization break down. Cloned myogenic rat L5/A10 cells also follow this developmental pattern, but in mature, contracting myotubes, Z-lines remain irregular and thin filaments are reduced. In all three types of muscle developing in vitro, thick filament aggregates are a common and predominant feature and as such appear to constitute an additional or alternate pathway to previously described models of myofibrillogenesis.

Animals↗
PubMed1998

Exchangeability of actin in cardiac myocytes and fibroblasts as determined by fluorescence photobleaching recovery.

Rhodamine (Rho)-labeled muscle and non-muscle actins were microinjected into cultured embryonic chicken cardiac myocytes and fibroblasts. After incorporation of the fluorescent actin analog into cellular structures, small areas of labeled structures were photobleached with a laser pulse, and fluorescence recovery (FR) was measured to determine the exchangeability of isoactins in these structures. With both Rho-muscle and Rho-non-muscle actins, the FR rate in any part of stress fibers was consistently faster than that observed in any part of myofibrils. Thus, although non-striated (proximal and terminal) portions of nascent myofibrils are similar in appearance and composition to stress fibers, our data clearly revealed differences in actin stability between these two structures. Further, although cardiomyocytes were incapable of discriminating between the incorporation of muscle and non-muscle actin isoforms into myofibrils, FR after photobleaching of Rho-muscle actin was faster than that of Rho-non-muscle actin in immature non-striated portions. This indicates that actin molecules in cardiac myofibrils cannot be readily exchanged by heterotypic non-muscle actin. Fluorescently labeled actin incorporated into non-striated (proximal and terminal) portions of myofibrils and terminal portions of stress fibers was found to be more stable than alpha-actinin. The relative stability of actin could facilitate the formation of nascent Z-bands of myofibrils and the reorganization of stress fibers at these portions.

Actinin↗
PubMed1994

Isoform specific interactions of troponin I and troponin C determine pH sensitivity of myofibrillar Ca2+ activation.

We investigated whether differences in isoforms of troponin I (TnI) and troponin C (TnC) can account for the greater inhibition of Ca(2+)-dependent MgATPase activity by acidic pH in cardiac (c) than in fast skeletal (fs) myofilaments. We studied fast skeletal myofibrils from which whole Tn was extracted by displacement with excess fsTnT (the tropomyosin binding subunit of Tn) followed by reconstitution with TnC-TnI. Exchange of fsTnI with cTnI did not alter the effect of a drop in pH from 7.0 to 6.5 on the relation between pCa (-log[Ca2+]) and MgATPase activity of fast skeletal myofibrils. Exchange of fsTnC with cTnC did, however, induce an increase in the effect of this same pH change on Ca2+ activation. Yet, the pH sensitivity of Ca2+ activation of fast skeletal myofibrils containing cTnC was not as great as that of native cardiac myofibrils. However, when both fsTnC and fsTnI of fast skeletal myofibrils were replaced by cTnC-cTnI, there was a pH-induced shift in Ca2+ sensitivity similar to that of cardiac myofibrils. In studies using fluorescent probes, both pure fsTnC and pure cTnC showed decreased Ca2+ binding as pH was lowered. This decrease was potentiated in the fsTnC-fsTnI and cTnC-cTnI complexes. However, the effect of acidic pH was the same in fsTnC and the hybrid complex, fsTnC-cTnI, and in cTnC and the hybrid complex, cTnC-fsTnI. Thus, isoform specific interactions between TnI and TnC appear important in the differential response of skeletal and cardiac myofilaments to acidosis.

Animals↗
PubMed1979

Titin: major myofibrillar components of striated muscle.

Electrophoretic analyses of protein components of striated muscle myofibril purified from various vertebrate and invertebrate species revealed that proteins much larger than myosin heavy chain are present in significant amounts. To define possible roles of these heretofore unidentified proteins, we purified a combination of two uncommonly large proteins, designated as titin, from chicken breast myofibrils. Chemical and immunological studies indicated that titin is distinct from myosin, actin, and filamin. Specific titin anti body crossreacts with similar protein in both skeletal and cardiac myofibrils of many vertebrate and invertebrate species. Immunofluorescent staining of glycerinated chicken breast myofibrils indicated that titin is present in M lines, Z lines, the junctions of A and I bands, and perhaps throughout the entire A bands. Similar staining studies of myofibrils from other species suggest that titinlike proteins may be organized in all myofibrils according to a common architectural plan. We conclude that titin is a structurally conserved myofibrillar component of vertebrate and invertebrate striated muscles.

Animals↗
PubMed1990

Myosin mRNA accumulation and myofibrillogenesis at the myotendinous junction of stretched muscle fibers.

Myofiber growth and myofibril assembly at the myotendinous junction (MTJ) of stretch-hypertrophied rabbit skeletal muscle was studied by in situ hybridization, immunofluorescence, and electron microscopy. In situ hybridization identified higher levels of myosin heavy chain (MHC) mRNA at the MTJ of fibers stretched for 4 d. Electron microscopy at the MTJ of these lengthening fibers revealed a large cytoplasmic space devoid of myofibrils, but containing polysomes, sarcoplasmic reticulum and T-membranes, mitochondria, Golgi complexes, and nascent filament assemblies. Tallies from electron micrographs indicate that myofibril assembly in stretched fibers followed a set sequence of events. (a) In stretched fiber ends almost the entire sarcolemmal membrane was electron dense but only a portion had attached myofibrils. Vinculin, detected by immunofluorescence, was greatly increased at the MTJ membrane of stretched muscles. (b) Thin filaments were anchored to the sarcolemma at the electron dense sites. (c) Thick filaments associated with these thin filaments in an unregistered manner. (d) Z-bodies splice into thin filaments and subsequently thin and thick filaments fall into sarcomeric register. Thus, the MTJ is a site of mRNA accumulation which sets up regional protein synthesis and myofibril assembly. Stretched muscles also lengthen by the addition of myotubes at their ends. After 6 d of stretch these myotubes make up the majority of fibers at the muscle ends. Essentially all these myotubes repeat the developmental program of primary myotubes and express slow MHC. MHC mRNA distribution in myotubes is disorganized as is the distribution of their myofibrils.

Animals↗
PubMed1957

An analysis of myogenesis by the use of fluorescent antimyosin.

Antibodies against myosin of adult chicken skeletal muscle were labelled with fluorescein and used as staining reagents to analyze the development of trunk myoblasts in the chick embryo. Myoblasts from the brachial myotomes were studied in three ways: (a) Specimens were fixed, sectioned, and stained with iron-hematoxylin. (b) Living myoblasts, and myoblasts prepared by glycerol extraction, were teased and examined by phase contrast microscopy. (c) Embryo trunks were treated with fluorescent antimyosin or with a control solution of fluorescent normal globulin, and were examined by fluorescence and phase contrast microscopy. Both glycerol-extracted and fixed materials were used. Cross-striated myofibrils appeared first in stage 16 to 17 embryos in the series studied by antimyosin staining and fluorescence microscopy. Striated myofibrils appeared first in stage 18 to 19 embryos, in the series stained by iron-hematoxylin, and at stage 22 to 23, in the series studied by glycerol extraction and phase contrast microscopy. In each series, myofibrils without apparent cross-striations were detected shortly before cross-striations were observed. Specific staining by antimyosin occurred only in differentiating myoblasts. Within the myoblasts antimyosin staining was confined to the A bands of the slender myofibrils. The following observations suggest that the first delicate striated structure to appear in the early 3 day myoblast was remarkably mature: (1) The sarcomere pattern both in length and in internal detail, was similar to that of adult muscle. (2) The distribution of myosin, as revealed by antimyosin staining, was the same in the embryonic as in the mature myofibril. (3) Glycerol-extracted myoblasts contracted vigorously on exposure to ATP. The changes in sarcomere band pattern were indistinguishable from those occurring during contraction of adult muscle induced by ATP. (4) ATP contraction was blocked by prior antimyosin staining in embryonic myoblasts as in mature muscle. It is suggested that the early myofibril grows laterally as a thin sheet associated with the sarcolemma, and that growth in length occurs in the growth tips of the elongating myoblast.

Animals↗
PubMed1996

Immunocytochemical localization of desmin in human fetal skeletal muscle.

The distribution of desmin filaments in the skeletal and cardiac muscles of experimental animals and cultured cells have been extensively studied. The purpose of this report is to identify the distribution of desmin filaments in vivo in human fetal skeletal muscle. Using 10nm gold-particle-labeling immunoelectron microscopy, desmin intermediate filaments were localized in skeletal muscles of 12, 20 and 29-week-old human fetuses. A number of desmin filaments were closely associated with the nascent nonstriated myofibrils. Nascent myofibrils were distributed in the subsarcolemmal space at all three developmental stages. Desmin, accompanying the nascent myofibrils, was most abundant after 12 weeks of gestation. An irregular network of desmin filaments was conspicuous in the subsarcolemmal space after 20 weeks of gestation. Desmin filaments penetrated the myofibrils and the Z-discs after 29 weeks of gestation. The developmental change in fine structural localization of desmin between 12, 20 and 29 weeks of gestation in human fetuses indicated that desmin filaments were involved in myofibril assembly in the subsarcolemmal space, and also in myofibrillogenesis in the inner space of the myofibrils.

Desmin↗
PubMed1969

Radial spread of contraction in frog muscle fibres.

1. The membrane potential of isolated muscle fibres in solutions containing tetrodotoxin (TTX) was controlled with a two-electrode voltage clamp. The striation pattern in the region of the electrodes was observed microscopically.2. With square steps of depolarization of increasing magnitude, contraction occurs first in the myofibrils just beneath the surface membrane, and then spreads inwards towards the axis of the fibre as the depolarization is increased.3. From the depolarizations which make the superficial and axial myofibrils contract it is possible to estimate a space constant (lambda(T)) for electrotonic spread in a transverse tubular network.4. lambda(T) was found to vary with fibre radius; for a 50 mu fibre it was about 60 mu. lambda(T) was not greatly affected by tetraethylammonium (TEA) chloride (111 mM), or by sucrose substitution of most of the sodium chloride in the Ringer solution.5. The ratio of the depolarization threshold for contraction of surface myofibrils and of central myofibrils was smaller for short (3 msec) than for long depolarization.6. Action potentials, recorded from a sartorius fibre, were used as the command signal for the voltage-clamped fibre in tetrodotoxin. The central myofibrils of this fibre did not appear to contract unless the imposed ;action potentials' were of normal size.7. The passive electrical characteristics of the transverse tubular system will just allow an action potential, at room temperature, to activate the myofibrils at the centre of a frog muscle fibre. An active potential change would be required to achieve a safety factor appreciably greater than one for this process.

Action Potentials↗
PubMed1971

Inward spread of activation in vertebrate muscle fibres.

1. A method for detecting the activation of individual myofibrils or groups of myofibrils within an isolated muscle fibre is described. It consists in making all the myofibrils wavy by setting the fibre in gelatine and compressing it longitudinally; active shortening of myofibrils can then be recognized by the straightening out of the waves.2. The time course of this straightening during a twitch was found by high-speed ciné micrography.3. There is a delay of activation between the superficial and central myofibrils, from which the velocity of inward spread of activation can be found.4. This velocity has a Q(10) of 2, and is about 7 cm/sec at 20 degrees C. The mechanism of the inward spread of activation is discussed.5. On relaxation the waves reappear, showing that there is a spontaneous elongation of the myofibrils.

Action Potentials↗
PubMed1985

Effect of endurance swimming on rat cardiac myofibrillar ATPase with experimental diabetes.

Diabetes is characterized by depressed cardiac functional properties attributed to Ca2+-activated ATPase activity. In contrast, endurance swimming enhances the cardiac functional properties and Ca2+-activated myofibril ATPase. Thus, the purpose of this study was to observe if the changes associated with experimental diabetes can be ameliorated with training. Diabetes was induced with a single i.v. injection of streptozotocin (60 mg/kg). Blood and urine glucose concentrations were 802 +/- 44 and 6965 +/- 617 mg/dL, respectively. The training control and training diabetic animals were made to swim (+/- 2% body weight) 4 days/week for 8 weeks. Cardiac myofibril, at 10 microM free Ca2+ concentration was reduced by 54% in the sedentary diabetics compared with sedentary control animals (p less than 0.05). Swim training enhanced the Ca2+-activated myofibril ATPase activities for the normal animals. The diabetic animals, which swam for 8 weeks, had further reduced their Ca2+-activated myofibril ATPase activity when compared with sedentary diabetics (p less than 0.05). Similarly, the Mg2+-stimulated myofibril ATPase activity was depressed by 31% in diabetics following endurance swimming. It is concluded that the depressed Ca2+-activated myofibril ATPase activity of diabetic hearts is not reversible with endurance swimming.

Animals↗
PubMed1981

Stretch-induced growth in chicken wing muscles: myofibrillar proliferation.

The patigialis muscle (PAT) in the wing of the chicken can be induced to grow rapidly in length and in diameter by passively stretching the muscle with a spring-loaded aluminum bar (Holly et al., Am. J. Physiol. 238 (Cell Physiol. 7): C62-C71, 1980). Rates of DNA, RNA, and protein synthesis are accelerated. Sarcomere length falls from 3.19 micrometers after 1 day of stretch to only 10% above control values at 7 days of stretch. Myofibrils are wavy and misaligned. Electron microscopy of cross-sectioned muscles shows that the fraction of cell volume occupied by myofibrils remains constant throughout the experimental period, even though cross-sectional area of the muscle fibers increases by 55%. The mean diameter of myofibrils in stretched muscle increases by more than 25%. The number of splitting myofibrils increases from 15% before stretching to 45% after 1 wk of stretch. Splits appear to originate in the center of the I band, and then progress to the A band and the periphery of the myofibril. Elements of the sarcotubular system develop quickly at the origin of the fractures. It is concluded that rapid growth of the myofibril is required for initiation of splitting. Neither neurally mediated active tension nor muscle contraction are required.

Aging↗
PubMed1990

Changes in myofibrillar activation and troponin C Ca2+ binding associated with troponin T isoform switching in developing rabbit heart.

Postnatal development of the mammalian heart is associated with changes in the population of isoforms of the thin filament proteins. We correlated the change in thin filament proteins, which occur in rabbit hearts between 5 days and 22 days of age, with changes in Ca2+ dependence of myofibrillar ATPase activity, force generation, and troponin C Ca2+ binding. The preparations derived from the 5-day-old animals exhibited a high molecular weight isoform of troponin T not found in the hearts of the 22-day-old animals. Other troponin T isoforms were also found to be present in different relative amounts. No other major differences in thin filament protein composition could be identified. Compared with the 5-day-old rabbit heart preparations, the ATPase activity of myofibrils from 22-day-old rabbit hearts exhibited a reduced Ca2+ sensitivity. The pCa50 (negative log of the half-maximal-activity free Ca2+) of the MgATPase activity was shifted by 0.15 pCa units with maturation. Maturation of the myofibrils was also associated with an increased effect of Mg2+ on pCa50. On increasing the Mg2+ from 2 to 10 mM at constant MgATP2-, the pCa50 of 5-day myofibrils was increased (shifted to the right) by 0.39 pCa units for 5-day-old rabbit hearts and 0.45 pCa units for 22-day-old rabbit hearts. Although similar changes in pCa50 of force developed by myofibrils were marginally significant, fibers from hearts of 5-day-old rabbits exhibited a greater Hill coefficient than hearts from 22-day-old rabbits (3.0 vs. 2.1). Despite the increased sensitivity of 5-day-old rabbit hearts to Ca2+, these hearts exhibited significantly less Ca2+ bound to myofibrillar troponin C than did the 22-day-old rabbit hearts. Moreover, the models that best described the Ca2+ binding data are different for the two age groups. Our data indicate that the Ca2+ activation and Ca2+ binding properties of myofibrillar troponin C are altered in developing cardiac myofibrils and that the changes in these properties may be influenced by changes in the troponin T isoforms present in the myofibril.

Animals↗
PubMed1992

Alterations in myofibrillar function and protein profiles after complete global ischemia in rat hearts.

We studied changes in myofibrillar function and protein profiles after complete global ischemia with anoxia in rat hearts. Hearts were exposed to global ischemia and anoxia (CGI) for 30 or 60 minutes at 37 degrees C, and myofibrils were prepared for measurement of Ca(2+)-dependent Mg(2+)-ATPase activity at pH 7.0 and 6.5. Hearts incubated in cold saline (1 +/- 1 degrees C) and nonincubated hearts served as controls. Maximum ATPase activity was unchanged at pH 7.0 and pH 6.5 in myofibrils from hearts treated with 30 or 60 minutes of CGI. At pH 7.0, the Hill coefficient, which is an index of cooperative interactions among thin-filament proteins, was unchanged after 30 minutes of CGI but was significantly increased after 60 minutes of CGI. A similar trend for increased cooperativity was observed when myofibrillar ATPase activity was measured at pH 6.5 in myofibrils from rat hearts made ischemic for 30 or 60 minutes. Both 30 and 60 minutes of CGI resulted in increased pCa50 values (half-maximally activating free [Ca2+]) at pH 7.0 and pH 6.5. Densitometric analysis of myofibrillar proteins separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that troponin I and troponin T were degraded during 60 minutes of CGI. Two new protein bands appearing in ischemia-treated myofibrils were identified as partially degraded troponin I and troponin T with Western blots. The troponin I fragment could be phosphorylated by cAMP-dependent protein kinase. In addition, we observed phosphorylation of a protein band that corresponded to myosin light chain-2 in myofibrils from CGI-treated hearts. These results suggest that degradation of thin-filament proteins may contribute to the changes in cooperativity of Ca2+ regulation of ATPase activity observed in the myofibrils from rat hearts exposed to CGI.

Animals↗
PubMed1992

The involvement of adherens junction components in myofibrillogenesis in cultured cardiac myocytes.

The distribution of adherens junction (AJ) components was investigated in cultured heart myocytes. These cells, derived from either newborn rats or chick embryos, develop elaborate arrays of myofibrils which become extensive and laterally aligned following several days in culture. The Z-disks in these cells, visualized by immunolabeling with antibodies to muscle-specific alpha-actinin, exhibit a characteristic periodicity of about 2 microns and are in register with those of neighboring myofibrils throughout the sarcoplasm. Vinculin, in these cells, associates with intercellular AJ and cell-matrix adhesions. In addition, this protein is detected in periodic bands located along the lateral cell membranes corresponding to "costamers" previously described by Pardo, J.V., Siliciano, J.D. and Craig, S.W. (Proc. Natn. Acad. Sci. USA, 80, 1008). Similarly, N-cadherin, which is predominantly associated with intercellular junctions, is also detected in periodic striations located mainly on the dorsal and lateral cell surfaces. Using computer-aided three-dimensional microscopy confirmed that these vinculin- and N-cadherin-containing structures are located in extrajunctional sites, apparently associated with Z-disks of peripheral myofibrils. Based on these findings an alternative pathway is proposed for the assembly of vinculin and N-cadherin, which is not triggered by adhesive interactions with extracellular surfaces but rather by interactions at the membrane-cytoplasm interphase with the periphery of the pre-assembled myofibrils. Moreover, we present evidence that antibodies to N-cadherin, which are capable of blocking AJ formation in culture, have an inhibitory effect also on the development and alignment of myofibrils. We discuss the functional significance of the "costameric" organization of vinculin and N-cadherin and consider its involvement both in the lateral alignment of neighboring muscle cells and in the stabilization of developing myofibrils.

Actins↗
PubMed2001

Characterization of heterokaryons between skeletal myoblasts and somatic cells formed by fusion with HVJ (Sendai virus); effects on myogenic differentiation.

In skeletal myogenic differentiation, myoblasts fuse with myogenic cells spontaneously, but do not fuse with non-myogenic cells either in vivo or in vitro, suggesting that the fusion of myoblasts with non-myogenic cells is unsuitable for differentiation. To understand the inevitability of the fusion among myoblasts, we prepared heterokaryons in crosses between quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) and rodent non-myogenic cells, such as tumor cells, fibroblasts, or neurogenic cells by HVJ (Sendai virus) and examined how myogenic differentiation was influenced in the prepared heterokaryons, focusing on myogenin expression and myofibril formation as markers of differentiation. When presumptive QM-RSV cells were fused with non-myogenic cells by HVJ and induced to differentiate, both myogenin expression and myofibril formation were suppressed. When myotubes of QM-RSV cells that had already expressed myogenin and formed myofibrils were fused with non-myogenic cells, both myogenin and myofibrils disappeared. Especially, fibrous structures of myofibrils were significantly lost and dots or aggregations of F-actin were formed within 24 hr after formation of heterokaryons. However, the fusion of presumptive or differentiated QM-RSV cells with rodent myoblasts did not disturb myogenin expression or myofibril formation. These results suggest that mutual fusion of myoblasts is indispensable for normal myogenic differentiation irrespective of the species, and that some factors inhibiting myogenic differentiation exist in the cytoplasm of non-myogenic cells, but not in myoblasts.

Animals↗
PubMed2004

Myofibrillogenesis in skeletal muscle cells in the presence of taxol.

We address the controversy of whether mature myofibrils can form in the presence of taxol, a microtubule-stabilizing compound. Previous electron microscopic studies reported the absence of actin filaments and Z-bands in taxol-treated myocytes [Antin et al., 1981: J Cell Biol 90:300-308; Toyoma et al., 1982: Proc Natl Acad Sci USA 79:6556-6560]. Quail skeletal myoblasts were isolated from 10-day-old embryos and grown in the presence or absence of taxol. Taxol inhibited the formation of multinucleated elongated myotubes. Myocytes cultured in the continual presence of taxol progressed from rounded to stellate shapes. Groups of myocytes that were clustered together after the isolation procedure fused in the presence of taxol but did not form elongated myotubes. Actin filaments and actin-binding proteins were detected with several different fluorescent probes in all myofibrils that formed in the presence of taxol. The Z-bands contained both alpha-actinin and titin, and the typical arrays of A-Bands were always associated with actin filaments in the myofibrils. Myofibril formation was followed by fixing cells each day in culture and staining with probes for actin, muscle-specific alpha-actinin, myosin II, nebulin, troponin, tropomyosin, and non-muscle myosin II. Small linear aggregates of alpha-actinin or Z-bodies, premyofibrils, were detected at the edges of the myocytes and in the arms of the taxol-treated cells and were always associated with actin filaments. Non-muscle myosin II was detected at the edges of the taxol-treated cells. Removal of the taxol drug led to the cells assuming a normal compact elongated shape. During the recovery process, additional myofibrils formed at the spreading edges of these elongated and thicker myotubes. Staining of these taxol-recovering cells with specific fluorescent reagents reveals three different classes of actin fibers. These results are consistent with a model of myofibrillogenesis that involves the transition of premyofibrils to mature myofibrils.

Animals↗